Polynucleotide synthesis methods, kits and systems

文档序号：927593 发布日期：2021-03-02 浏览：17次中文

阅读说明：本技术 多核苷酸合成方法、试剂盒和系统 (Polynucleotide synthesis methods, kits and systems ) 是由约翰·米尔顿索比亚·纳亚简·里德尔大柿亮祐于 2019-07-19 设计创作，主要内容包括：本发明涉及根据预定的核苷酸序列合成多核苷酸分子的新方法。本发明还涉及合成后组装合成多核苷酸的方法,以及用于进行合成和/或组装方法的系统和试剂盒。(The present invention relates to a novel method for synthesizing polynucleotide molecules based on a predetermined nucleotide sequence. The invention also relates to methods of post-synthesis assembly of synthetic polynucleotides, as well as systems and kits for performing the methods of synthesis and/or assembly.)

1. An in vitro method of synthesizing a double stranded polynucleotide having a predetermined sequence, the method comprising performing repeated cycles of synthesis wherein in each cycle:

(A) elongating a first strand of a double-stranded polynucleotide by adding a first nucleotide of a predetermined sequence by the action of a ligase in a blunt-end ligation reaction;

(B) extending a second strand of the double-stranded polynucleotide hybridized to the first strand by adding a second nucleotide of the predetermined sequence by a nucleotidyl transferase or a polymerase; and

Wherein the first and second nucleotides of the predetermined sequence of each cycle remain in the double-stranded polynucleotide after cleavage.

2. The method of claim 1, wherein, in each cycle, the first nucleotide is a partner nucleotide of the second nucleotide, and wherein, when incorporated into the double-stranded polynucleotide, the first and second nucleotides form a nucleotide pair.

3. The method of claim 1 or claim 2, wherein the cleavage site is defined by a polynucleotide sequence comprising universal nucleotides.

4. A method according to any one of the preceding claims, wherein in each cycle a cleavage site is created in the double stranded polynucleotide prior to extension of the second strand.

5. The method of claim 4, wherein during step (A), a universal nucleotide is incorporated into the first strand of the double-stranded polynucleotide by the action of a ligase to define the cleavage site.

6. The method according to claim 5, wherein the first nucleotide and the universal nucleotide are components of a polynucleotide linker molecule, and wherein, in step (A), in the blunt-end ligation reaction, the polynucleotide linker molecule is ligated to the double-stranded polynucleotide by the action of a ligase, and wherein, when the polynucleotide linker molecule is ligated to the double-stranded polynucleotide, the first strand of the double-stranded polynucleotide is extended and the cleavage site is generated.

7. The method of any one of the preceding claims, wherein, in a given cycle of synthesis, the cycled second nucleotide added to the second strand of the double-stranded polynucleotide comprises a reversible terminator group that prevents further extension by the enzyme, and wherein the reversible terminator group is removed from the pooled second nucleotides of the cycle prior to addition in the next cycle of synthesis of the second nucleotide of the next cycle.

8. The method of any one of claims 2 to 7, comprising performing a first synthesis loop comprising:

(1) providing a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, wherein the synthetic strand comprises a primer strand portion, and wherein the support strand is a first strand of the double-stranded polynucleotide and the synthetic strand is a second strand of the double-stranded polynucleotide;

(2) ligating a double-stranded polynucleotide linker molecule to a scaffold polynucleotide by the action of a ligase in a blunt-end ligation reaction, the polynucleotide linker molecule comprising a support strand and an auxiliary strand hybridized thereto, and further comprising a complementary ligation end comprising:

(i) a universal nucleotide and a first nucleotide of a predetermined sequence in the support strand; and

(ii) a non-ligatable terminal nucleotide in the helper strand;

wherein upon ligation, a first strand of the double-stranded polynucleotide is extended with a first nucleotide and a cleavage site is generated by combining universal nucleotides into the first strand;

(3) extending the ends of the primer strand portion of the synthetic strand of the double-stranded scaffold polynucleotide by combining second nucleotides of the predetermined sequence under the action of the nucleotidyl transferase or polymerase, the second nucleotides comprising a reversible terminator group that prevents further extension by the enzyme, wherein the second nucleotides are partners of the first nucleotides, and wherein, upon combination, the second nucleotides and the first nucleotides form nucleotide pairs;

(4) Cleaving the ligated scaffold polynucleotide at a cleavage site, wherein cleaving comprises cleaving the support strand and removing the universal nucleotides from the scaffold polynucleotide to provide a cleaved double stranded scaffold polynucleotide comprising the combined nucleotide pairs; and

(5) removing the reversible terminator group from the second nucleotide;

the method further comprises performing a further synthesis cycle comprising:

(6) ligating an additional double stranded polynucleotide linker molecule to the cleaved scaffold polynucleotide by the action of a ligase in a blunt end ligation reaction, the polynucleotide linker molecule comprising a support strand and a helper strand hybridized thereto, and further comprising a complementary ligation end, the ligation end comprising:

(i) universal nucleotides in the support strand and the first nucleotides of further synthesis cycles; and

(ii) a non-ligatable terminal nucleotide in the helper strand;

wherein upon ligation, a first strand of the double-stranded polynucleotide is extended with a first nucleotide of a further synthesis cycle and a cleavage site is generated by combining universal nucleotides into the first strand;

(7) extending the ends of the primer strand portions of the synthetic strands of the double-stranded scaffold polynucleotide by combining the second nucleotides of the further synthesis cycles under the action of the nucleotidyl transferase enzyme or polymerase, the second nucleotides comprising a reversible terminator group that prevents further extension by the enzyme, wherein the second nucleotides of the further synthesis cycles are partners for the first nucleotides of the further synthesis cycles, and wherein, upon combination, the second and first nucleotides of the further cycles form further nucleotide pairs;

(8) Cleaving the ligated scaffold polynucleotide at a cleavage site, wherein cleaving comprises cleaving the support strand and removing the universal nucleotide from the scaffold polynucleotide to provide a cleaved double stranded scaffold polynucleotide comprising the incorporated first and additional nucleotide pairs;

(9) removing the reversible terminator group from the second nucleotide; and

(10) repeating steps 6 to 9 a plurality of times to provide the double-stranded polynucleotide having a predetermined nucleotide sequence.

9. The method of claim 8, wherein the reversible terminator group is alternatively removed from the second nucleotide prior to the step of cleaving the linked scaffold polynucleotide at the cleavage site in any one, more or all cycles of synthesis.

10. The method of claim 8 or claim 9, wherein:

(a) in the first cycle of ligation steps (step 2) and all further cycles of ligation steps, the complementary linkers of the polynucleotide linker molecule are configured such that:

i. the first nucleotide of the predetermined sequence of the cycle is the terminal nucleotide of the support strand, is located at nucleotide position n in the support strand, and is paired with the terminal nucleotide of the auxiliary strand;

The universal nucleotide is the penultimate nucleotide of the support strand, paired with the penultimate nucleotide of the auxiliary strand at nucleotide position n +1 of the support strand; and

the terminal nucleotide of the auxiliary strand is a non-ligatable nucleotide;

wherein position n is the nucleotide position relative to the second nucleotide of the predetermined sequence of the cycles after the second nucleotide is combined, and wherein position n +1 is the next nucleotide position in the support strand relative to position n in a direction away from the complementary linker; and wherein, upon ligation, the terminal nucleotide of the support strand of the polynucleotide linker molecule is ligated to the terminal nucleotide of the scaffold polynucleotide adjacent to the primer strand portion of the synthetic strand and a single-stranded break is created between the terminal nucleotide of the helper strand and the primer strand portion of the synthetic strand;

(b) in the extension step (step 3) of the first cycle and all other cycles, the second nucleotide of the cycle is incorporated into and paired with a second strand opposite to the first nucleotide in the first strand;

(c) in the cleavage step of the first cycle (step 4) and in all other cycles, the support strand of the ligated scaffold polynucleotide is cleaved between positions n +1 and n, thereby releasing the polynucleotide linker molecule from the scaffold polynucleotide and leaving the first nucleotide of the cycle ligated to the first strand of the cleaved scaffold polynucleotide and paired with the second nucleotide of the cycle, whereby the position occupied by the first nucleotide of the cycle in the support strand of the cleaved scaffold polynucleotide is defined as nucleotide position n-1 in the next synthesis cycle.

11. The method of claim 8 or claim 9, wherein:

(a) in the first cycle of ligation steps (step 2) and all further cycles of ligation steps, the complementary linkers of the polynucleotide linker molecule are configured such that:

the universal nucleotide occupies nucleotide position n +2 in the support strand and pairs with a partner nucleotide in the helper strand; and

the terminal nucleotide of the auxiliary strand is a non-ligatable nucleotide;

wherein position n is the nucleotide position relative to the second nucleotide of the predetermined sequence of the cycles after the second nucleotide is merged, and wherein position n +2 is the second nucleotide position relative to position n in the support strand in a direction away from the complementary linker; and wherein, upon ligation, the terminal nucleotide of the support strand of the polynucleotide linker molecule is ligated to the terminal nucleotide of the scaffold polynucleotide adjacent to the primer strand portion of the synthetic strand and a single-stranded break is created between the terminal nucleotide of the helper strand and the primer strand portion of the synthetic strand;

(b) In the extension step (step 3) of the first cycle and all other cycles, the second nucleotide of the cycle is incorporated into and paired with a second strand opposite to the first nucleotide in the first strand;

12. The method of claim 8 or claim 9, wherein:

(a) in the first cycle of ligation steps (step 2) and all further cycles of ligation steps, the complementary linkers of the polynucleotide linker molecule are configured such that:

The universal nucleotide occupies nucleotide position n +2+ x in the support strand and pairs with a partner nucleotide in the helper strand; and

the terminal nucleotide of the auxiliary strand is a non-ligatable nucleotide;

wherein position n is the nucleotide position relative to the second nucleotide of the predetermined sequence of the cycles after the second nucleotide is merged, and wherein position n +2 is the second nucleotide position in the support strand relative to position n in the direction away from the complementary ligation end, and wherein x is the number of nucleotide positions relative to position n +2 in the direction away from the complementary ligation end, wherein the number is an integer of 1 to 10 or more; and wherein, upon ligation, the terminal nucleotide of the support strand of the polynucleotide linker molecule is ligated to the terminal nucleotide of the scaffold polynucleotide adjacent to the primer strand portion of the synthetic strand and a single-stranded break is created between the terminal nucleotide of the helper strand and the primer strand portion of the synthetic strand;

(c) In the cleavage step of the first cycle (step 4) and in all other cycles, the support strand of the ligated scaffold polynucleotide is cleaved between positions n +1 and n, thereby releasing the polynucleotide linker molecule from the scaffold polynucleotide and leaving the first nucleotide of the cycle ligated to the first strand of the cleaved scaffold polynucleotide and paired with the second nucleotide of the cycle, whereby the position occupied by the first nucleotide of the cycle in the support strand of the cleaved scaffold polynucleotide is defined as nucleotide position n-1 in the next synthesis cycle.

13. The method according to any one of claims 8 and 10 to 12, wherein the method is modified such that

(i) In step (2), the polynucleotide linker molecule has a complementary linker comprising a first nucleotide of the predetermined sequence of the first cycle and further comprising one or more additional nucleotides of the predetermined sequence of the first cycle;

(ii) in step (3), extending the ends of the primer strand portion of the synthetic strand of the double-stranded scaffold polynucleotide by combining second nucleotides of the predetermined sequence of the first cycle under the action of the nucleotidyl transferase or polymerase, and wherein the ends of the primer strand portions are further extended by combining one or more additional nucleotides of the predetermined sequence of the first cycle under the action of the nucleotidyl transferase enzyme or polymerase, wherein each of the second nucleotide and the additional nucleotide of the first cycle comprises a reversible terminator group that prevents further extension by the enzyme, and wherein, after each further extension, removing the reversible terminator group from the nucleotide before merging the next nucleotide;

(iii) In step (4), after cleaving, retaining the first, second and other nucleotides of the first cycle of the predetermined sequence in the cleaved scaffold polynucleotide;

(iv) in step (6), the polynucleotide linker molecule has a complementary linker comprising the additional cyclic predetermined sequence of first nucleotides and further comprising the additional cyclic predetermined sequence of one or more additional nucleotides;

(v) in step (6), extending the ends of the primer strand portion of the synthetic strand of the double-stranded scaffold polynucleotide by combining the further cycles of the second nucleotide of the predetermined sequence under the action of the nucleotidyl transferase or polymerase, and wherein the ends of the primer strand portions are further extended by combining one or more additional nucleotides of the predetermined sequence of the further cycles under the action of the nucleotidyl transferase enzyme or polymerase, wherein each of the second nucleotide and the additional nucleotide of the further cycle comprises a reversible terminator group that prevents further extension by the enzyme, and wherein, after each further extension, removing the reversible terminator group from the nucleotide before merging the next nucleotide;

(vi) In step (8), after cleaving, the additional cycles of the first, second and additional nucleotides of the predetermined sequence are retained in the cleaved scaffold polynucleotide.

14. The method according to claim 13, wherein the complementary linking end of the polynucleotide linking molecule is configured such that in steps (4) and (8) prior to cleavage, the universal nucleotide occupies a position in the support strand that is: the position is the next nucleotide position in the support strand after the nucleotide positions of the first and further nucleotides in a direction away from the complementary linkers, and the support strand is cleaved between the position occupied by the last further nucleotide and the position occupied by the universal nucleotide.

15. The method according to claim 13, wherein the complementary linking end of the polynucleotide linking molecule is configured such that in steps (4) and (8) prior to cleavage, the universal nucleotide occupies a position in the support strand that is: the position is the next nucleotide position in the support strand after the nucleotide positions of the first and further nucleotides in a direction away from the complementary linkers, and the support strand is cleaved between the position occupied by the last further nucleotide in the support strand and the position occupied by the next nucleotide.

16. The method of claim 14 or claim 15, wherein the reversible terminator group of the last additional nucleotide of another cycle to be combined is alternatively removed from the last nucleotide prior to the step of cleaving the linked scaffold polynucleotides at the cleavage site.

17. The method of any one of the preceding claims, wherein the partner nucleotide paired with the first nucleotide of the predetermined sequence in any one, more or all cycles of synthesis is a nucleotide complementary to the first nucleotide, preferably a nucleotide naturally complementary to the first nucleotide.

18. A method according to any one of claims 8 to 17, wherein the scaffold polynucleotide is provided prior to steps (3) and/or (7) in any one, more or all cycles of synthesis, the scaffold polynucleotide comprising a synthetic strand and a support strand hybridised to the synthetic strand, and wherein the synthetic strand provided is free of an auxiliary strand.

19. The method of any one of claims 8 to 18, wherein the auxiliary strand portion of the synthetic strand is removed from the scaffold polynucleotide prior to steps (3) and/or (7) in any one, more or all cycles of synthesis.

20. The method of claim 19, wherein the auxiliary strand portion of the synthetic strand is removed from the scaffold polynucleotide by: (i) heating the scaffold polynucleotide to a temperature of about 80 ℃ to about 95 ℃ and separating the auxiliary strand portion from the scaffold polynucleotide, (ii) treating the scaffold polynucleotide with a urea solution, such as 8M urea, and separating the auxiliary strand portion from the scaffold polynucleotide, (iii) treating the scaffold polynucleotide with a formamide or a formamide solution, such as 100% formamide, and separating the auxiliary strand portion from the scaffold polynucleotide, or (iv) contacting the scaffold polynucleotide with a single stranded polynucleotide molecule comprising a nucleotide sequence region complementary to the sequence of the auxiliary strand portion, thereby competitively inhibiting hybridization of the auxiliary strand portion to the scaffold polynucleotide.

21. The method of any one of claims 1 to 10, 13, 14, and 16 to 20, wherein each lysis step comprises a two-step lysis process, wherein each lysis step comprises: a first step comprising removing the universal nucleotide thereby forming an abasic site; and a second step comprising cleaving the support strand at the base-free site.

22. The method of claim 21, wherein the first step is performed with a nucleotide excising enzyme.

23. The method of claim 22, wherein the nucleotidic excisionase is 3-methyladenine DNA glycosylase.

24. The method of claim 23, wherein the nucleotide excising enzyme is:

i. human alkyl adenine DNA glycosylase (hAAG); or

Uracil DNA Glycosylase (UDG).

25. The process according to any one of claims 21 to 24, wherein the second step is carried out using a chemical substance which is a base.

26. The method of claim 25, wherein the base is NaOH.

27. The method of any one of claims 21 to 24, wherein the second step is performed using an organic chemical having no base site cleavage activity.

28. The method of claim 27, wherein the organic chemical is N, N' -dimethylethylenediamine.

29. The method of any one of claims 21 to 24, wherein the second step is performed with an enzyme having no base site lyase activity, optionally wherein the enzyme having no base site lyase activity is:

(i) AP endonuclease 1;

(ii) endonuclease iii (nth); or

(iii) Endonuclease VIII.

30. The method of any one of claims 1 to 10, 13, 14 and 16 to 20, wherein each cleavage step comprises a one-step cleavage process comprising removing the universal nucleotide with a cleaving enzyme, wherein the enzyme is

(i) Endonuclease III;

(ii) an endonuclease VIII;

(iii) formamidopyrimidine DNA glycosylase (Fpg); or

(iv) 8-oxoguanine DNA glycosylase (hOGG 1).

31. The method of any one of claims 1 to 9, 11, 13, and 15 to 20, wherein the cleaving step comprises cleaving the support strand with an enzyme.

32. The method of claim 31, wherein the enzyme cleaves the support strand between nucleotide positions n +1 and n.

33. A method according to claim 31 or claim 32, wherein the enzyme is endonuclease V.

34. A method according to any one of the preceding claims wherein both strands of the synthetic double stranded polynucleotide are DNA strands.

35. The method of claim 34, wherein the pooled nucleotides are dntps.

36. The method of claim 35, wherein the incorporated nucleotides are dntps comprising a reversible terminator group.

37. The method of claim 36, wherein the one or more combined nucleotides comprising a reversible terminator group is a 3' -O-allyl-dNTP.

38. The method of claim 36, wherein the one or more combined nucleotides comprising a reversible terminator group is 3' -O-azidomethyl-dNTP.

39. The method of any one of claims 1 to 33, wherein one strand of the synthetic double-stranded polynucleotide is a DNA strand and the other strand of the synthetic double-stranded polynucleotide is an RNA strand.

40. The method of claim 39, wherein the synthetic strand is an RNA strand and the support strand is a DNA strand.

41. The method of claim 40, wherein the combined nucleotides are NTPs.

42. The method of claim 41, wherein the incorporated nucleotide is an NTP comprising a reversible terminator group.

43. The method of claim 42, wherein the combined nucleotides comprising a reversible terminator group are 3' -O-allyl-NTPs.

44. The method of claim 42, wherein the combined nucleotides comprising a reversible terminator group are 3' -O-azidomethyl-NTPs.

45. The method according to any one of claims 1 to 38, wherein the polymerase is a DNA polymerase, preferably a modified DNA polymerase, having an enhanced ability to incorporate dntps comprising a reversible terminator group compared to an unmodified polymerase.

46. A method according to claim 45, wherein the polymerase is a variant of a native DNA polymerase from Pyrococcus species 9 ° N, preferably species 9 ° N-7.

47. The method of any one of claims 1 to 33 and 39 to 44, wherein the polymerase is an RNA polymerase, such as T3 or T7 RNA polymerase, optionally a modified RNA polymerase, having an enhanced ability to incorporate NTPs comprising a reversible terminator group as compared to the unmodified polymerase.

48. The method of any one of the preceding claims, wherein the transferase has terminal transferase activity, optionally wherein the enzyme is terminal nucleotidyl transferase, terminal deoxynucleotidyl transferase (TdT), pol lambda, pol micro or Φ 29DNA polymerase.

49. The method of any one of claims 7 to 48, wherein the step of removing the reversible terminator group from the second nucleotide is performed using tris (carboxyethyl) phosphine (TCEP).

50. The method of any one of the preceding claims, wherein the ligase is T3 DNA ligase or T4 DNA ligase.

51. The method of any one of claims 8 to 50, wherein, in any one, more or all cycles of synthesis in steps (1)/(6), the synthetic strand comprising the primer strand portion and the portion of the support strand to which it hybridises in the scaffold polynucleotide are connected by a hairpin loop.

52. A method according to any one of claims 8 to 50, wherein, in the polynucleotide linker molecule, the portions of the auxiliary strand and the supporting strand which hybridise to the auxiliary strand are linked by a hairpin loop at the end opposite the complementary linking end, during any one, more or all cycles of synthesis in steps (2)/(6).

53. A method according to any one of claims 8 to 50, wherein, in any one, more or all synthesis cycles:

c) in the step (1)/(6) of the scaffold polynucleotide, the synthetic strand comprising the primer strand portion and the support strand portion hybridized therewith are linked by a hairpin loop; and

d) in step (2)/(6), in the polynucleotide linker molecule, the auxiliary strand and the part of the supporting strand hybridizing with the auxiliary strand are ligated by a hairpin loop at the end opposite to the complementary linker.

54. The method according to any one of claims 8 to 50, wherein, in steps (1) and (6), the synthetic strand comprising the primer strand portion and/or the portion of the support strand to which it hybridizes are tethered to a common surface.

55. The method of claim 54, wherein the primer strand portion and the portion of the support strand to which the primer strand portion hybridizes comprise a cleavable linker, wherein, after synthesis, the linker is cleavable to detach the double-stranded polynucleotide from the surface.

56. The method of claims 52 and 53, wherein in steps (1)/(2) and (6) the hairpin loops in the scaffold polynucleotide are tethered to a surface.

57. The method of claim 56, wherein the hairpin loop is tethered to a surface via a cleavable linker, wherein after synthesis the linker is cleavable to detach the double stranded polynucleotide from the surface.

58. The method of claim 55 or claim 57, wherein the cleavable linker is a UV cleavable linker.

59. The method of any one of claims 54 to 58, wherein the surface is a microparticle.

60. The method of any one of claims 54 to 58, wherein the surface is a flat surface.

61. The method of any one of claims 56-60, wherein the surface comprises a gel.

62. The method of claim 61, wherein the surface comprises a polyacrylamide surface, such as about 2% polyacrylamide, preferably wherein the polyacrylamide surface is coupled to a solid support, such as glass.

63. The method of any one of claims 54 to 62, wherein the synthetic strand comprising the primer strand portion and the portion of the support strand to which it hybridizes are tethered to the common surface via one or more covalent bonds.

64. The method of claim 63, wherein the one or more covalent bonds are formed between functional groups on the common surface and functional groups on the scaffold molecules, wherein the functional groups on the scaffold molecules are amine groups, thiol groups, thiophosphate groups, or thioamide groups.

65. The method of claim 64, wherein the functional group on the common surface is a bromoacetyl group, optionally wherein the bromoacetyl group is disposed on a polyacrylamide surface derivatized with N- (5-bromoacetamidopentyl) acrylamide (BRAPA).

66. A method according to any one of the preceding claims, wherein the synthesis cycle is performed in a droplet within a microfluidic system.

67. The method of claim 66, the microfluidic system being an electrowetting system.

68. The method of claim 67, wherein the microfluidic system is an electrowetting on dielectric system (EWOD).

69. A method according to any one of the preceding claims wherein, following synthesis, the strands of the double stranded polynucleotide are separated to provide a single stranded polynucleotide having a predetermined sequence.

70. The method according to any one of the preceding claims, wherein the double stranded polynucleotide or a region of the double stranded polynucleotide is amplified after synthesis, preferably by PCR.

71. A method of assembling a polynucleotide having a predetermined sequence, the method comprising: performing the method according to any one of the preceding claims to synthesize a first polynucleotide having a predetermined sequence and one or more additional polynucleotides having a predetermined sequence; and ligating the first polynucleotide and the one or more additional polynucleotides together.

72. The method of claim 71, wherein the first polynucleotide and the one or more additional polynucleotides are double stranded.

73. The method of claim 71, wherein the first polynucleotide and the one or more additional polynucleotides are single stranded.

74. A method according to any one of claims 71 to 73, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved to produce compatible ends and ligated together, preferably by a ligation reaction.

75. The method of claim 74, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved at a cleavage site by a restriction enzyme.

76. The method of any one of claims 67 to 75, wherein the synthesizing and/or assembling steps are performed in droplets within a microfluidic system.

77. The method of claim 76, wherein the assembling step comprises: providing a first droplet comprising a first synthetic polynucleotide having a predetermined sequence and a second droplet comprising one or more additional synthetic polynucleotides having a predetermined sequence, wherein the droplets are brought into contact with each other, and wherein the synthetic polynucleotides are ligated together, thereby assembling a polynucleotide comprising the first nucleotide and the one or more additional polynucleotides.

78. The method of claim 77, wherein the synthesizing step is performed by providing a plurality of droplets, each droplet comprising a reactive agent corresponding to a step of the synthesis cycle, and sequentially delivering the droplets to the scaffold polynucleotide according to the steps of the synthesis cycle.

79. The method of claim 78, wherein after delivering a droplet and before delivering the next droplet, a washing step is performed to remove excess reactive agent.

80. The method according to claim 78 and 79, wherein the microfluidic system is an electrowetting system.

81. The method of claim 80, wherein the microfluidic system is an electrowetting on dielectric system (EWOD).

82. The method of any one of claims 77 to 81, wherein the synthesizing and assembling steps are performed in the same system.

83. A polynucleotide synthesis system for performing the method of any one of claims 1 to 82, the system comprising: (a) an array of reaction regions, wherein each reaction region comprises at least one scaffold polynucleotide; and (b) means for delivering a reactive agent to the reaction zone; and optionally (c) a means for cleaving the synthesized double stranded polynucleotide from the scaffold polynucleotide.

84. The system of claim 83, further comprising: means for providing the reaction reagents in the form of droplets; and means for delivering the droplets to the scaffold polynucleotide according to a synthesis cycle.

85. A kit for use with the system of claim 83 or 84 and for carrying out the method of any one of claims 1 to 82, the kit comprising volumes of reaction reagents corresponding to steps of the synthesis cycle.

86. A method of preparing a polynucleotide microarray, wherein the microarray comprises a plurality of reaction regions, each region comprising one or more polynucleotides having a predetermined sequence, the method comprising:

a) providing a surface comprising a plurality of reaction regions, each region comprising one or more double-stranded anchor or scaffold polynucleotides, and

b) performing a synthesis cycle at each reaction region according to the method of any one of claims 1 to 68, thereby synthesizing one or more double stranded polynucleotides having a predetermined sequence at each region.

87. The method of claim 86, wherein after synthesis, the strands of the double stranded polynucleotides are separated to provide a microarray, wherein each region comprises one or more single stranded polynucleotides having a predetermined sequence.

Technical Field

The present invention relates to a novel method for synthesizing polynucleotide molecules based on a predetermined nucleotide sequence. The invention also relates to methods of post-synthesis assembly of synthetic polynucleotides, as well as systems and kits for performing the methods of synthesis and/or assembly.

Background

There are two main methods for the synthesis and assembly of polynucleotide molecules, particularly DNA.

Phosphoramidite chemistry is a synthetic method of assembling chemically activated T, C, A or G monomers into oligonucleotides of approximately 100/150 bases in length by a stepwise process. The chemical reaction steps are highly sensitive and the conditions alternate between completely anhydrous (completely absent water), aqueous oxidation and acidic conditions (Roy and carothers, Molecules,2013,18, 14268-. If the reagents from the previous reaction step have not been completely removed, this will be detrimental to future synthesis steps. Thus, this synthetic method is limited to producing polynucleotides of about 100 nucleotides in length.

The polymerase synthesis method uses a polymerase to synthesize the complementary strand of the DNA template using T, C, A and G triphosphate. The reaction conditions are aqueous and mild, and the method can be used to synthesize DNA polynucleotides thousands of bases in length. The main disadvantage of this method is that single-and double-stranded DNA cannot be synthesized de novo by this method, which requires a DNA template from which copies are made. (Kosuri and Church, Nature Methods,2014,11, 499-507).

Thus, previous methods cannot be used to synthesize double-stranded DNA de novo without the aid of a copy of a pre-existing template molecule.

The present inventors have developed novel methods by which single-and double-stranded polynucleotide molecules can be synthesized de novo in a stepwise manner without the need to replicate pre-existing template molecules. These methods also avoid the extreme conditions associated with phosphoramidite chemistry, but instead proceed under mild aqueous conditions near neutral pH. Such methods also enable de novo synthesis of single-or double-stranded polynucleotide molecules with current synthetic methods>The 100 mer nucleotide length has the potential of 10 compared to the entire genome⁸Improved, provides a wide range of possible applications in synthetic biology.

Disclosure of Invention

The present invention provides an in vitro method of synthesizing a double stranded polynucleotide having a predetermined sequence, the method comprising performing repeated cycles of synthesis wherein in each cycle:

(A) elongating a first strand of a double-stranded polynucleotide by adding a first nucleotide of a predetermined sequence by the action of a ligase in a blunt-end ligation reaction;

Wherein the first and second nucleotides of the predetermined sequence of each cycle remain in the double-stranded polynucleotide after cleavage.

In any of the above methods, in each cycle, the first nucleotide can be a partner nucleotide of the second nucleotide, and wherein the first and second nucleotides form a nucleotide pair when combined in a double-stranded polynucleotide.

In any of the above methods, the cleavage site may be defined by a polynucleotide sequence comprising universal nucleotides.

In any of the above methods, in each cycle, a cleavage site may be created in the double-stranded polynucleotide prior to extension of the second strand.

In any of the above methods, during step (a), a universal nucleotide may be incorporated into the first strand of the double-stranded polynucleotide by the action of a ligase to define the cleavage site.

In any of the above methods, the first nucleotide and the universal nucleotide may be components of a polynucleotide linker molecule, and wherein, in step (a), the polynucleotide linker molecule is ligated to the double stranded polynucleotide by the action of a cleaving enzyme in a blunt end ligation reaction, and wherein, upon ligation of the polynucleotide linker molecule to the double stranded polynucleotide, the first strand of the double stranded polynucleotide is extended and a cleavage site is created.

In any of the above methods, in a given synthesis cycle, the second nucleotide of the cycle added to the second strand of the double-stranded polynucleotide may comprise a reversible terminator group, which prevents further extension of the enzyme, and wherein the reversible terminator group is removed from the second nucleotide incorporated in the cycle prior to addition in the next cycle of synthesis of the second nucleotide of the next cycle.

In any of the above methods, the method may include performing a first synthesis loop comprising:

(2) Ligating a double-stranded polynucleotide linker molecule to a scaffold polynucleotide by the action of a ligase in a blunt-end ligation reaction, the polynucleotide linker molecule comprising a support strand and an auxiliary strand hybridized thereto, and further comprising a complementary ligation end comprising:

(i) a universal nucleotide and a first nucleotide of a predetermined sequence in the support strand; and

(ii) a non-ligatable terminal nucleotide in the helper strand;

(4) cleaving the ligated scaffold polynucleotide at a cleavage site, wherein cleaving comprises cleaving the support strand and removing the universal nucleotides from the scaffold polynucleotide to provide a cleaved double stranded scaffold polynucleotide comprising the combined nucleotide pairs; and

(5) Removing the reversible terminator group from the second nucleotide;

the method further comprises performing a further synthesis cycle comprising:

(i) universal nucleotides in the support strand and the first nucleotides of further synthesis cycles; and

(ii) a non-ligatable terminal nucleotide in the helper strand;

(9) removing the reversible terminator group from the second nucleotide; and

(10) repeating steps 6 to 9 a plurality of times to provide the double-stranded polynucleotide having a predetermined nucleotide sequence. In any such method, the reversible terminator group may alternatively be removed from the second nucleotide prior to the step of cleaving the linked scaffold polynucleotide at the cleavage site in any one, more or all cycles of synthesis.

In such a method as described above:

(a) in the first cycle of the ligation step (step 2) and in all other cycles of the ligation step, the complementary linkers of the polynucleotide linker molecules may be configured such that:

the universal nucleotide is the penultimate nucleotide of the support strand, paired with the penultimate nucleotide of the auxiliary strand at nucleotide position n +1 of the support strand; and

The terminal nucleotide of the auxiliary strand is a non-ligatable nucleotide;

Alternatively, in such a method as described above:

(a) in the first cycle of the ligation step (step 2) and in all other cycles of the ligation step, the complementary linkers of the polynucleotide linker molecules may be configured such that:

the universal nucleotide occupies nucleotide position n +2 in the support strand and pairs with a partner nucleotide in the helper strand; and

the terminal nucleotide of the auxiliary strand is a non-ligatable nucleotide;

Alternatively, in such a method as described above:

(a) in the first cycle of the ligation step (step 2) and in all other cycles of the ligation step, the complementary linkers of the polynucleotide linker molecules may be configured such that:

The universal nucleotide occupies nucleotide position n +2+ x in the support strand and pairs with a partner nucleotide in the helper strand; and

the terminal nucleotide of the auxiliary strand is a non-ligatable nucleotide;

wherein position n is the nucleotide position relative to the second nucleotide of the predetermined sequence of the cycles after the second nucleotide is merged, and wherein position n +2 is the second nucleotide position in the support strand relative to position n in the direction away from the complementary ligation end, and wherein x is the number of nucleotide positions relative to position n +2 in the direction away from the complementary ligation end, wherein the number is an integer of 1 to 10 or more; and wherein, upon ligation, the terminal nucleotide of the support strand of the polynucleotide linker molecule is ligated to the terminal nucleotide of the scaffold polynucleotide adjacent to the primer strand portion of the synthetic strand and a single-stranded break is created between the terminal nucleotide of the helper strand and the primer strand portion of the synthetic strand;

In any of the above methods, the method may be modified to:

(i) in step (2), the polynucleotide linker molecule has a complementary linker comprising a first nucleotide of the predetermined sequence of the first cycle and further comprising one or more additional nucleotides of the predetermined sequence of the first cycle;

(iii) In step (4), after cleaving, retaining the first, second and other nucleotides of the first cycle of the predetermined sequence in the cleaved scaffold polynucleotide;

(vi) In step (8), after cleaving, the additional cycles of the first, second and additional nucleotides of the predetermined sequence are retained in the cleaved scaffold polynucleotide.

In any such method, the complementary linking end of the polynucleotide linking molecule may be configured such that in steps (4) and (8) prior to cleavage, the universal nucleotide occupies a position in the support strand that is: the position is the next nucleotide position in the support strand after the nucleotide positions of the first and further nucleotides in a direction away from the complementary linkers, and the support strand is cleaved between the position occupied by the last further nucleotide and the position occupied by the universal nucleotide. Alternatively, in any such method, the complementary ligation end of the polynucleotide ligation molecule may be configured such that the complementary ligation end of the polynucleotide ligation molecule is configured such that, in steps (4) and (8) prior to cleavage, the universal nucleotide occupies such a position in the support strand that: the position is the next nucleotide position in the support strand after the nucleotide positions of the first and further nucleotides in a direction away from the complementary linkers, and the support strand is cleaved between the position occupied by the last further nucleotide in the support strand and the position occupied by the next nucleotide. In any such method, the reversible terminator group of the last nucleotide of the additional cycle to be bound may be removed from the last nucleotide prior to the step of cleaving the linked scaffold polynucleotide at the cleavage site.

In any of the methods described above and herein, the partner nucleotide that pairs with the first nucleotide of the predetermined sequence in any one, more or all cycles of synthesis may be a nucleotide that is complementary to the first nucleotide, preferably naturally complementary.

In any of the methods described above and herein, a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, and wherein the synthetic strand is provided without an auxiliary strand, may be provided prior to step (2) and/or (6) in any one, more or all of the synthesis cycles. The synthetic strand may be removed from the scaffold polynucleotide prior to steps (2) and/or (6) in any one, more or all cycles of synthesis.

In any of the methods described above and herein, in any one, more or all cycles of synthesis, the helper strand portion of the synthetic strand may be removed from the scaffold polynucleotide after the step of ligating the double-stranded polynucleotide linker molecule to the cleaved scaffold polynucleotide and before incorporating the next nucleotide of the predetermined nucleotide sequence into the synthetic strand of the scaffold polynucleotide. In any such method, the auxiliary strand portion of the synthetic strand may be removed from the scaffold polynucleotide by: (i) heating the scaffold polynucleotide to a temperature of about 80 ℃ to about 95 ℃ and separating the auxiliary strand portion from the scaffold polynucleotide, (ii) treating the scaffold polynucleotide with a urea solution, such as 8M urea, and separating the auxiliary strand portion from the scaffold polynucleotide, (iii) treating the scaffold polynucleotide with a formamide or a formamide solution, such as 100% formamide, and separating the auxiliary strand portion from the scaffold polynucleotide, or (iv) contacting the scaffold polynucleotide with a single stranded polynucleotide molecule comprising a nucleotide sequence region complementary to the sequence of the auxiliary strand portion, thereby competitively inhibiting hybridization of the auxiliary strand portion to the scaffold polynucleotide.

In any such method described above and herein, the support strand of the scaffold polynucleotide is cleaved, in the orientation relative to the universal nucleotide, in the orientation adjacent the primer, a portion of the synthetic strand between the position occupied by the universal nucleotide and the position occupied by the next nucleotide in the support strand, each cleavage step may comprise a two-step cleavage process, wherein each cleavage step may comprise a first step comprising removing the universal nucleotide to form an abasic site, and a second step comprising cleaving the support strand at the abasic site. In any such method, the first step may be performed with a nucleotide excising enzyme. The nucleotidic excisionase may be 3-methyladenine DNA glycosylase. The nucleotidectomy enzyme may be human alkyl adenine DNA glycosylase (hAAG) or Uracil DNA Glycosylase (UDG). In any such method, the second step may be performed with chemicals that act as bases. The base may be NaOH. In any such method, the second step may be performed with an organic chemical having abasic site cleavage activity. The organic chemical may be N, N' -dimethylethylenediamine. In any such method, the second step may be performed with an enzyme having a base-site-free lyase activity, such as an AP endonuclease, endonuclease III (Nth), or endonuclease VIII.

In any such method described above and herein, wherein the support strand of the scaffold polynucleotide is cleaved relative to the universal nucleotide in a direction adjacent to the primer strand portion of the synthetic strand between the position occupied by the universal nucleotide and the position occupied by the next nucleotide in the support strand, each cleavage step may comprise a one-step cleavage process comprising removal of the universal nucleotide with a cleaving enzyme, wherein the enzyme is: endonuclease III, endonuclease VIII, formamidopyrimidine DNA glycosylase (Fpg) or 8-oxoguanine DNA glycosylase (hOGG 1).

In any such method described above and herein, wherein the support strand of the scaffold polynucleotide is cleaved between the position occupied by the next nucleotide in the support strand relative to the universal nucleotide in the direction adjacent to the primer strand portion and the position occupied by the second nucleotide in the support strand relative to the universal nucleotide in the direction adjacent to the primer strand portion, the cleaving step can comprise cleaving the support strand with an enzyme. Such an enzyme may be endonuclease V.

In any of the methods described above and herein, both strands of the synthesized double-stranded polynucleotide can be DNA strands. The synthetic strand and the support strand may be DNA strands. In this case, the incorporated nucleotide is preferably a dNTP, preferably a dNTP comprising a reversible terminator group. In any such method, any one or more or all of the combined nucleotides comprising a reversible terminator group may comprise 3 '-O-allyl-dNTP or 3' -O-azidomethyl-dNTP.

In any of the methods described above and herein, one strand of the synthetic double-stranded polynucleotide can be a DNA strand and the other strand of the synthetic double-stranded polynucleotide can be an RNA strand. The synthetic strand may be an RNA strand and the support strand may be an RNA or DNA strand. In this case, the nucleotide incorporated by the transferase or polymerase is preferably an NTP, preferably an NTP comprising a reversible terminator. In any such method, any one or more or all of the combined nucleotides comprising a reversible terminator group may be 3 '-O-allyl-NTP or 3' -O-azidomethyl-NTP.

In any of the methods described above and herein that involve incorporating nucleotides into a synthesized strand comprising DNA (e.g., incorporating one or more dntps), the enzyme may be a polymerase, preferably a DNA polymerase, more preferably a modified DNA polymerase, that has an enhanced ability to incorporate dntps comprising a reversible terminator group as compared to an unmodified polymerase. The polymerase may be a variant of a native DNA polymerase from a Thermococcus species (Thermococcus) at 9 ℃ N, preferably at 9 ℃ N-7.

In any of the methods described above and herein that involve incorporating nucleotides into a synthetic strand comprising RNA (e.g., incorporating one or more NTPs), the enzyme may be a polymerase, preferably an RNA polymerase, such as T3 or T7 RNA polymerase, more preferably a modified RNA polymerase, that has an enhanced ability to incorporate NTPs comprising a reversible terminator group as compared to an unmodified polymerase.

In any of the methods described above and herein, the first strand of the synthesized double-stranded polynucleotide can be a DNA strand and the second strand of the synthesized double-stranded polynucleotide can be an RNA strand. Alternatively, the first strand of the synthetic double-stranded polynucleotide can be an RNA strand and the second strand of the synthetic double-stranded polynucleotide can be a DNA strand.

In any of the methods described above and herein, the transferase has terminal transferase activity, optionally wherein the enzyme is a terminal nucleotidyl transferase, a terminal deoxynucleotidyl transferase (TdT), pol lambda, pol micro, or Φ 29DNA polymerase.

In any of the methods described above and herein, the step of removing the reversible terminator group from the first/next nucleotide can be performed with tris (carboxyethyl) phosphine (TCEP).

In any of the methods described above and herein, the step of ligating double stranded polynucleotide linker molecules to the cleaved scaffold polynucleotides is preferably performed using a ligase. The ligase can be T3 DNA ligase or T4 DNA ligase.

In any of the methods described above and herein, the synthetic strand, including the primer strand portion and the support strand portion hybridized thereto, may be joined by a hairpin loop in any one, more or all of the synthesis cycles of steps (1)/(6) in the scaffold polynucleotide.

In any of the methods described above and herein, in any one, more or all of the synthesis cycles in steps (2)/(6), the portions of the auxiliary strand and the support strand that hybridize to the auxiliary strand in the polynucleotide linker molecule may be joined by a hairpin loop at the end opposite the complementary joining end.

In any of the methods described above and herein, in any one, more, or all of the synthesis cycles:

a) in the step (1)/(6) of the scaffold polynucleotide, the synthetic strand comprising the primer strand portion and the support strand portion hybridized therewith are linked by a hairpin loop; and

b) in step (2)/(6), in the polynucleotide linker molecule, the auxiliary strand and the part of the supporting strand hybridizing with the auxiliary strand are ligated by a hairpin loop at the end opposite to the complementary linker.

In any of the methods described above and herein, at least one or more or all of the polynucleotide linker molecules may be provided as a single molecule comprising a hairpin loop connecting the support strand and the auxiliary strand at the end opposite the complementary linker end. In any of the methods described above and herein, the polynucleotide linker molecule of each synthesis cycle may be provided as a single molecule, each molecule comprising a hairpin loop connecting the support strand and the auxiliary strand at the end opposite the complementary linker end.

In any of the methods described above and herein, in steps (1) and (6), the synthetic strands of the scaffold polynucleotide comprising the primer strand portions and/or the portions of the support strand hybridized thereto may be tethered to a common surface. The synthetic strand of the scaffold polynucleotide comprising the portion of the primer strand portion and/or the support strand that hybridizes to the primer strand portion may comprise a cleavable linker, wherein, after synthesis, the linker may be cleaved to detach the double-stranded polynucleotide from the surface.

In any of the methods described above and herein, in steps (1) and (6), the portion of the primer strand of the synthetic strand and the portion of the support strand hybridized thereto may be linked by a hairpin loop, and wherein the hairpin loop is tethered to a surface.

In any of the methods described above and herein, the hairpin loop may be tethered to a surface via a cleavable linker, wherein, after synthesis, the linker may be cleaved to detach the double stranded polynucleotide from the surface. The cleavable linker may be a UV cleavable linker.

In any of the methods described above and herein, the surface to which the polynucleotide is attached can be the surface of a microparticle or a flat surface.

In any of the methods described above and herein, the surface to which the polynucleotide is attached can comprise a gel. The surface may comprise a polyacrylamide surface, for example about 2% polyacrylamide, preferably wherein the polyacrylamide surface is attached to a solid support such as glass.

In any of the methods described above and herein, the synthetic strands comprising the primer strand portion and the support strand portion hybridized thereto may be tethered to a common surface by one or more covalent bonds. One or more covalent bonds may be formed between functional groups on the common surface and functional groups on the scaffold molecules, where the functional groups on the scaffold molecules may be amine groups, thiol groups, phosphorothioate groups, or thioamide groups. The functional group on the common surface may be a bromoacetyl group, optionally wherein the bromoacetyl group is provided on a polyacrylamide surface derivatized with N- (5-bromoacetylpentyl) acrylamide (BRAPA).

In any of the methods described above and herein, the reactions associated with any of the synthesis cycles described above and herein can be performed as droplets within a microfluidic system. The microfluidic system may be an electrowetting system. The microfluidic system may be an electrowetting on dielectric (EWOD).

In any of the methods described above and herein, after synthesis, the strands of the double-stranded polynucleotide can be separated to provide a single-stranded polynucleotide having a predefined sequence.

In any of the methods described above and herein, after synthesis, the double-stranded polynucleotide or region thereof is amplified, preferably by PCR.

The invention also provides a method of assembling a polynucleotide having a predetermined sequence, the method comprising performing any of the synthetic methods described above and herein to synthesize a first polynucleotide having the predetermined sequence and one or more additional polynucleotides having the predetermined sequence and ligating the first polynucleotide and the one or more additional polynucleotides together. The first polynucleotide and the one or more further polynucleotides may preferably comprise different predefined sequences. The first polynucleotide and the one or more additional polynucleotides may be double stranded or may be single stranded. The first polynucleotide and one or more additional polynucleotides may be first cleaved to produce compatible ends, which are then ligated together, e.g., by ligation. The first polynucleotide and the one or more additional polynucleotides may be cleaved at the cleavage site by a restriction enzyme to produce compatible ends.

Any of the in vitro methods described above and herein for synthesizing double-stranded polynucleotides having a predefined sequence, and/or any of the in vitro methods described above and herein for assembling polynucleotides having a predefined sequence, may be performed in droplets within a microfluidic system. In any such method, the method of assembly may comprise an assembly step comprising providing a first droplet comprising a first synthetic polynucleotide having a predefined sequence and a second droplet comprising another one or more synthetic polynucleotides having a predefined sequence, wherein the droplets are brought into contact with each other and wherein the synthetic polynucleotides are linked together, thereby assembling a polynucleotide comprising the first nucleotide and the other one or more polynucleotides. In any such method, the step of synthesizing may be performed by providing a plurality of droplets, each droplet comprising a reactive agent corresponding to a step of a synthesis cycle, and sequentially delivering the droplets to the scaffold polynucleotide according to the steps of the synthesis cycle. In any such method, after delivery of a droplet and before delivery of the next droplet, a washing step may be performed to remove excess reactive agent. In any such method, the microfluidic system may be an electrowetting system. In any such method, the microfluidic system may be an electrowetting on dielectric system (EWOD). In any such method, the synthesis and assembly steps may be performed within the same system.

In any in vitro method for synthesizing a double stranded polynucleotide having a predetermined sequence as described above and herein, the universal nucleotide is inosine or an analogue, variant or derivative thereof and the partner nucleotide of the universal nucleotide in the auxiliary strand is cytosine.

In a related aspect, the invention also provides an in vitro method of extending a double stranded polynucleotide to synthesize a double stranded polynucleotide having a predetermined sequence, the method comprising one or more synthesis cycles, wherein in each synthesis cycle, a universal nucleotide and a first nucleotide of the predetermined sequence are added to a first strand of a double stranded scaffold polynucleotide in a blunt end ligation reaction, a second nucleotide of the predetermined sequence is added to the opposite strand of the scaffold polynucleotide, and the scaffold polynucleotide is cleaved at a cleavage site defined by a sequence comprising the universal nucleotide, wherein, upon cleavage, the universal nucleotide is released from the scaffold polynucleotide and the first and second nucleotides remain in the scaffold polynucleotide.

In a related aspect, the invention also provides the use of universal nucleotides in an in vitro method of extending a double stranded polynucleotide to synthesize a double stranded polynucleotide having a predetermined sequence, wherein, in a synthesis cycle, the universal nucleotide is added to a double-stranded scaffold polynucleotide in a blunt-end ligation reaction to generate a polynucleotide cleavage site in the scaffold polynucleotide, and wherein the scaffold polynucleotide is cleaved to provide sites in the first strand of the scaffold polynucleotide for combining the first nucleotide of the predetermined sequence and optionally one or more further nucleotides of the predetermined sequence in the next cycle, and providing a site in the opposite strand of the scaffold polynucleotide for incorporation of the second nucleotide of the predetermined sequence and optionally one or more further nucleotides of the predetermined sequence in the next cycle.

In a related aspect, the invention also provides the use of a universal nucleotide in an in vitro method of synthesizing a double stranded polynucleotide having a predetermined sequence, wherein the universal nucleotide is used in synthesis cycles to generate polynucleotide cleavage sites in a double stranded scaffold polynucleotide, and wherein cleavage of the scaffold polynucleotide provides sites in each strand of the scaffold polynucleotide for merging one or more nucleotides of the predetermined sequence, wherein, in each synthesis cycle, the use comprises: providing a double stranded scaffold polynucleotide comprising a synthetic strand and a support strand hybridized to the synthetic strand, wherein the synthetic strand comprises a primer strand portion; providing a double stranded polynucleotide ligation molecule comprising a support strand and an auxiliary strand hybridized to the support strand, and further comprising a complementary ligation end, wherein the terminal nucleotide of the auxiliary strand at the complementary ligation end comprises a non-ligatable nucleotide, wherein the terminal nucleotide of the support strand at the complementary ligation end comprises a ligatable first nucleotide of the predetermined sequence, and wherein the support strand comprises a universal nucleotide for generating a polynucleotide cleavage site; ligating the support strand of the polynucleotide linker molecule to the support strand of the scaffold polynucleotide in a blunt end ligation reaction, thereby extending the support strand of the scaffold polynucleotide with the first nucleotide of the predetermined sequence and creating a single-stranded break between the helper strand and the primer strand portion of the synthesis strand, and then optionally removing the helper strand; adding a second nucleotide of the predetermined sequence comprising a reversible terminator group to an end of the primer strand portion of the synthetic strand of the scaffold polynucleotide by a polymerase or transferase; and cleaving a support strand of the scaffold polynucleotide at a cleavage site defined by a sequence comprising the universal nucleotide, whereby the polynucleotide linker molecule comprising the universal nucleotide is removed from the scaffold polynucleotide and the first and second nucleotides of the predetermined sequence are retained in the cleaved scaffold polynucleotide; wherein the reversible terminator group is removed from the second nucleotide before or after the cleaving step.

In any such method of synthesizing a double stranded polynucleotide having a predetermined sequence, the support strand of the polynucleotide linker molecule may further comprise one or more additional nucleotides of the predetermined sequence immediately adjacent to the first nucleotide of the predetermined sequence, wherein the first nucleotide of the predetermined sequence is a terminal nucleotide of the support strand of the polynucleotide linker molecule and is linked to a terminal nucleotide of the support strand of the scaffold polynucleotide; and wherein, after adding the second nucleotide of the predetermined sequence, the method further comprises: adding one or more additional nucleotides of the predetermined sequence to the end of the primer strand portion of the synthesized strand under the action of a polymerase or transferase by performing one or more cycles of adding additional nucleotides comprising a reversible terminator group, and then removing the reversible terminator group, and wherein, after cleavage, the first, second and additional nucleotides of the predetermined sequence remain in the cleaved scaffold polynucleotide.

Such use of universal nucleotides in a method of synthesizing a double-stranded polynucleotide having a predetermined sequence can be carried out using any of the specific methods defined and described above and herein.

In a related aspect, the invention also provides an in vitro method of extending each strand of a double-stranded polynucleotide molecule at the same end with a predetermined nucleotide, the method comprising: providing a double stranded scaffold polynucleotide comprising a synthetic strand and a support strand hybridized to the synthetic strand, wherein the synthetic strand comprises a primer strand portion; adding a first nucleotide of the predetermined sequence to an end of a support strand of the scaffold polynucleotide under the action of a ligase in a blunt end ligation reaction, wherein the first nucleotide is a terminal nucleotide in a support strand of a double-stranded polynucleotide linker molecule, the support strand further comprising a universal nucleotide, wherein the first nucleotide is ligated to a terminal nucleotide of a support strand of the scaffold polynucleotide and a single-stranded break is created between the opposite/auxiliary strand of the polynucleotide linker molecule and the primer strand portion of the synthetic strand, and optionally removing the auxiliary strand of the polynucleotide linker molecule; adding a second nucleotide of the predetermined sequence comprising a reversible terminator group to the end of the primer strand portion of the synthesized strand under the action of a polymerase or transferase; cleaving a support strand of the scaffold polynucleotide at a cleavage site defined by a sequence comprising the universal nucleotide, whereby the polynucleotide linker molecule comprising the universal nucleotide is removed from the scaffold polynucleotide, wherein, after cleavage, the first nucleotide is retained in the support strand and the second nucleotide is retained in the primer strand portion; and removing the reversible terminator group from the second nucleotide, wherein the removing is performed before or after cleavage.

In any such method of extending each strand of a double-stranded polynucleotide molecule, the support strand of the polynucleotide linker molecule may further comprise one or more additional nucleotides of the predetermined sequence immediately adjacent to the first nucleotide of the predetermined sequence, wherein the first nucleotide of the predetermined sequence is a terminal nucleotide of the support strand of the polynucleotide linker molecule and is linked to a terminal nucleotide of the support strand of the scaffold polynucleotide; and wherein, after adding the second nucleotide of the predetermined sequence, the method further comprises: adding one or more additional nucleotides of the predetermined sequence to the end of the primer strand portion of the synthesized strand under the action of a polymerase or transferase by performing one or more cycles of adding additional nucleotides comprising a reversible terminator group, and then removing the reversible terminator group, and wherein, after cleavage, the first, second and additional nucleotides of the predetermined sequence remain in the cleaved scaffold polynucleotide.

The present invention provides an in vitro method of synthesizing a double stranded polynucleotide having a predetermined sequence, the method comprising pre-forming one or more extension cycles according to the extension method described previously.

In any such method of extending each strand of a double-stranded polynucleotide molecule with a predetermined nucleotide, or in any such method of synthesizing a double-stranded polynucleotide having a predetermined sequence, the method may be effected using any particular method as defined and described above and herein.

In a related aspect, the invention also provides an in vitro method of ligating a polynucleotide linker molecule comprising a universal nucleotide to a double stranded scaffold polynucleotide during cycles of extending each strand of said double stranded scaffold polynucleotide with a predetermined nucleotide at the same end, said method comprising: providing a double stranded scaffold polynucleotide comprising a support strand and a synthetic strand hybridized to the support strand, wherein the synthetic strand comprises a primer strand portion; and ligating a double stranded polynucleotide ligation molecule to the double stranded scaffold polynucleotide, wherein the polynucleotide ligation molecule comprises a support strand and an auxiliary strand hybridized to the support strand, and the polynucleotide ligation molecule further comprises a complementary ligation end, wherein the terminal nucleotide of the auxiliary strand at the complementary ligation end comprises a non-ligatable nucleotide, wherein the terminal nucleotide of the support strand at the complementary ligation end comprises a ligatable first nucleotide of the predetermined sequence, and wherein the support strand comprises a universal nucleotide for generating a polynucleotide cleavage site, wherein the ligation reaction comprises: ligating the support strand of the polynucleotide linker molecule to the support strand of the double-stranded scaffold polynucleotide in a blunt-end ligation reaction, thereby extending the support strand of the scaffold polynucleotide with the first nucleotide of the predetermined sequence and creating a single-stranded break between the helper strand and the primer strand portion of the synthesis strand, and then, optionally removing the helper strand; the method further comprises the following steps: adding a second nucleotide of the predetermined sequence comprising a reversible terminator group to an end of the primer strand portion of the synthetic strand of the scaffold polynucleotide by a polymerase or transferase; and cleaving a support strand of the scaffold polynucleotide at a cleavage site defined by a sequence comprising the universal nucleotide, whereby the polynucleotide linker molecule comprising the universal nucleotide is removed from the scaffold polynucleotide and the first and second nucleotides of the predetermined sequence are retained in the cleaved scaffold polynucleotide; wherein the reversible terminator group is removed from the second nucleotide before or after the cleaving step.

In any such method of ligating a polynucleotide linker molecule comprising a universal nucleotide to a double stranded scaffold polynucleotide, the support strand of the polynucleotide linker molecule may further comprise one or more additional nucleotides of the predetermined sequence immediately adjacent to the first nucleotide of the predetermined sequence, wherein the first nucleotide of the predetermined sequence is a terminal nucleotide of the support strand of the polynucleotide linker molecule and is ligated to a terminal nucleotide of the support strand of the scaffold polynucleotide; and wherein, after adding the second nucleotide of the predetermined sequence, the method further comprises: adding one or more additional nucleotides of the predetermined sequence to the end of the primer strand portion of the synthesized strand under the action of a polymerase or transferase by performing one or more cycles of adding additional nucleotides comprising a reversible terminator group, and then removing the reversible terminator group, and wherein, after cleavage, the first, second and additional nucleotides of the predetermined sequence remain in the cleaved scaffold polynucleotide.

The present invention provides an in vitro method of synthesizing a double stranded polynucleotide having a predetermined sequence, the method comprising pre-forming one or more cycles of extension according to the ligation method described above.

In any such method of ligating a linking polynucleotide comprising universal nucleotides to a double stranded polynucleotide during a cycle of synthesizing the double stranded polynucleotide having a predetermined sequence, the method may be carried out using any particular method as defined and described above and herein.

The present invention further provides a polynucleotide synthesis system for carrying out any of the synthesis and/or assembly methods described above and herein, the system comprising: (a) an array of reaction regions, wherein each reaction region comprises at least one scaffold polynucleotide; and (b) means for delivering a reactive agent to the reaction zone; and optionally (c) a means for cleaving the synthesized double stranded polynucleotide from the scaffold polynucleotide. Such a system may further comprise means for providing the reaction reagents in the form of droplets and means for delivering the droplets to the scaffold polynucleotide according to a synthesis cycle.

The present invention further provides a kit for use with any of the systems described above and herein and for carrying out any of the synthesis methods described above and herein, the kit comprising volumes of reaction reagents corresponding to steps of a synthesis cycle.

The present invention also provides a method of preparing a polynucleotide microarray, wherein the microarray comprises a plurality of reaction regions, each region comprising one or more polynucleotides having a predefined sequence, the method comprising:

a) providing a surface comprising a plurality of reaction regions, each region comprising one or more double-stranded anchor or scaffold polynucleotides, and

b) a synthesis cycle is performed at each reaction region according to any of the methods described above and herein, whereby one or more double-stranded polynucleotides having a predefined sequence are synthesized at each region.

In such methods, after synthesis, the strands of double-stranded polynucleotides may be separated to provide a microarray, wherein each region comprises one or more single-stranded polynucleotides having a predefined sequence.

Drawings

The relevant figures provided herein and described below show some or all of the steps of a synthesis cycle using a method comprising the methods of the invention, as well as the means for carrying out aspects of the methods, such as oligonucleotides, surfaces, surface attachment chemistry, linkers, and the like. These figures, as well as all descriptions thereof and all related methods, reagents, and protocols, are presented for purposes of illustration only and are not to be construed as limiting.

The related figures, such as, for example, fig. 6, 7, 8, 9, 10, 13a, 14a, 15a, etc., illustrate some or all of the steps of a synthesis cycle, including: combining nucleotides (e.g., nucleotides comprising a reversible terminator group), cleaving (e.g., cleaving a scaffold polynucleotide into a first portion and a second portion, wherein the first portion comprises universal nucleotides and the second portion comprises combined nucleotides), ligating (e.g., ligating a polynucleotide construct comprising a single-stranded portion to a second portion of a cleaved scaffold polynucleotide comprising combined nucleotides, wherein the single-stranded portion comprises a partner nucleotide complementary to the combined nucleotides), and deprotecting (e.g., removing the reversible terminator group from the combined nucleotides). These methods are provided for illustrative support only and are not within the scope of the claimed invention. The process schemes shown in figures 1 to 5 and 52 are the process of the present invention.

Fig. 1. an exemplary method version 1 arrangement of the present invention.

A scheme of a first synthesis loop according to exemplary method version 1 of the present invention is shown.

The method comprises the following cycles: providing a scaffold polynucleotide; ligating a polynucleotide linker molecule to the scaffold polynucleotide; combining nucleotides comprising a reversible terminator group or a blocking group; deprotection; and lysing.

The scheme shows providing a scaffold polynucleotide (101, 106) comprising a support strand (labelled "a") and a synthesis strand hybridised to the support strand (labelled "b"). The synthetic strand comprises a primer strand portion (dashed line). The terminal nucleotide of the support strand adjacent to the primer strand portion includes a ligatable group, preferably a terminal phosphate group as depicted in the figure. The terminal nucleotide of the primer strand portion and the nucleotide paired therewith are described as "X". The two nucleotides can be any two nucleotides or analogues or derivatives thereof, and are not limited to natural complementary nucleotide pairs.

This scheme shows the provision of polynucleotide linker molecules (102, 107; structure at the top right of the figure). The polynucleotide linker molecule comprises an auxiliary strand (dotted line), a support strand hybridized thereto and a complementary linker end. The terminal nucleotide of the support strand of the complementary linker is the first nucleotide of the predetermined sequence and is described as "a" (adenosine). The terminal nucleotide of the auxiliary strand of the complementary linker is described as "T" (thymine). The terminal nucleotide of the auxiliary strand of the complementary linker comprises a non-ligatable nucleotide. The complementary linkers contain a universal nucleotide (described as "Un") in the support strand and pair with a partner nucleotide (described as "X") in the auxiliary strand. A and T are for illustration only and may be any nucleotide or analog or derivative thereof. X may be any nucleotide or an analogue or derivative thereof. The paired nucleotides need not comprise naturally complementary nucleotides.

This scheme shows the ligation of the support strand of a polynucleotide linker molecule (102, 107) to the support strand of a scaffold polynucleotide and the generation of a single-stranded break ("nick") in the synthetic strand between the helper strand and primer strand portions.

The scheme shows the pooling of the second nucleotide of the predetermined sequence (103, 108). The nucleotide contains a reversible termination group (triangle) and is described as a "T" (thymine) for illustrative purposes only, and it may be any nucleotide or an analog or derivative thereof.

The scheme shows a deprotection step (104, 109) comprising the removal of a reversible terminator group from a second nucleotide of the predetermined sequence.

The scheme shows a cleavage step (105, 110) comprising cleavage of the support strand at a cleavage site defined by a sequence comprising universal nucleotides (jagged arrow). The cleavage releases the polynucleotide linker molecule comprising the universal nucleotide and causes the first and second nucleotides to remain in the scaffold polynucleotide. In the synthesis method of version 1 of the present invention, the support strand is cleaved between the position occupied by the universal nucleotide and the nucleotide occupying the next nucleotide position in the support strand in the direction toward/away from the auxiliary strand portion.

In the synthesis method of version 1 of the present invention, in each cycle, a first nucleotide of the predetermined sequence and a second nucleotide of the predetermined sequence form a nucleotide pair.

Fig. 2. an exemplary method version 2 arrangement of the present invention.

This scheme shows a first synthesis cycle according to exemplary method version 2 of the present invention.

The scheme shows that a scaffold polynucleotide (201, 206) comprising a support strand (labeled "a") and a synthetic strand hybridized thereto (labeled "b") is provided. The synthetic strand comprises a primer strand portion (dashed line). The terminal nucleotide of the support strand adjacent to the primer strand portion includes a ligatable group, preferably a terminal phosphate group as depicted in the figure. The terminal nucleotide of the primer strand portion and the nucleotide paired therewith are described as "X". The two nucleotides can be any two nucleotides or analogues or derivatives thereof, and are not limited to natural complementary nucleotide pairs.

This scheme shows the provision of polynucleotide linker molecules (202, 207; structure at the top right of the figure). The polynucleotide linker molecule comprises an auxiliary strand (dotted line), a support strand hybridized thereto and a complementary linker end. The terminal nucleotide of the support strand of the complementary linker is the first nucleotide of the predetermined sequence and is described as "a" (adenosine). The terminal nucleotide of the auxiliary strand of the complementary linker is described as "T" (thymine). The terminal nucleotide of the auxiliary strand of the complementary linker comprises a non-ligatable nucleotide. The complementary linkers contain a universal nucleotide (described as "Un") in the support strand and pair with a partner nucleotide (described as "X") in the auxiliary strand. The penultimate nucleotides of both the support and auxiliary strands at the complementary junction are described as "X". A and T are for illustration only and may be any nucleotide or analog or derivative thereof. The nucleotide depicted as "X" may be any nucleotide or an analog or derivative thereof. The paired nucleotides need not comprise naturally complementary nucleotides.

This scheme shows the ligation of the support strand of a polynucleotide linker molecule (202, 207) to the support strand of a scaffold polynucleotide and the creation of a single-stranded break ("nick") in the synthetic strand between the helper strand and the primer strand portions.

The scheme shows pooling of the second nucleotide of the predetermined sequence (203, 208). The nucleotide contains a reversible termination group (triangle) and is described as a "T" (thymine) for illustrative purposes only, and it may be any nucleotide or an analog or derivative thereof.

The scheme shows a deprotection step (204, 209) comprising the removal of a reversible terminator group from a second nucleotide of the predetermined sequence.

The protocol shows a cleavage step (205, 210) comprising cleavage of the support strand at a cleavage site defined by a sequence comprising universal nucleotides (jagged arrow). The cleavage releases the polynucleotide linker molecule comprising the universal nucleotide and causes the first and second nucleotides to remain in the scaffold polynucleotide. In the synthesis method of version 2 of the present invention, the support strand is cleaved between the nucleotide occupying the next nucleotide position in the direction closer to/away from the primer strand portion with respect to the universal nucleotide and the nucleotide occupying the second nucleotide position in the direction closer to/away from the auxiliary strand portion with respect to the universal nucleotide.

In the synthesis method of version 2 of the present invention, in each cycle, a first nucleotide of the predetermined sequence and a second nucleotide of the predetermined sequence form a nucleotide pair.

Fig. 3 shows a variant of the exemplary method version 2 of the invention.

This scheme shows a first synthesis cycle according to a variation of the exemplary method version 2 of the present invention.

The scheme shows that a scaffold polynucleotide (301, 306) comprising a support strand (labelled "a") and a synthetic strand hybridised thereto (labelled "b") is provided. The synthetic strand comprises a primer strand portion (dashed line). The terminal nucleotide of the support strand adjacent to the primer strand portion includes a ligatable group, preferably a terminal phosphate group as depicted in the figure. The terminal nucleotide of the primer strand portion and the nucleotide paired therewith are described as "X". The two nucleotides can be any two nucleotides or analogues or derivatives thereof, and are not limited to natural complementary nucleotide pairs.

The scheme shows the provision of a polynucleotide linker molecule (302, 307; structure at the top right of the figure). The polynucleotide linker molecule comprises an auxiliary strand (dotted line), a support strand hybridized thereto and a complementary linker end. The terminal nucleotide of the support strand of the complementary linker is the first nucleotide of the predetermined sequence and is described as "a" (adenosine). The terminal nucleotide of the auxiliary strand of the complementary linker is described as "T" (thymine). The terminal nucleotide of the auxiliary strand of the complementary linker comprises a non-ligatable nucleotide. The complementary linkers contain a universal nucleotide (described as "Un") in the support strand and pair with a partner nucleotide (described as "X") in the auxiliary strand. At the complementary junction, two nucleotides, denoted "X", are located between the universal nucleotide and the terminal nucleotide of the support strand and pair with the partner nucleotide in the auxiliary strand, also referred to as "X". A and T are for illustration only and may be any nucleotide or analog or derivative thereof. The nucleotide depicted as "X" may be any nucleotide or an analog or derivative thereof. The paired nucleotides need not comprise naturally complementary nucleotides.

This scheme shows the ligation of the support strand of the polynucleotide linker molecule (302, 307) to the support strand of the scaffold polynucleotide and the generation of single-stranded breaks ("gaps") between the helper strand and primer strand portions in the synthetic strand.

The scheme shows the pooling of the second nucleotide of the predetermined sequence (303, 308). The nucleotide contains a reversible termination group (triangle) and is described as a "T" (thymine) for illustrative purposes only, and it may be any nucleotide or an analog or derivative thereof.

The scheme shows a deprotection step (304, 309) comprising the removal of a reversible terminator group from a second nucleotide of the predetermined sequence.

The protocol shows a cleavage step (305, 310) which involves cleavage of the support strand at a cleavage site defined by a sequence comprising universal nucleotides (jagged arrow). The cleavage releases the polynucleotide linker molecule comprising the universal nucleotide and causes the first and second nucleotides to remain in the scaffold polynucleotide. In these variants of the synthesis method of version 2 of the invention, the support strand is always cleaved between the position occupied by the first nucleotide of the predetermined sequence and the position occupied by the next nucleotide in the support strand in the direction closer to the primer strand portion/further from the auxiliary strand portion.

In all these particular variants of the synthesis method of version 2 of the invention, in each cycle the first nucleotide of the predetermined sequence and the second nucleotide of the predetermined sequence form a nucleotide pair.

FIG. 4. this scheme shows an exemplary version of the method of the invention involving the incorporation of more than two nucleotides per cycle Variants of the invention 1.

The scheme showing a first synthesis cycle for variants according to exemplary method version 1 of the invention comprises combining polynucleotides in two steps, ligation and combining.

The method comprises the following cycles: providing a scaffold polynucleotide; ligating a polynucleotide linker molecule to the scaffold polynucleotide; the method comprises the following steps: (a) combining nucleotides comprising a reversible terminator group or blocking group, followed by (b) deprotection; and then finally lysed.

The scheme shows that a scaffold polynucleotide (401, 406) comprising a support strand (labeled "a") and a synthetic strand hybridized thereto (labeled "b") is provided. The synthetic strand comprises a primer strand portion (dashed line). The terminal nucleotide of the support strand adjacent to the primer strand portion includes a ligatable group, preferably a terminal phosphate group as depicted in the figure. The terminal nucleotide of the primer strand portion and the nucleotide paired therewith are described as "X". The two nucleotides can be any two nucleotides or analogues or derivatives thereof, and are not limited to natural complementary nucleotide pairs.

This scheme shows the provision of a polynucleotide linker molecule (402, 407; structure at the top right of the figure). The polynucleotide linker molecule comprises an auxiliary strand (dotted line), a support strand hybridized thereto and a complementary linker end. The terminal nucleotide of the support strand of the complementary linker is the first nucleotide of the predetermined sequence and is described as "a" (adenosine). The terminal nucleotide of the auxiliary strand of the complementary linker is described as "T" (thymine). The terminal nucleotide of the auxiliary strand of the complementary linker comprises a non-ligatable nucleotide. The second-most nucleotide of the support strand is another nucleotide of the predetermined sequence, depicted as "G" (guanine), and pairs with the second-most nucleotide of the auxiliary strand, depicted as "C" (cytosine). The complementary linkers contain a universal nucleotide (described as "Un") in the support strand and pair with a partner nucleotide (described as "X") in the auxiliary strand. A. T, G and C are for illustration only and can be any nucleotide or analog or derivative thereof. X may be any nucleotide or an analogue or derivative thereof. The paired nucleotides need not comprise naturally complementary nucleotides.

This scheme shows the ligation of the support strand of the polynucleotide linker molecule (402, 407) to the support strand of the scaffold polynucleotide and the generation of single-stranded breaks ("gaps") between the helper strand and the primer strand portions in the synthetic strand.

The scheme shows the pooling of the second nucleotide of the predetermined sequence (403, 408). The nucleotide contains a reversible termination group (triangle) and is described as a "T" (thymine) for illustrative purposes only, and it may be any nucleotide or an analog or derivative thereof. After combination, the second nucleotide forms a nucleotide pair with the first nucleotide.

The scheme shows a deprotection step after incorporation of a second nucleotide (404, 409), which step comprises removal of a reversible terminator group from the second nucleotide of the predetermined sequence.

The protocol shows the incorporation of another nucleotide of the predetermined sequence (403', 408'). The nucleotide contains a reversible terminating group (triangle) and is described as "C" (cytosine). After pooling, the additional nucleotides form nucleotide pairs, depicted as "G" (guanine), with the additional nucleotides provided by the polynucleotide linker molecule in step (2). Cytosine and guanine are depicted for illustration only, and these nucleotides can be any nucleotide or analog or derivative thereof and are not limited to naturally complementary nucleotide pairs.

The scheme shows a second deprotection step after incorporation of further nucleotides (404', 409'), comprising removal of the reversible terminator group from the further nucleotides of the predetermined sequence.

The scheme shows a cleavage step (405, 410) which involves cleavage of the support strand at a cleavage site defined by a sequence comprising universal nucleotides (jagged arrow). The cleavage releases the polynucleotide linker molecule comprising the universal nucleotide and causes the first nucleotide, the second nucleotide, and the other nucleotides to remain in the scaffold polynucleotide. In this particular variant of the synthesis method of version 1 of the invention, the support strand is cleaved between the position occupied by the universal nucleotide and the nucleotide in the support strand occupying the next nucleotide position in the direction proximal to the primer strand portion/distal to the auxiliary strand portion.

In all these particular variants of the synthetic method of version 1 of the invention, in each cycle the first nucleotide of the predetermined sequence and the second nucleotide of the predetermined sequence form a nucleotide pair, the first further nucleotide provided by the polynucleotide linker molecule in step (2) forms a nucleotide pair with the first further nucleotide combined in step (3'), and so on.

FIG. 5 shows this scheme an exemplary version of the method of the invention involving the incorporation of more than two nucleotides per cycle Variants of the instant 2.

The scheme showing a first synthesis cycle for variants according to exemplary method version 2 of the invention comprises combining polynucleotides in two steps, ligation and combining.

The scheme shows that a scaffold polynucleotide (501, 506) comprising a support strand (labeled "a") and a synthetic strand (labeled "b") hybridized thereto is provided. The synthetic strand comprises a primer strand portion (dashed line). The terminal nucleotide of the support strand adjacent to the primer strand portion includes a ligatable group, preferably a terminal phosphate group as depicted in the figure. The terminal nucleotide of the primer strand portion and the nucleotide paired therewith are described as "X". The two nucleotides can be any two nucleotides or analogues or derivatives thereof, and are not limited to natural complementary nucleotide pairs.

This scheme shows the provision of polynucleotide linker molecules (502, 507; structure at the top right of the figure). The polynucleotide linker molecule comprises an auxiliary strand (dotted line), a support strand hybridized thereto, and a complementary linker end. The terminal nucleotide of the support strand of the complementary linker is the first nucleotide of the predetermined sequence and is described as "a" (adenosine). The terminal nucleotide of the auxiliary strand of the complementary linker is described as "T" (thymine). The terminal nucleotide of the auxiliary strand of the complementary linker comprises a non-ligatable nucleotide. The second-most nucleotide of the support strand is another nucleotide of the predetermined sequence, depicted as "G" (guanine), and pairs with the second-most nucleotide of the auxiliary strand, depicted as "C" (cytosine). The complementary linkers contain a universal nucleotide (described as "Un") in the support strand and pair with a partner nucleotide (described as "X") in the auxiliary strand. Additional nucleotides depicted as "X" are located between the universal nucleotide and the additional nucleotides of the predetermined sequence in the support strand. This additional nucleotide pairs with a partner nucleotide in the helper strand, which is also described as "X". A. T, G and C are for illustration only and can be any nucleotide or analog or derivative thereof. X may be any nucleotide or an analogue or derivative thereof. The paired nucleotides need not comprise naturally complementary nucleotides.

This scheme shows the ligation of the support strand of the polynucleotide linker molecule (502, 507) to the support strand of the scaffold polynucleotide and the generation of single-stranded breaks ("gaps") between the helper strand and the primer strand portions in the synthetic strand.

The scheme shows pooling of the second nucleotide of the predetermined sequence (503, 508). The nucleotide contains a reversible termination group (triangle) and is described as a "T" (thymine) for illustrative purposes only, and it may be any nucleotide or an analog or derivative thereof. After combination, the second nucleotide forms a nucleotide pair with the first nucleotide.

The scheme shows a deprotection step after incorporation of a second nucleotide (504, 509) comprising removal of a reversible terminator group from the second nucleotide of the predetermined sequence.

The scheme shows the pooling of another nucleotide of the predetermined sequence (503', 508'). The nucleotide contains a reversible terminating group (triangle) and is described as "C" (cytosine). After pooling, the additional nucleotides form nucleotide pairs, depicted as "G" (guanine), with the additional nucleotides provided by the polynucleotide linker molecule in step (2). Cytosine and guanine are depicted for illustration only, and these nucleotides can be any nucleotide or analog or derivative thereof and are not limited to naturally complementary nucleotide pairs.

The scheme shows a second deprotection step after incorporation of further nucleotides (504', 509'), comprising removal of the reversible terminator group from the further nucleotides of the predetermined sequence.

The protocol shows a cleavage step (505, 510) which involves cleavage of the support strand at a cleavage site defined by a sequence comprising universal nucleotides (jagged arrows). The cleavage releases the polynucleotide linker molecule comprising the universal nucleotide and causes the first nucleotide, the second nucleotide, and the other nucleotides to remain in the scaffold polynucleotide. In the synthesis method of version 2 of the present invention, the support strand is cleaved between the nucleotide occupying the next nucleotide position in the direction closer to/away from the primer strand portion with respect to the universal nucleotide and the nucleotide occupying the second nucleotide position in the direction closer to/away from the auxiliary strand portion with respect to the universal nucleotide.

In all these particular variants of the synthetic method of version 2 of the invention, in each cycle the first nucleotide of the predetermined sequence and the second nucleotide of the predetermined sequence form a nucleotide pair, the first further nucleotide provided by the polynucleotide linker molecule in step (2) forms a nucleotide pair with the first further nucleotide combined in step (3'), and so on.

Fig. 6 illustrates an exemplary method version 1 scheme.

The figure shows a first synthesis loop of exemplary method version 1 according to the embodiments section. This method is provided for illustrative support only and is not within the scope of the claimed invention. The method includes providing cycles of scaffold polynucleotide, pooling, cleavage, ligation, and deprotection. The scheme shows the pooling of thymine nucleotides in the first synthesis cycle (101, 102) and their pairing against the partner adenine nucleotide (104), and the provision of a scaffold polynucleotide (106) for the next synthesis cycle. This pair is shown for illustrative purposes only and is not limiting, and may be any pair depending on the desired predefined sequence. The nucleotide Z may be any nucleotide. The nucleotide X may be any suitable nucleotide. The figure also shows reference signs corresponding to the second synthesis cycle.

Fig. 7 illustrates an exemplary method version 2 scheme.

The figure shows a first synthesis loop of exemplary method version 2 according to the examples section. This method is provided for illustrative support only and is not within the scope of the claimed invention. The method includes providing cycles of scaffold polynucleotide, pooling, cleavage, ligation, and deprotection. The protocol shows that thymine nucleotides and their counterparts to the adenine nucleotides are combined (204) in the first cycle (201, 202), and a scaffold polynucleotide comprising guanine paired with cytosine is provided (206) in the next synthesis cycle. These pairs are for illustrative purposes only and are not limiting, and they may be any pair depending on the desired predefined sequence. The nucleotide Z may be any nucleotide. The nucleotide X may be any suitable nucleotide. The figure also shows reference signs corresponding to the second synthesis cycle.

Fig. 8 illustrates an exemplary method version 3 scheme.

The figure shows a first synthesis loop of exemplary method version 3 according to the embodiments section. This method is provided for illustrative support only and is not within the scope of the claimed invention. The method includes providing cycles of scaffold polynucleotide, pooling, cleavage, ligation, and deprotection. The protocol shows incorporation of thymine nucleotides and their pairing with the partner adenine nucleotides (304) in the first cycle (301, 302), and provision of scaffold polynucleotides (306) for the next synthesis cycle. This pair is shown for illustrative purposes only and is not limiting, and may be any pair depending on the desired predefined sequence. The protocol also shows that cytosine-guanine pairs are components of the scaffold polynucleotide and are not part of the predefined sequence. This pair is also shown for illustrative purposes only, and is not limiting, which can be any pair. The nucleotide Z may be any nucleotide. The nucleotide X may be any suitable nucleotide.

Fig. 9 illustrates an exemplary method version 4 scheme.

The figure shows a first synthesis loop of exemplary method version 4 according to the embodiments section. This method is provided for illustrative support only and is not within the scope of the claimed invention. The method includes providing cycles of scaffold polynucleotide, pooling, cleavage, ligation, and deprotection. The protocol shows that thymine nucleotides and their pairs as opposed to partner universal nucleotides are combined (404) in a first cycle (401, 402) and a scaffold polynucleotide comprising guanine paired with cytosine is provided (406) in the next synthesis cycle. These pairs are for illustrative purposes only and are not limiting, and they may be any pair depending on the desired predefined sequence. Nucleotides X, Y and Z can be any nucleotide.

Fig. 10 illustrates an exemplary method version 5 scheme.

The figure shows a first synthesis loop of exemplary method version 5 according to the embodiments section. This method is provided for illustrative support only and is not within the scope of the claimed invention. The method includes providing cycles of scaffold polynucleotide, pooling, cleavage, ligation, and deprotection. The protocol shows that thymine nucleotides and their counterparts to the partner adenine nucleotides are combined in the first cycle (501, 502) (504), and that in the next synthesis cycle a scaffold polynucleotide comprising guanine paired with cytosine is provided (506). The protocol also shows that the cytosine-guanine pair (position n-2) is a component of the scaffold polynucleotide and is not part of the predefined sequence. These pairs are for illustrative purposes only and are not limiting, and they may be any pair depending on the desired predefined sequence. Nucleotides X, Y and Z can be any nucleotide.

FIG. 11 shows a scheme for surface immobilization of scaffold polynucleotides.

The scheme shows (a to h) possible exemplary hairpin loop configurations of the scaffold polynucleotide and its fixation to the surface.

Protocols (i and j) show examples of surface chemistries for attaching polynucleotides to a surface. The examples show double stranded embodiments in which both strands are joined by a hairpin, but the same chemistry can be used to attach one or both strands of an unligated double stranded polynucleotide.

FIG. 12. without auxiliary strand-merging.

a) The scheme of the merging step is highlighted in a dashed box.

b) DNA polymerase was evaluated for dTTP incorporating 3' -O-modification as opposed to inosine. The figure depicts a gel showing Mn at 50 deg.C²⁺As a result of 3' -O-modified dTTP incorporation by various DNA polymerases (Bst, Deep Vent (Exo-), terminator I and terminator IX) in the presence of ions. Lane 1: bst DNA polymerase was used to combine 3' -O-allyl-dTTP. Lane 2: bst DNA polymerase was used to incorporate 3' -O-azidomethyl-dTTP. Lane 3: 3' -O-allyl-dTTP was combined using Deep vent (exo-) DNA polymerase. Lane 4: 3' -O-azidomethyl-dTTP was combined using Deep vent (exo-) DNA polymerase. Lane 5: terminator I DNA polymerase was used to incorporate 3' -O-allyl-dTTP. Lane 6: terminator I DNA polymerase was used to incorporate 3' -O-azidomethyl-dTTP. Lane 7: terminator IX DNA polymerase was used to incorporate 3' -O-allyl-dTTP. Lane 8: terminator IX DNA polymerase was used to incorporate 3' -O-azidomethyl-dTTP.

c) DNA polymerase was evaluated for dTTP incorporating 3' -O-modification as opposed to inosine. Results were combined using various DNA polymerases.

d) The terminator IX DNA polymerase was used to assess the pooling temperature. The figure depicts a gel showing the use of terminator IX DNA polymerase at Mn at different temperatures ²⁺Results of combining 3' -modified dTTP in the presence of ions as opposed to inosine. Lane 1: combine 3' -O-allyl dTTP at 37 ℃. Lane 2: 3' -O-azidomethyl dTTP was combined at 37 ℃. Lane 3: the 3' -O-allyl dTTP was combined at 50 ℃. Lane 4: 3' -O-azidomethyl dTTP was combined at 50 ℃. Lane 5: the 3' -O-allyl dTTP was combined at 65 ℃. Lane 6: 3' -O-azidomethyl dTTP was combined at 65 ℃.

e) The terminator IX DNA polymerase was used to assess the pooling temperature. Combined results at different temperatures.

f) Evaluation of Mn at incorporation Using terminator IX DNA polymerase²⁺Is present. The figure depicts a gel showing results at 65 ℃ relative to inosine incorporation with 3' -O-modified dTTP. Lane S: and (4) standard. Lane 1: no Mn²⁺Incorporation of ionic 3' -O-allyl-dTTP. Lane 2: no Mn²⁺Incorporation of ionic 3' -O-azidomethyl-dTTP. Lane 3: in Mn²⁺3' -O-allyl-dTTP is combined in the presence of ions. Lane 4: in Mn²⁺Combining 3' -O-azidomethyl-dTTP in the presence of ions.

g) Evaluation of Mn at incorporation Using terminator IX DNA polymerase²⁺Is present. In the presence and absence of Mn²⁺Combined results in the case of ions.

h) Oligonucleotides used to study the incorporation step.

FIG. 13. No auxiliary strand-cleavage.

a) Shows a scheme for cleaving hybridized polynucleotide strands in the absence of auxiliary strands. The cleavage step is highlighted by the dashed box.

b) The gel shows cleavage of the oligonucleotide with hAAG and 0.2M NaOH (strong base) at 37 ℃ and 24 ℃ at room temperature, respectively. Lane 1. starting oligonucleotide lane 2, a positive control containing two full-length strands, shows a 90% ratio of cleaved to uncleaved DNA: higher yield of 10%. Lane 3, containing the cleavage reaction without the helper strand, shows a 10% ratio of cleaved to uncleaved DNA: low percent yield of 90%.

c) The gel shows that the oligonucleotides are cleaved with hAAG and Endo VIII at 37 ℃. Lane 2, a positive control containing two full long strands, shows a ratio of cleaved to uncleaved DNA of about 90%: higher yield of 10%. Lane 3, which contains the cleavage reaction without the helper strand, shows a ratio of cleaved to uncleaved DNA of about 7%: low percent yield of 93%.

d) Summary of oligonucleotide cleavage with hAAG/Endo VIII and hAAG/chemical base.

e) Oligonucleotides used to study the cleavage step.

FIG. 14. No auxiliary strand-link.

a) Shows a scheme for linking hybridized polynucleotide strands in the absence of auxiliary strands. The linking step is highlighted in the dashed box.

b) The gel shows the ligation of oligonucleotides with Quick T4 DNA ligase at room temperature (24 ℃) without the helper strand. Lane 1 contains a mixture of 36-mer TAMRA single strand oligonucleotides and 18-mer TAMRA single strand oligonucleotides. These oligonucleotides were used as reference bands.

c) Oligonucleotides used to study ligation steps.

FIG. 15 version 1 chemical-merger with auxiliary strand.

a) The scheme of the merging step is highlighted in a dashed box.

b) Oligonucleotides suitable for use in the study of the incorporation step.

FIG. 16. version 1 chemical-cleavage with helper strand.

a) Shows a scheme for cleaving hybridized polynucleotide strands in the absence of auxiliary strands. The cleavage step is highlighted by the dashed box.

c) Endonuclease VIII was evaluated for cleavage of abasic sites. The gel shows that the oligonucleotides are cleaved with hAAG and Endo VIII at 37 ℃. Lane 2, a positive control containing two full long strands, shows a ratio of cleaved to uncleaved DNA of about 90%: higher yield of 10%. Lane 3, which contains the cleavage reaction without the helper strand, shows a ratio of cleaved to uncleaved DNA of about 7%: low percent yield of 93%. Lane 4 of the cleavage reaction containing the helper strand shows a 10% ratio of cleaved to uncleaved DNA: low percent yield of 90%.

d) Evaluation of N, N' -two methyl ethylene diamine cleavage of abasic sites. The gel showed that the oligonucleotides were cleaved with hAAG and 100mM N, N' -dimethylethylenediamine at 37 ℃. Lane 1. starting oligonucleotide lane 2, a positive control containing two full-length strands, shows 100% cleaved DNA. Lane 3 of the cleavage reaction containing the helper strand shows a 90% ratio of cleaved to uncleaved DNA: higher percentage yield of 10%.

e) Summary of oligonucleotide cleavage with hAAG/Endo VIII, hAAG/chemical base and hAAG/alternative chemical base.

f) Oligonucleotides used to study the cleavage step.

FIG. 17 version 1 chemistry with auxiliary strand-ligation.

a) Shows a scheme for linking hybridized polynucleotide strands in the presence of an auxiliary strand. The linking step is highlighted in the dashed box.

b) The gel shows the ligation of oligonucleotides with Quick T4 DNA ligase in the presence of the helper strand at room temperature (24 ℃). Lane 1 contains a mixture of 36-mer TAMRA single strand oligonucleotides and 18-mer TAMRA single strand oligonucleotides. These oligonucleotides were used as reference bands. In lane 2, there is an observable ligation product with an expected band size of 36 mers after 20 minutes.

c) The gel shows that after overnight incubation in the presence of the helper strand, oligonucleotides were ligated with Quick T4 DNA ligase at room temperature (24 ℃). Lane 1 contains a mixture of 36-mer TAMRA single strand oligonucleotides and 18-mer TAMRA single strand oligonucleotides. These oligonucleotides serve as reference bands. In lane 2, there is an observable fully ligated product with an expected band size of 36 mers.

d) Oligonucleotides used to study ligation steps.

FIG. 18 version 2 chemistry with auxiliary chain-incorporation.

a) The scheme showing the merging step is highlighted with an orange dashed box

b) The gel shows the results of incorporation of 3' -O-modified dTTP by terminator IX DNA polymerase at 27 ℃. Lane 1: starting materials. Lane 2: after 1 min the mixtures were combined and the conversion was 5%. Lane 3: after 2 minutes, the mixtures were combined and the conversion was 10%. Lane 4: after 5 minutes, the two are combined, the conversion being 20%. Lane 5: after 10 minutes, the mixtures were combined and the conversion was 30%. Lane 6: after 20 minutes, the two are combined, the conversion being 35%.

c) The figure depicts a gel showing the results of incorporation of 3' -O-modified dTTP by terminator IX DNA polymerase at 37 ℃. Lane 1: starting materials. Lane 2: after 1 minute, the mixtures were combined and the conversion was 30%. Lane 3: after 2 minutes, the mixtures were combined and the conversion was 60%. Lane 4: after 5 minutes the mixtures were combined and the conversion was 90%. Lane 5: after 10 minutes the mixtures were combined and the conversion was 90%. Lane 6: after 20 minutes the fractions were combined and the conversion was 90%.

d) The gel shows the results of incorporation of 3' -O-modified dTTP by terminator IX DNA polymerase at 47 ℃. Lane 1: starting materials. Lane 2: after 1 minute, the mixtures were combined and the conversion was 30%. Lane 3: after 2 minutes, the mixtures were combined and the conversion was 65%. Lane 4: after 5 minutes the mixtures were combined and the conversion was 90%. Lane 5: after 10 minutes the mixtures were combined and the conversion was 90%. Lane 6: after 20 minutes the fractions were combined and the conversion was 90%.

e) The gel shows the results of incorporation of 3' -O-modified dTTP by terminator IX DNA polymerase at 27 ℃. Lane 1: starting materials. Lane 2: after 1 minute, the mixtures were combined and the conversion was 70%. Lane 3: after 2 minutes, the mixtures were combined and the conversion was 85%. Lane 4: after 5 minutes the fractions were combined and the conversion was 92%. Lane 5: after 10 minutes, the mixtures were combined and the conversion was 96%. Lane 6: after 20 minutes, the mixtures were combined and the conversion was 96%.

f) The gel shows the results of incorporation of 3' -O-modified dTTP by terminator IX DNA polymerase at 37 ℃. Lane 1: starting materials. Lane 2: after 1 min the mixtures were combined and the conversion was 85%. Lane 3: after 2 minutes, the mixtures were combined and the conversion was 95%. Lane 4: after 5 minutes, the mixtures were combined and the conversion was 96%. Lane 5: after 10 minutes, the mixtures were combined and the conversion was 96%. Lane 6: after 20 minutes, the mixtures were combined and the conversion was 96%.

g) The gel shows the results of incorporation of 3' -O-modified dTTP by terminator IX DNA polymerase at 47 ℃. Lane 1: starting materials. Lane 2: after 1 min the mixtures were combined and the conversion was 85%. Lane 3: after 2 minutes the mixtures were combined and the conversion was 90%. Lane 4: after 5 minutes, the mixtures were combined and the conversion was 96%. Lane 5: after 10 minutes, the mixtures were combined and the conversion was 96%. Lane 6: after 20 minutes, the mixtures were combined and the conversion was 96%.

h) At various temperatures and Mn²⁺A summary of the incorporation of 3' -O-azidomethyl-dTTP in the presence of ions.

i) The gel showed Mn at 37 ℃²⁺By the terminator IX DNA polymerase with complementary base-relatively 3' -O-modified dNTPs. Lane 1: starting materials. Lane 2: 3' -O-azidomethyl-dTTP was pooled for 5 min. Lane 3: 3' -O-azidomethyl-dATP was combined for 5 minutes. Lane 4: 3' -O-azidomethyl-dCTP was combined for 5 minutes. Lane 5: 3' -O-azidomethyl-dGTP is combined for 5 minutes.

j) Oligonucleotides used to study the incorporation step.

Figure 19 version 2 chemistry with auxiliary strand-lysis.

a) Shows a scheme for cleaving hybridized polynucleotide strands in the presence of the helper strand. The cleavage step is highlighted by an orange dashed box.

b) The gel shows cleavage of the oligonucleotide with Endo V at 37 ℃. Lane 1. starting oligonucleotide lane 2, a positive control containing two full-length strands, shows a 80% ratio of cleaved to uncleaved DNA: yield of 20%. Lane 3, which contains the cleavage reaction without the helper strand, shows a much higher yield of cleaved DNA > 99%. Lane 4, which contains the helper strand cleavage reaction, also shows > 99% DNA cleavage yield.

c) Summary of cleavage studies with endonuclease V.

d) Oligonucleotides used to study the cleavage step.

FIG. 20 version 2 chemistry with auxiliary strand-ligation.

a) Shows a scheme for linking hybridized polynucleotide strands in the absence of auxiliary strands. The linking step is highlighted by an orange dashed box.

b) Oligonucleotides used to study ligation steps.

Figure 21 version 2 chemistry with auxiliary chain-deprotection.

a) The scheme showing the deprotection step is highlighted in the orange dashed box.

b) The figure depicts a gel showing the results of deprotection of 3 '-O-azidomethyl by 50mM TCEP after incorporation of 3' -O-azidomethyl-dTTP. Lane 1: initial primer

Lane 2: in Mn²⁺3' -O-azidomethyl-dTTP is combined in the presence. Lane 3: the product was extended in lane 2 by the addition of all native dntps. Lane 4: the product in lane 2 (0.5. mu.M) was deprotected by 50mM TCEP. Lane 5: the product was extended in lane 4 by the addition of all native dntps.

c) The figure depicts a gel showing the results of deprotection of 3 '-O-azidomethyl by 300mM TCEP after incorporation of 3' -O-azidomethyl-dTTP. Lane 1: initial primer lane 2: in the presence of Mn²⁺Then 3-O-azidomethyl-dTTP is combined. Lane 3: the product was extended in lane 2 by the addition of all native dntps. Lane 4: the product in lane 2 (0.5. mu.M) was deprotected by 300mM TCEP. Lane 5: the product was extended in lane 4 by the addition of all native dntps.

d) The figure depicts a gel showing the results of deprotection of 3 '-O-azidomethyl by 50mM TCEP after incorporation of 3' -O-azidomethyl-dCTP. Lane 1: initial primer lane 2: in the presence of Mn²⁺Next, 3-O-azidomethyl-dCTP was combined. Lane 3: by passing Addition of all native dntps extends the product in lane 2. Lane 4: the product in lane 2 (0.5. mu.M) was deprotected by 300mM TCEP. Lane 5: the product was extended in lane 4 by the addition of all native dntps.

e) The figure depicts a gel showing the results of deprotection of 3 '-O-azidomethyl by 300mM TCEP after incorporation of 3' -O-azidomethyl-dCTP. Lane 1: initial primer

Lane 2: in the presence of Mn²⁺Next, 3-O-azidomethyl-dCTP was combined. Lane 3: the product was extended in lane 1 by the addition of all native dntps. Lane 4: the product in lane 1 (0.5. mu.M) was deprotected by 300mM TCEP. Lane 5: the product was extended in lane 3 by the addition of all native dntps.

f) The figure depicts a gel showing the results of deprotection of 3 '-O-azidomethyl by 300mM TCEP after incorporation of 3' -O-azidomethyl-dATP.

Lane 1: initial primer

Lane 2: in the presence of Mn²⁺The following 3-O-azidomethyl-dATP was combined. Lane 3: the product was extended in lane 2 by the addition of all native dntps. Lane 4: the product in lane 2 (0.5. mu.M) was deprotected by 300mM TCEP. Lane 5: the product was extended in lane 4 by the addition of all native dntps.

g) The figure depicts a gel showing the results of deprotection of 3 '-O-azidomethyl by 300mM TCEP after incorporation of 3' -O-azidomethyl-dGTP. Lane 1: initial primer

Lane 2: in the presence of Mn²⁺And combining the 3-O-azidomethyl-dGTP. Lane 3: the product was extended in lane 2 by the addition of all native dntps. Lane 4: the product in lane 2 (0.5. mu.M) was deprotected by 300mM TCEP. Lane 5: the product was extended in lane 4 by the addition of all native dntps.

h) Efficiency of deprotection of TCEP on 0.2. mu.M DNA.

i) Oligonucleotides used to study the cleavage step.

Figure 22. version 2 chemistry with double hairpin model-incorporation.

a) The scheme of the merging step is highlighted in a dashed box.

b) DNA polymerase was evaluated for dTTP incorporating a 3' -O-modification as opposed to its natural counterpart. The figure depicts a gel showing the results of incorporation of 3' -O-modified dTTP by terminator IX DNA polymerase at 37 ℃. Lane 1: starting materials. Lane 2: the native dNTP mix was pooled. Lane 3: 3' -O-azidomethyl-dTTP was pooled by terminator IX DNA polymerase. Lane 4: the product was extended in lane 3 by the addition of all native dntps.

c) DNA polymerase was evaluated for dTTP incorporating a 3' -O-modification as opposed to its natural counterpart. Oligonucleotides suitable for use in the study of the incorporation step.

Figure 23 version 2 chemistry with double hairpin model-lysis.

a) The scheme for hairpin oligonucleotide cleavage is shown. The cleavage step is highlighted by the dashed box.

b) The gel shows cleavage of the hairpin oligonucleotide with Endo V at 37 ℃. Lane 1. initial hairpin oligonucleotide Lane 2, as cleaved hairpin oligonucleotide after 5 minutes, shows high yield of digested DNA, at a rate of about 98%. Lane 3, which is a cleaved hairpin oligonucleotide after 10 minutes, shows high yields of digested DNA at a rate of about 99%. Lane 4, which is a cleaved hairpin oligonucleotide after 30 minutes, shows a high yield of digested DNA at a rate of about 99%, and lane 5, which is a cleaved hairpin oligonucleotide after 1 hour, shows a high yield of digested DNA at a rate of about 99%.

c) Oligonucleotides used to study the cleavage step.

Figure 24. version 2 chemistry-ligation using the double hairpin model.

a) The scheme for ligation of hybrid hairpins is shown. The linking step is highlighted in the dashed box.

b) The gel shows the hairpin oligonucleotides were ligated with Blunt/TA DNA ligase at room temperature (24 ℃) in the presence of the helper strand. Lane 1 contains the initial hairpin oligonucleotide. Lane 2 of the ligated hairpin oligonucleotide after 1 min showed a high yield of ligated DNA product at a rate of about 85%. Lane 3 of the ligated hairpin oligonucleotide after 2 min showed high yields of digested DNA at a rate of about 85%. Lane 4 of the ligated hairpin oligonucleotide after 3 min showed a high yield of ligated DNA product at a rate of about 85%. Lane 5 of the ligated hairpin oligonucleotide after 4 min shows high yield of ligated DNA product at a rate of about > 85%.

c) Hairpin oligonucleotides used to study ligation steps.

Figure 25. version 2 chemistry-complete cycle of the double hairpin model.

a) A complete cycle protocol involving enzyme pooling, cleavage, ligation and deprotection steps is shown.

b) DNA polymerase was evaluated for dTTP incorporating a 3' -O-modification as opposed to its natural counterpart. The figure depicts a gel showing the results of incorporation of 3' -O-modified dTTP by terminator IX DNA polymerase at 37 ℃. Lane 1: starting materials. Lane 2: 3' -O-azidomethyl-dTTP was pooled by terminator IX DNA polymerase. Lane 3: the product was extended in lane 2 by the addition of all native dntps. Lane 4: the product was cleaved by endonuclease V in lane 2. Lane 5: the products were ligated by a blunt TA ligase kit in lane 4.

c) Oligonucleotides suitable for use in the study of the incorporation step.

Figure 26. version 2 chemistry-complete cycle of single hairpin model using helper strand.

a) A complete cycle protocol involving enzyme pooling, cleavage, ligation and deprotection steps is shown.

b) Oligonucleotides suitable for use in the study of the incorporation step.

Figure 27. version 3 chemistry-complete cycle of the double hairpin model.

a) A complete cycle protocol involving enzyme pooling, cleavage, ligation and deprotection steps is shown.

b) Oligonucleotides suitable for use in the study of the incorporation step.

Figure 28. version 2 chemistry-complete double cycle of double hairpin model.

a) A scheme involving the first complete cycle of enzymatic pooling, deprotection, cleavage and ligation steps is shown.

b) The protocol for the second complete cycle after the first complete cycle is shown, involving enzymatic pooling, deprotection, cleavage and ligation steps.

c) The figure depicts a gel showing a complete two-cycle experiment including: combining, deprotecting, cleaving and linking.

Lane 1 starting material.

Lane 2. extension of the starting material with native dNTPs.

Lane 3. incorporation of 3' -O-azidomethyl-dTTP by terminator IX DNA polymerase.

Lane 4. the product was extended in lane 3 by the addition of all native dNTPs.

Lane 5.TCEP deprotects the product in lane 3.

Lane 6. the product was extended in lane 5 by the addition of all native dNTPs.

Lane 7. endonuclease V cleaves the product in lane 5.

Lane 8. ligation products were ligated by a blunt TA ligase kit in lane 7.

Lane 9. the product in lane 8 was cleaved by lambda exonuclease.

Lane 10 starting material for the second cycle-same as in lane 9.

Lane 11. 3' -O-azidomethyl-dTTP was pooled by terminator IX DNA polymerase.

Lane 12. the product was extended in lane 11 by the addition of all native dNTPs.

Lane 13.TCEP deprotects the product in lane 11.

Lane 14. the product was extended in lane 13 by the addition of all native dNTPs.

Lane 15. endonuclease V cleaves the product in lane 13.

Lane 16. ligation products were ligated by a blunt TA ligase kit in lane 15.

d) Oligonucleotides for study.

FIG. 29.

Examples of scaffold polynucleotide release mechanisms from polynucleotides of predefined sequences synthesized according to the methods described herein are shown.

FIG. 30.

Schematic representation of an exemplary method for synthesizing RNA according to the present invention. Exemplary methods show synthesis in the absence of auxiliary chains.

FIG. 31.

Schematic representation of an exemplary method for synthesizing RNA according to the present invention. Exemplary methods show synthesis in the presence of an auxiliary chain.

FIG. 32.

Schematic representation of an exemplary method for synthesizing RNA according to the present invention. Exemplary methods show synthesis in the presence of an auxiliary chain.

FIG. 33.

Schematic of the 1 st full cycle of an exemplary method of synthesizing DNA according to synthesis method version 2 with single hairpin model, including a step of denaturing the helper strand prior to the step of combining.

FIG. 34.

Schematic of the 2 nd full cycle of an exemplary method for synthesizing DNA according to synthesis method version 2 with single hairpin model, including a step of denaturing the helper strand prior to the step of combining.

FIG. 35 is a schematic view.

Schematic of the 3 rd full cycle of an exemplary method for synthesizing DNA according to synthesis method version 2 with single hairpin model, comprising a step of denaturing the helper strand prior to the step of combining.

FIG. 36.

The oligonucleotides used in the experiments detailed in example 9.

FIG. 37.

A gel corresponding to the reaction product of a complete three-cycle experiment detailed in example 9 is shown.

The figure depicts a gel showing the results of a complete three-cycle experiment, including: combining, deblocking, splitting and connecting.

Lane 1: starting materials.

Lane 2 extension of starting Material with native dNTPs

Lane 3: 3' -O-azidomethyl-dTTP was pooled by terminator X DNA polymerase.

Lane 4: extension of the product in lane 3 by addition of all native dNTPs

Lane 5: TCEP in lane 3 deblocks the product

Lane 6: the product in lane 5 was extended by adding all native dntps.

Lane 7: cleavage of the product in lane 5 by endonuclease V.

Lane 8: ligation of the products by T3 DNA ligase in lane 7

Lane 9: starting material for cycle 2-same as in lane 9.

Lane 10: the product was extended in lane 9 by the addition of all native dntps.

Lane 11: 3' -O-azidomethyl-dTTP was pooled by terminator X DNA polymerase.

Lane 12: the product was extended in lane 11 by the addition of all native dntps.

Lane 13: TCEP deblocks the product in lane 11

Lane 14: the product was extended in lane 13 by the addition of all native dntps.

Lane 15: endonuclease V cleavage product in lane 13

Lane 16: ligation of the products by T3 DNA ligase in lane 15

Lane 17: starting material for cycle 3-same as in lane 16.

Lane 18: the product was extended in lane 17 by the addition of all native dntps.

Lane 19: 3' -O-azidomethyl-dTTP was pooled by terminator X DNA polymerase.

Lane 20: the product was extended in lane 19 by the addition of all native dntps.

Lane 21: TCEP in lane 19 deblocks the product

Lane 22: the product was extended in lane 21 by the addition of all native dntps.

Lane 23: endonuclease V cleavage product in lane 21

Lane 24: ligation of the products in lane 23 by T3 DNA ligase

FIG. 38.

The fluorescent signal from the surface of the polyacrylamide gel combined with varying amounts of BRAPA, which was exposed to FITC-PEG-SH and FITC-PEG-COOH.

FIG. 39.

The fluorescence signal from the fluorescein lane on the surface of the polyacrylamide gel was measured, which combined with varying amounts of BRAPA, exposed to FITC-PEG-SH and FITC-PEG-COOH.

FIG. 40 is a schematic view.

(a) The sequences of the hairpin DNA without the linker immobilized on the different samples are shown.

(b) The sequences of the hairpin DNA with the linker immobilized on different samples are shown.

FIG. 41.

Fluorescence signals from hairpin DNA oligomers with and without linkers immobilized onto a bromoacetyl-functionalized polyacrylamide surface.

FIG. 42.

Measured fluorescence from hairpin DNA oligomers with and without linker immobilized onto bromoacetyl functionalized polyacrylamide surface.

FIG. 43.

After the triphosphate is combined, the fluorescent signal from the hairpin DNA oligomer, with and without linker, is immobilized on the bromoacetyl-functionalized polyacrylamide surface.

FIG. 44.

After combining the triphosphates, fluorescence from the hairpin DNA oligomers was measured with and without the linker immobilized on the bromoacetyl-functionalized polyacrylamide surface.

FIG. 45.

(a) Experimental summary and results for each reaction step as detailed in example 12.

(b) The oligonucleotides used in the experiments detailed in example 12.

FIG. 46.

The fluorescent signal from the hairpin DNA oligomer before and after the cleavage reaction is shown (example 12).

FIG. 47.

The fluorescence signals from the hairpin DNA oligomers measured before and after the cleavage reaction are shown (example 12).

FIG. 48 is a schematic view.

The sequences of the inosine-containing strand and the complementary "helper" strand for the ligation reaction are shown (example 12).

FIG. 49 is a schematic view.

Results relating to fluorescence signals from the hairpin DNA oligo corresponding to the monitoring of the ligation reaction (example 12).

FIG. 50.

Results related to the measured fluorescence from the hairpin DNA oligo corresponding to ligation monitoring (example 12).

FIG. 51.

Results of the incorporation of 3' -O-modified-dNTPs by the terminator X DNA polymerase using the incorporation step of the method of the invention, e.g., the synthesis methods of invention 1 and 2 and variants thereof, by the terminator X DNA polymerase (FIGS. 1 to 5 and example 13).

FIG. 51a provides the nucleic acid sequences of the primer strand (primer strand portion of the synthesis strand; SEQ ID NO: 68) and the template strand (support strand; SEQ ID NO: 69).

FIG. 51b depicts a gel showing the result of incorporating 3' -O-modified-dNTPs by terminator X DNA polymerase in the presence of Mn2+ ion at 37 ℃.

Lane 1: starting the oligonucleotide.

Lane 2: combined 3' -O-azidomethyl-dTTP (efficiency > 99%)

Lane 3: 3' -O-azidomethyl-dATP incorporation (efficiency > 99%).

Lane 4: 3' -O-azidomethyl-dCTP was pooled (> 90% efficiency).

Lane 5: 3' -O-azidomethyl-dGTP was pooled (> 99% efficiency).

After addition, the newly added 3' -O-modified-dNTP occupies position n in the primer strand portion. The next nucleotide position in the primer strand portion is referred to as n-1.

FIG. 52 is a schematic view.

The figure shows a scheme describing the DNA synthesis reaction cycle described in example 14.

FIG. 53 is a schematic view.

The figure shows the oligonucleotides used in the experiment described in example 14.

FIG. 54 results of the ligation experiment as described in example 14.

The figure shows a photograph of a gel demonstrating the result of the step of cleaving the hairpin scaffold polynucleotide at the cleavage site defined by 2-deoxyuridine used as the universal nucleotide, followed by ligation of a polynucleotide linker molecule comprising 2-deoxyuridine to the cleaved scaffold polynucleotide. Lanes of the gel are as follows:

lane 1: a starting hairpin scaffold polynucleotide.

Lane 2: hairpin-scaffold polynucleotides were cleaved using a mixture of uracil DNA glycosylase and endonuclease VIII.

Lane 3: the cleaved hairpin scaffold polynucleotide is linked to a polynucleotide linker molecule.

Explanation of the drawings.

The structures depicted in fig. 11, 12a, 13a, 14a, 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a, 26a, 27a, 28b, 29, 30, 31, 32, 33, 34 and 35 will be explained in correspondence with the structures depicted in fig. 6, 7, 8, 9 and 10. Thus, in these figures, each left hand strand of a double stranded scaffold polynucleotide molecule is associated with a support strand (corresponding to strand "a" in figures 6 to 10); each right-hand strand of the double-stranded scaffold polynucleotide molecule is associated with a synthetic strand (corresponding to strand "b" in figures 6 to 10); all scaffold polynucleotide molecules comprise a lower synthetic strand corresponding to the strand comprising the primer strand portion (corresponding to the solid and dotted lines of strand "b" of fig. 6-10); before incorporation of the new nucleotides, certain scaffold polynucleotide molecules are shown (e.g., fig. 15a and 23a) having an upper synthetic strand corresponding to the strand comprising the helper strand portion (corresponding to the dashed line for strand "b" in fig. 6-10); certain scaffold polynucleotide molecules (e.g., in fig. 12a, 13a and 14 a) are shown without auxiliary strand portions (corresponding to the dashed line where strand "b" is absent in fig. 6-10); after the ligation step, certain scaffold polynucleotide molecules are shown (e.g., fig. 33, 34 and 35) whose upper synthetic strand corresponds to the strand comprising the auxiliary strand portion (corresponding to the dashed line of strand "b" in fig. 6-10), and where the auxiliary strand portion is removed prior to incorporation of the new nucleotides in the next synthesis cycle.

Furthermore, in these figures, where appropriate, each new nucleotide is shown bound to a reversible terminator group, labeled rtNTP, depicted as a small circular structure (corresponding to the small triangular structures in fig. 6 to 10), and the terminal phosphate group is labeled "p" and depicted as a small elliptical structure.

FIGS. 11c, 11d, 11g, 11h, 22a, 23a, 24a, 25a, 27a, 28b and 29 show scaffold polynucleotide molecules in which the strands comprising the auxiliary strand portion and the support strand are connected by hairpin loops. FIGS. 11b, 22a, 23a, 24a, 25a, 26a, 27a, 28b, 29, 33, 34 and 35 show scaffold polynucleotide molecules in which strands comprising a primer strand portion and a support strand are connected by a hairpin loop.

For example, the diagrams of FIGS. 27a and 28a show a scaffold polynucleotide molecule in which strands comprising an auxiliary strand portion (upper right strand) and a support strand (upper left strand) are joined by a hairpin loop, and in the same molecule, the strands of a primer strand portion (lower right strand) and support strand (lower left strand) are joined by a hairpin loop.

Detailed Description

The present invention provides methods for de novo synthesis of polynucleotide molecules based on predetermined nucleotide sequences. The synthetic polynucleotide is preferably DNA, and preferably a double-stranded polynucleotide molecule. The present invention provides advantages over existing synthetic methods. For example, all reaction steps can be carried out under aqueous conditions of mild pH without extensive protection and deprotection procedures. In addition, synthesis is not dependent on copying a pre-existing template strand comprising the predetermined nucleotide sequence.

The present inventors have determined that the use of universal nucleotides as defined herein allows for the creation of polynucleotide cleavage sites within the synthesis region that facilitate cleavage and repeated cycles of synthesis. The present invention provides general methods for synthesizing polynucleotides and for assembling large fragments comprising such synthetic polynucleotides.

Certain embodiments of the synthetic methods of the present invention will be described more generally herein by reference to exemplary methods that include two exemplary method versions of the invention and certain variants thereof (fig. 1-5). It should be understood that all exemplary methods, including exemplary method versions and variations thereof, are not intended to limit the present invention. The invention provides an in vitro method of synthesizing a double stranded polynucleotide molecule having a predetermined sequence, the method comprising performing synthesis cycles wherein in each cycle a first polynucleotide strand is extended by pooling first nucleotides of the predetermined sequence and then a second polynucleotide strand hybridized to the first strand is extended by pooling second nucleotides of the predetermined sequence. Preferably, the method is used for synthesizing DNA. The specific methods described herein are provided as examples of the invention.

Reaction conditions

In one aspect, the invention provides methods of synthesizing double-stranded polynucleotides having a predefined sequence.

In some embodiments, the synthesis is performed under conditions suitable for hybridization of nucleotides within a double-stranded polynucleotide. The polynucleotide is typically contacted with the agent under conditions that permit hybridization of the nucleotide to a complementary nucleotide. Conditions that allow hybridization are well known in the art (e.g., Sambrook et al, 2001, Molecular Cloning: a Laboratory manual,3rd edition, Cold Spring harbor Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley-lnterscice, New York (1995)).

Nucleotides can be incorporated into a polynucleotide under suitable conditions, for example, using a polymerase (e.g., terminator IX polymerase) or terminal deoxynucleotidyl transferase (TdT), or a functional variant thereof, to incorporate modified nucleotides (e.g., 3' -O-modified-dntps) at a suitable temperature (e.g., about 65 ℃) in the presence of a suitable buffer solution. In one embodiment, the buffer solution may comprise 2mM Tris-HCl, 1mM (NH)₄)₂SO₄、1mM KCl、0.2mM MgSO₄And 0.01 percentX-100。

Cleavage of the polynucleotide may be performed under suitable conditions, for example using a polynucleotide lyase (e.g., an endonuclease) in the presence of a suitable buffer solution at a temperature compatible with the enzyme (e.g., 37 ℃). In one embodiment, the buffer solution may comprise 5mM potassium acetate, 2mM Tris-acetate, 1mM magnesium acetate and 0.1mM DTT.

Ligation of polynucleotides may be performed under suitable conditions, such as using a ligase (e.g., T4 DNA ligase) in the presence of a suitable buffer solution at a temperature compatible with the enzyme (e.g., room temperature). In one embodiment, the buffer solution may comprise 4.4mM Tris-HCl, 7mM MgCl₂0.7mM dithiothreitol, 0.7mM ATP, 5% polyethylene glycol (PEG 6000).

Deprotection may be carried out under suitable conditions, for example using a reducing agent (e.g. TCEP). For example, deprotection can be performed using TCEP in Tris buffer (e.g., at a final concentration of 300 mM).

Anchor polynucleotides and scaffold polynucleotides

Double-stranded polynucleotides having a predetermined sequence are synthesized by the methods of the invention by incorporating the predetermined nucleotides into a pre-existing polynucleotide, referred to herein as a scaffold polynucleotide, which is attachable or capable of being attached to a surface, as described herein. As described in more detail herein, the scaffold polynucleotide forms a support structure to accommodate the newly synthesized polynucleotide, and as is apparent from the description herein, it does not include a preexisting template strand that replicates as in conventional synthetic methods. If the scaffold polynucleotide is attached to a surface, the scaffold polynucleotide may be referred to as an anchor polynucleotide. Surface attachment chemistry for attaching scaffold polynucleotides to a surface to form anchor polynucleotides is described in more detail herein.

In one embodiment, the scaffold polynucleotide comprises a synthetic strand hybridized to a complementary support strand. The synthetic strand comprises a primer strand portion (see, e.g., FIGS. 1 to 5). A synthetic strand may be provided that hybridizes to the complementary support strand. Alternatively, the support strand and the synthetic strand may be provided separately and then hybridized.

A scaffold polynucleotide may be provided in which each support and synthesis strand is not joined at adjacent ends. The scaffold polynucleotide may be provided with support and synthesis strands at both ends of the scaffold polynucleotide, which are linked at adjacent ends, for example by hairpin loops. The scaffold polynucleotide may be provided with a support and synthesis strand at one end of the scaffold polynucleotide or any other suitable linker, which are connected at adjacent ends, for example by hairpin loops.

As described in more detail herein (see fig. 11), a scaffold polynucleotide with or without a hairpin can be immobilized to a solid support or surface.

The term "hairpin" or "hairpin loop" is commonly used in the art. The term "hairpin loop" is also commonly referred to as "stem loop". These terms refer to regions of secondary structure in a polynucleotide that include a loop of unpaired nucleobases that is formed when one strand of a polynucleotide molecule hybridizes to another portion of the same strand due to intramolecular base pairing. The hair clip may thus resemble a U-shaped structure. An example of such a structure is shown in fig. 11.

In certain methods described herein, a new synthesis is initiated by incorporating a first nucleotide of a predetermined sequence into a scaffold polynucleotide by the action of a ligase. Thus, as further described herein, the first nucleotide of the predetermined sequence is linked to a terminal nucleotide of the support strand of the scaffold polynucleotide. The first nucleotide of the predefined sequence is provided by a polynucleotide linker molecule comprising a support strand, an auxiliary strand and a complementary linker end. The first nucleotide of the predetermined sequence is provided as the terminal nucleotide of the support strand of the complementary linker.

The terminal nucleotide of the auxiliary strand at the complementary linker is a non-ligatable nucleotide and is typically provided lacking a phosphate group. This prevents the terminal nucleotide of the helper strand from ligating to the terminal nucleotide of the primer strand portion of the scaffold polynucleotide and, upon ligation, creates a single-stranded break site between the helper strand and the primer strand portion. The generation and maintenance of single-strand breaks may be achieved in other ways. For example, the terminal nucleotide of the auxiliary strand portion may have a suitable blocking group that prevents attachment to the primer strand portion.

In certain methods described herein, the second nucleotide of the predetermined sequence is incorporated into the scaffold polynucleotide by the action of a polymerase or transferase. Thus, the polymerase or transferase will act to extend the terminal nucleotide of the primer strand portion.

Further details of the general process scheme of the exemplary process are provided further herein.

Nucleotides and universal nucleotides

The nucleotides in the synthetic polynucleotides that may be combined by any of the methods described herein may be nucleotides, nucleotide analogs, and modified nucleotides.

Nucleotides can include natural nucleobases or non-natural nucleobases. Nucleotides may contain natural nucleobases, sugars and phosphate groups. Natural nucleobases include adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C). One component of the nucleotide may be further modified.

Nucleotide analogs are nucleotides that are structurally modified in the base, sugar or phosphate or a combination thereof and are still acceptable to polymerases as substrates for the merged oligonucleotide strands.

The non-natural nucleobase can be a nucleobase that to some extent bonds, e.g., hydrogen, to all nucleobases in the target polynucleotide. The non-natural nucleobases are preferably nucleobases that to some extent bond, e.g., hydrogen, to nucleotides including the nucleosides adenosine (a), thymine (T), uracil (U), guanine (G), and cytosine (C).

The non-natural nucleotides may be Peptide Nucleic Acids (PNA), Locked Nucleic Acids (LNA) and Unlocked Nucleic Acids (UNA), Bridged Nucleic Acids (BNA) or morpholinos, phosphorothioates or methylphosphonates.

The non-natural nucleotide can include a modified sugar and/or a modified nucleobase. Modified sugars include, but are not limited to, 2' -O-methyl ribose. Modified nucleobases include but are not limited to methylated nucleobases. Methylation of nucleobases is a recognized form of epigenetic modification that has the ability to alter the expression of genes and other elements, such as micrornas. Methylation of nucleobases occurs at discrete loci that are primarily dinucleotides consisting of CpG motifs, but can also occur at CHH motifs (where H is A, C or T). Typically, during methylation, a methyl group is added to the fifth carbon of the cytosine base to produce methylcytosine. Thus, modified nucleobases include but are not limited to 5-methylcytosine.

The nucleotides of the predetermined sequence can be combined with the partner nucleotide pair to form a nucleotide pair. The partner nucleotide may be a complementary nucleotide. Complementary nucleotides are nucleotides that are capable of binding, for example hydrogen, to some extent to a nucleotide of the predefined sequence.

Typically, the nucleotide of the predetermined sequence is incorporated into the polynucleotide opposite the natural complementary partner nucleobase. Thus, adenosine can be combined as opposed to thymine, and vice versa. Guanine may be incorporated as opposed to cytosine, and vice versa. Alternatively, nucleotides of a predetermined sequence may be combined as opposed to the nucleobase which is the partner to which the linkage, e.g., hydrogen, is to some extent.

Alternatively, the partner nucleotide may be a non-complementary nucleotide. Non-complementary nucleotides are nucleotides that are not capable of binding, e.g., hydrogen, to a nucleotide of the predefined sequence. Thus, nucleotides of the predetermined sequence can be combined opposite to partner nucleotides to form mismatches, provided that the synthesized polynucleotide is generally double-stranded, and wherein the first strand is linked to the second strand by hybridization.

The term "relative" is to be understood as relating to the normal use of the term in the field of nucleic acid biochemistry and in particular to conventional Watson-Crick base pairing. Thus, a first nucleic acid molecule of sequence 5'-ACGA-3' may form a duplex with a second nucleic acid molecule of sequence 5'-TCGT-3', wherein the G of the first molecule will be located opposite and hydrogen bonded to the C of the second molecule. A first nucleic acid molecule of sequence 5'-ATGA-3' may form a duplex with a second nucleic acid molecule of sequence 5'-TCGT-3', wherein the T of the first molecule is mismatched with the G of the second molecule but still located opposite thereto and will serve as a partner nucleotide. The principles apply to any nucleotide pair relationship disclosed herein, including partner pairs comprising universal nucleotides.

In all of the methods described herein, the position in the support chain and the relative position in the synthesis chain are designated as position numbers "n". This position refers to the position of a nucleotide in the support strand of the scaffold polynucleotide that is opposite the nucleotide position in the synthetic strand that is occupied by or will be occupied by the second or other nucleotide of the predetermined sequence in any given synthesis cycle (when it is added to the end of a primer strand portion in that cycle or in a subsequent cycle of the pooling step). Position "n" also refers to the position of the polynucleotide linker molecule in the support strand prior to the ligation step, which is the nucleotide position that will be opposite to the second or other nucleotide of the predetermined sequence upon ligation with the polynucleotide linker molecule scaffold polynucleotide and incorporation of the second or other nucleotide of the predetermined sequence by the action of a polymerase or transferase.

Both the position in the support chain and the relative position in the synthesis chain may be referred to as position n.

Further details regarding the definition of location "n" are provided with reference to fig. 1-5 and their descriptions regarding exemplary synthesis method versions of the present invention and variations thereof described in greater detail herein.

Nucleotides and nucleotide analogs may preferably be provided as nucleoside triphosphates. Thus, in any of the methods of the present invention, nucleotides may be incorporated from 2 '-deoxyribonucleoside-5' -O-triphosphate (dNTPs) for the synthesis of a DNA polynucleotide, for example by the action of a DNA polymerase or by the action of an enzyme having deoxynucleotide terminal transferase activity. In any of the methods of the invention, to synthesize an RNA polynucleotide, the nucleotides can be combined with ribonucleoside 5' -O-triphosphates (NTPs), for example by the action of an RNA polymerase or for example by the action of an enzyme having nucleotide terminal transferase activity. The triphosphate may be substituted by tetraphosphoric acid or by pentaphosphoric acid (generally, oligophosphoric acid). These oligomeric phosphoric acids may be substituted by other alkyl or acyl groups:

The method of the present invention may use universal nucleotides. Universal nucleotides can be used as components of the support strand of the scaffold molecule to facilitate the newly incorporated nucleotides to be correctly paired with their desired partner nucleotides during each cycle of synthesis. If desired, universal nucleotides may also be incorporated into the synthetic strand as a component of the predetermined nucleotide sequence.

Universal nucleotides are nucleotides in which the nucleobase will to some extent bond, e.g. hydrogen bond, to the nucleobase of any nucleotide of the predefined sequence. The universal nucleotide is preferably one that will be bonded to some extent with nucleotides including the nucleosides adenosine (a), thymine (T), uracil (U), guanine (G) and cytosine (C), e.g., hydrogen bonded. Universal nucleotides may bond more strongly to some nucleotides than to others. For example, a universal nucleotide (I) comprising the nucleoside 2' -deoxyinosine would show a preferential pairing order of I-C > I-a > I-G-I-T.

Examples of possible universal nucleotides are inosine or nitroindole. The universal nucleotide preferably comprises one of the following nucleobases: hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole or phenyl (C6-aromatic ring). More preferably, the universal nucleotide comprises one of the following nucleosides: 2 '-deoxyinosine, inosine, 7-deaza-2' -deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 4-nitroindole 2 '-deoxyribonucleoside, 4-nitroindole ribonucleoside, 5-nitroindole 2' -deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2 '-deoxyribonucleoside, 6-nitroindole ribonucleoside, 3-nitropyrrole 2' -deoxyribonucleoside, 3-nitropyrrole ribonucleoside, acyclic sugar analogs of inosine, nitroimidazole 2 '-deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole 2' -deoxyribonucleoside, 4-nitropyrazole ribonucleoside, deoxyriboside, and pharmaceutically acceptable salts thereof, 4-nitrobenzimidazole 2 'deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside, 5-nitroindazole 2' deoxyribonucleoside, 5-nitroindazole ribonucleoside, 4-aminobenzimidazole 2 'deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside or phenyl C-2' -deoxyribonucleoside.

Some examples of universal bases are shown below:

universal nucleotides incorporating a cleavable base, including photocleavable bases and enzymatically cleavable bases, some examples of which are shown below, may also be used.

Photocleavable base:

base analogues cleavable by endonuclease III:

base analogues cleavable by formamidopyrimidine DNA glycosylase (Fpg):

base analogues cleavable by 8-oxoguanine DNA glycosylase (hOGG 1):

base analogues cleavable by hNeil 1:

base analogues cleavable by Thymine DNA Glycosylase (TDG):

base analogues cleavable by human alkyl adenine DNA glycosylase (hAAG):

bases cleavable by uracil DNA glycosylase:

a base cleavable by human single-strand selective monofunctional uracil-DNA glycosylase (SMUG 1):

bases cleavable by 5-methylcytosine DNA glycosylase (ROS 1):

(see S.S.David, S.D.Williams Chemical reviews 1998,98, 1221-.

In any method involving a scaffold polynucleotide, the universal nucleotide most preferably comprises 2' -deoxyinosine.

Examples of epigenetic bases that can be incorporated using any of the synthetic methods described herein include the following:

examples of modified bases that can be incorporated using any of the synthetic methods described herein include the following:

examples of halogenated bases that can be combined using any of the synthetic methods described herein include the following:

wherein R1 ═ F, Cl, Br, I, alkyl, aryl, fluorescent label, aminopropargyl, aminoallyl.

Examples of amino-modified bases that can be incorporated using any of the synthetic methods described herein that can be used, for example, in attachment/linker chemistry, include the following:

wherein the base is A, T, G or C, with an alkyne or alkene linker.

Examples of modified bases that can be incorporated using any of the synthetic methods described herein that can be used, for example, in click chemistry, include the following:

examples of biotin-modified bases that can be incorporated using any of the synthetic methods described herein include the following:

wherein the base is A, T, G or C, with an alkyne or alkene linker.

Examples of bases bearing fluorophores and quenchers that can be combined using any of the synthetic methods described herein include the following:

nucleotide incorporating enzymes

Available enzymes are capable of extending the single-stranded polynucleotide portion of a double-stranded polynucleotide molecule by the addition of nucleotides and/or are capable of extending one strand of a blunt-ended double-stranded polynucleotide molecule. This comprises an enzyme having a template-independent enzyme activity, such as a template-independent polymerase or a template-independent transferase activity.

Thus, in any of the methods described herein, the enzyme is used to add a second nucleotide of the predetermined sequence and/or another nucleotide of the predetermined sequence to the end of the primer strand portion of the synthetic strand of the scaffold polynucleotide that has template-independent enzymatic activity, e.g., a template-independent polymerase or template-independent transferase activity.

Any suitable enzyme may be employed to add the predetermined nucleotide using the methods described herein. Thus, in all the methods defined and described herein, with reference to the use of a polymerase or transferase, the polymerase or transferase may be replaced by another enzyme capable of performing the same function as the polymerase or transferase in the context of the methods of the present invention.

Polymerases can be used in the methods described herein. Polymerases, particularly nucleotides having an attached reversible terminator group, can be selected based on their ability to incorporate modified nucleotides, as described herein. In the exemplary methods described herein, all polymerases acting on DNA must not have 3 'to 5' exonuclease activity. The polymerase may have strand displacement activity.

Thus, preferably, the polymerase is a modified polymerase having an enhanced ability to incorporate nucleotides comprising a reversible terminator group compared to an unmodified polymerase. The polymerase is more preferably a genetically engineered variant of a native DNA polymerase from a Thermococcus species (Thermococcus) 9 ℃ N, preferably species 9 ℃ N-7. Examples of modified polymerases are terminator IX DNA polymerase and terminator X DNA polymerase available from New England BioLabs. The enzyme has enhanced ability to incorporate 3' -O-modified dNTPs.

Examples of other polymerases that can be used to incorporate a reversible terminator dNTP in any of the methods of the present invention are Deep Vent (Exo-), 9 ℃ N DNA polymerase, terminator DNA polymerase, Therminator IX DNA polymerase, terminator X DNA polymerase, Klenow fragment (Exo-), Bst DNA polymerase, Bsu DNA polymerase, Sulfolobus DNA polymerase I, and Taq polymerase.

Examples of other polymerases for which the reversible terminator NTP incorporation can be used in any of the methods of the present invention are T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, pol lambda, pol micro or Φ 29DNA polymerase.

For extension of such polynucleotide synthesis molecules, including DNA, DNA polymerases may be used. Any suitable DNA polymerase may be used.

The DNA polymerase can be, for example, Bst DNA polymerase full length, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, E.coli DNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reverse transcriptase, phi29 DNA polymerase, sulfolobus DNA polymerase IV, Taq DNA polymerase, T4 DNA polymerase, T7 DNA polymerase and enzymes with reverse transcriptase activity, such as M-MuLV reverse transcriptase.

The DNA polymerase may lack 3 'to 5' exonuclease activity. Any such suitable polymerase may be used. Such DNA polymerases can be, for example, Bst DNA polymerase full length, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3 '→ 5' in vitro-), M-MuLV reverse transcriptase, sulfolobus DNA polymerase IV, Taq DNA polymerase.

The DNA polymerase may have strand displacement activity. Any such suitable polymerase may be used. Such DNA polymerases may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3 '→ 5' in vitro-), M-MuLV reverse transcriptase, phi29 DNA polymerase.

The DNA polymerase may lack 3 'to 5' exonuclease activity and may have strand displacement activity. Any such suitable polymerase may be used. Such DNA polymerases may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, E.coli DNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reverse transcriptase.

The DNA polymerase may lack 5 'to 3' exonuclease activity. Any such suitable polymerase may be used. Such DNA polymerases may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3 '→ 5' in vitro-), M-MuLV reverse transcriptase, phi29 DNA polymerase, sulfolobus solfataricus DNA polymerase IV, T4 DNA polymerase, T7 DNA polymerase.

DNA polymerases may lack both 3 'to 5' and 5 'to 3' exonuclease activity and may have strand displacement activity. Any such suitable polymerase may be used. Such DNA polymerases may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3 '→ 5' in vitro-), M-MuLV reverse transcriptase.

The DNA polymerase may also be a genetically engineered variant. For example, the DNA polymerase may be a genetically engineered variant of a native DNA polymerase from a Thermococcus species, 9 ° N, e.g., species 9 ° N-7. One such example of a modified polymerase is terminator IX DNA polymerase or terminator X DNA polymerase available from New England Biolabs. Other engineered or variant DNA polymerases include Deep Vent (Exo-), Vent (Exo-), 9 ℃ N DNA polymerase, terminator DNA polymerase, Klenow fragment (Exo-), Bst DNA polymerase, Bsu DNA polymerase, sulfolobus DNA polymerase I, and Taq polymerase.

To extend such polynucleotide synthetic molecules comprising RNA, any suitable enzyme may be used. For example, RNA polymerase can be used. Any suitable RNA polymerase may be used.

The RNA polymerase can be T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, and Escherichia coli RNA polymerase holoenzyme.

The enzyme may have terminal transferase activity, e.g., the enzyme may be a terminal nucleotidyl transferase or a terminal deoxynucleotidyl transferase, and wherein the polynucleotide synthesis molecule is extended to form a polynucleotide molecule comprising DNA or RNA, preferably DNA. Any of these enzymes may be used in the methods of the invention, wherein it is desired to extend the polynucleotide synthesis molecule.

One such enzyme is a terminal nucleotidyl transferase, such as terminal deoxynucleotidyl transferase (TdT) (see, e.g., Motea et al, 2010; MinhazUd-Dean, Syst. Synth. biol.,2008,2(3-4), 67-73). TdT is capable of catalyzing the addition of a nucleotide molecule (nucleoside monophosphate) from a nucleoside triphosphate substrate (NTP or dNTP) to a polynucleotide synthesis molecule. TdT is capable of catalyzing the addition of natural and non-natural nucleotides. It is also capable of catalyzing the addition of nucleotide analogs (Motea et al, 2010). Pol λ and Pol μ enzymes (Ramadan K, et al, J.mol.biol.,2004,339(2), 395-.

Techniques for extending both single-stranded polynucleotide molecules DNA and RNA in the absence of a template by the action of a terminal transferase (e.g., terminal deoxynucleotidyl transferase; TdT) to produce artificially synthesized single-stranded polynucleotide molecules have been widely discussed in the art. Such techniques are disclosed in, for example, patent application publications WO2016/034807, WO 2016/128731, WO2016/139477, and WO2017/009663, as well as US2014/0363852, US2016/0046973, US2016/0108382, and US 2016/0168611. These documents describe controlled extension of single stranded polynucleotide synthetic molecules to produce artificially synthesized single stranded polynucleotide molecules by the action of TdT. The use of such enzymes is described for extension by natural and non-natural/artificial nucleotides, such as by extension of modified nucleotides, for example nucleotides incorporating blocking groups. Any terminal transferase disclosed in these documents and any enzyme fragment, derivative, analogue or functional equivalent thereof may be used in the method of the invention, provided that the terminal transferase function is preserved in the enzyme.

Directed evolution techniques, routine screening, rational or semi-rational engineering/mutagenesis methods, or any other suitable method can be used to alter any such enzyme to provide and/or optimize a desired function. Any other enzyme capable of extending a portion of a single-stranded polynucleotide molecule without the use of a template may be used, such as a molecule comprising DNA or RNA, or one strand of a blunt-ended molecule having nucleotides.

Thus, in any of the methods defined herein, a single-stranded polynucleotide synthesis molecule comprising DNA or a blunt-ended double-stranded polynucleotide comprising DNA may be extended by an enzyme having template-independent enzyme activity, such as template-independent polymerase or transferase activity. The enzyme may have nucleotidyl transferase activity, for example a deoxynucleotidyl transferase, for example terminal deoxynucleotidyl transferase (TdT) or an enzymatic fragment, derivative, analogue or functional equivalent thereof. The polynucleotide-synthesized molecules extended by the action of this enzyme include DNA.

In any of the methods defined herein, the single-stranded portion of the RNA-containing polynucleotide synthesis molecule or the RNA-containing blunt-ended double-stranded polynucleotide may be extended with a nucleotidyl transferase (e.g., comprising TdT) or enzyme fragment, derivative, analog, or functional equivalent thereof. The polynucleotide-synthesized molecule extended by the action of such an enzyme may comprise RNA. For the synthesis of single stranded polynucleotide synthetic molecules comprising RNA or single stranded portions of polynucleotide synthetic molecules comprising RNA, any suitable nucleotidyl transferase can be used. Nucleotide transferases, such as poly (U) polymerases and poly (a) polymerases (e.g., from e.coli), are capable of template-independent addition of nucleotide monophosphate units to polynucleotide synthesis molecules. Any of these enzymes, as well as any enzyme fragment, derivative, analogue or functional equivalent thereof, may be used in the method of the invention, provided that nucleotidyl transferase function is preserved in the enzyme. Directed evolution techniques, routine screening, rational or semi-rational engineering/mutagenesis methods, or any other suitable method can be used to alter any such enzyme to provide and/or optimize a desired function.

Reversible terminator groups

In any of the synthetic methods of the invention defined and described herein, the nucleotides incorporated into the synthetic strand by the action of a polymerase or transferase, also referred to herein as reversible terminating groups, are preferably incorporated as nucleotides comprising one or more reversible blocking groups.

The function of these groups is to prevent further extension of the enzyme in a given synthesis cycle, so that only a predetermined sequence of nucleotides can be controllably used to extend the synthesized strand, thus preventing non-specific nucleotide incorporation. Any function that achieves the described effect may be used in any of the methods defined and described herein. Reversible blocking group/reversible terminator group attached to the nucleotide and the deblocking step are preferred methods to achieve this effect. However, this effect may be achieved by suitable alternatives.

Any suitable reversible protecting group may be attached to the nucleotide to prevent further extension of the enzyme after incorporation of the nucleotide into the synthetic strand in a given cycle and to limit the incorporation of one nucleotide per step into the synthetic strand. In any of the methods of the present invention, the reversible blocking group is preferably a reversible terminator group, which acts to prevent further extension by the polymerase. Examples of reversible terminators are provided below.

Propargyl reversible terminator:

allyl reversible terminator:

cyclooctene reversible terminator:

cyanoethyl reversible terminator:

nitrobenzyl reversible terminator:

disulfide reversible terminator:

azidomethyl reversible terminator:

aminoalkoxy reversible terminator:

nucleoside triphosphates with bulky groups attached to the base can serve as a substitute for the reversible terminator group on the 3' -hydroxyl group and can prevent further incorporation. The group can be deprotected by TCEP or DTT to yield a natural nucleotide.

For the synthesis of a DNA polynucleotide according to any of the methods of the present invention, a preferred modified nucleoside is a 3' -O-modified-2 ' -deoxyribonucleoside-5 ' -O-triphosphate. For the synthesis of RNA polynucleotides according to any of the methods of the present invention, a preferred modified nucleoside is a 3 '-O-modified-ribonucleoside-5' -O-triphosphate.

Preferred modified dntps are modified dntps which are 3 '-O-allyl-dNTP and 3' -O-azidomethyl-dNTP.

The 3' -O-allyl-dNTPs are shown below.

The methods of the invention described and defined herein may involve a deprotection or deblocking step. Such a step involves removal of the reversible blocking group (e.g., reversible terminator group) by any suitable method, or otherwise reversing the function of the blocking/terminator group to inhibit further extension by the enzyme/polymerase.

The reversible terminator group may be removed in a deprotection step using any suitable reagent.

A preferred deprotection agent is tris (carboxyethyl) phosphine (TCEP). TCEP can be used to synthesize 3' -O-allyl-nucleotide (and Pd)⁰Binding) and 3' -O-azidomethyl-nucleotide.

Examples of deprotection reagents are provided below.

Propargyl reversible terminator:

with Pd catalyst-Na₂PdCl₄、PdCl₂And (6) processing.

Ligands may be used, for example: triphenylphosphine-3, 3',3 "-trisodium trisulfonate.

Allyl reversible terminator:

with Pd catalyst-Na₂PdCl₄、PdCl₂And (6) processing.

Ligands may be used, for example: triphenylphosphine-3, 3',3 "-trisodium trisulfonate.

Azidomethyl reversible terminator:

treatment with thiol (mercaptoethanol or dithiothreitol) or tris (2-carboxyethyl) phosphine-TCEP.

Cyanoethyl reversible terminator:

treatment with fluoride-ammonium fluoride, tetrabutylammonium chloride (TBAF).

Nitrobenzyl reversible terminator:

exposure to UV light.

Disulfide reversible terminator:

treatment with thiol (mercaptoethanol or dithiothreitol) or tris (2-carboxyethyl) phosphine-TCEP.

Aminoalkoxy reversible terminator:

with Nitrite (NO)₂ ^-、HNO₂) Treatment at pH 5.5

Reversible protecting groups (e.g., reversible terminator groups) may be removed by a step performed immediately after the combining step, provided that the undesired agent is removed from the combining step to prevent further combining after removal of the reversible terminator groups.

Polynucleotide linker molecules

In methods requiring the presence of a scaffold polynucleotide and a ligation step, the choice of configuration and structure of the polynucleotide linker molecule will also depend on the particular method employed. The polynucleotide linker molecule typically comprises a support strand as described herein and an auxiliary strand as described herein. The polynucleotide linker molecule comprises a complementary linker at one end of the molecule. The complementary linker ends of the polynucleotide linker molecules will be attached to the ends of the scaffold polynucleotide.

The complementary linker end of the polynucleotide linker molecule has a non-ligatable terminal nucleotide, typically a non-phosphorylated terminal nucleotide, in the auxiliary strand. This prevents the auxiliary strand portion of the synthetic strand from being linked to the primer strand portion of the synthetic strand, and thus a single-strand break is generated in the synthetic strand after the linkage. Alternative methods for preventing ligation in the synthesis chain may be used. For example, the blocking moiety may be linked to the terminal nucleotide in the helper strand. Furthermore, the auxiliary strands may be removed from the scaffold molecule prior to cleavage, for example by denaturation, as further described herein. The complementary linking end of the polynucleotide linker molecule has a ligatable terminal nucleotide in the support strand adjacent to the non-ligatable terminal nucleotide in the auxiliary strand. The ligatable terminal nucleotide of the support strand is the first nucleotide of the predetermined sequence incorporated into the scaffold molecule by the action of the ligase. The complementary linking end of the polynucleotide linker molecule also has a universal nucleotide in the support strand. The exact positioning of the universal nucleotides in the support strand with respect to the ligatable terminal nucleotides of the support strand will depend on the specific reaction chemistry employed, as will be apparent from the description of the specific method version and variants thereof.

By reference to the exemplary methods described herein and their depictions in the figures, the appropriate structure of a polynucleotide linker molecule can be readily determined.

Connection of

In the method of the invention involving the step of joining, the joining may be achieved using any suitable method. Preferably, the ligation step will be performed by a ligase enzyme. The ligase may be a modified ligase. The ligase can be T3 DNA ligase or T4 DNA ligase. The ligase may be blunt-ended TA ligase (blunt TA ligase). Blunt-ended TA ligase is available, for example, from New England Biolabs (NEB). This is a ready-to-use premix of T4 DNA ligase, ligation enhancer and optimized reaction buffer specifically formulated to improve ligation and conversion of blunt-ended substrates. Molecules, enzymes, chemicals and methods for ligating (ligating) single-and double-stranded polynucleotides are well known to those skilled in the art.

Cleavage of scaffold polynucleotides

In methods where the presence of scaffold polynucleotide and a cleavage step is desired, the choice of reagents to perform the cleavage step will depend on the particular method employed. In the support strand, the cleavage site is defined by the specific position of the universal nucleotide. Thus, the configuration of the desired cleavage site and the selection of an appropriate cleavage reagent will depend on the particular chemistry employed in the method, as will be readily apparent by reference to the exemplary methods described herein.

Some examples of DNA cleaving enzymes that recognize modified bases are shown in the table below.

Synthetic chain

In the methods of synthesizing polynucleotides or oligonucleotides described herein, including but not limited to fig. 1-5 and synthetic method versions 1 and 2 of the invention and variants thereof further described herein, the scaffold polynucleotide has a synthetic strand. The synthetic strand comprises a primer strand portion. During the synthesis cycle, the first nucleotide of the predetermined sequence is incorporated into the support strand by extending the primer strand portion, incorporating each new second nucleotide of the predetermined sequence into the synthesis strand. Enzymes such as polymerases or enzymes with terminal transferase activity may be used to catalyze the incorporation/addition of each new second nucleotide. Each newly bound second nucleotide of the predetermined sequence will serve as a terminal nucleotide of the primer strand portion for priming binding in the next binding step. Thus, in any given synthesis cycle, the primer strand portion of the synthesized strand will contain sufficient polynucleotide sequence to allow priming by the appropriate enzyme. In certain embodiments further described herein, in a given synthesis cycle, a second nucleotide of the predetermined sequence is incorporated into the synthetic strand, and then one or more additional nucleotides are incorporated into the synthetic strand. In such embodiments, the second nucleotide and the other nucleotides of the predetermined sequence comprise a reversible terminator group, and the method further comprises the step of removing the reversible terminator group from the nucleotide after combining the next nucleotide and before combining the next nucleotide.

The terms "incorporation", "extension" and "addition" of nucleotides have the same meaning herein.

Auxiliary chain

Auxiliary strands may be provided in the polynucleotide linker molecule to facilitate ligation of the polynucleotide linker molecule to the scaffold polynucleotide during the ligation step. The helper strand may also facilitate the binding of the lyase during the cleavage step. The auxiliary strand may be omitted, provided that alternative means are provided to ensure binding of the lyase in the cleavage step and, if desired, ligation in the ligation step. In a preferred method of the invention, the synthetic strand has an auxiliary strand.

There is no particular requirement on the length, sequence and structural parameters of the helper strand, provided that the helper strand is adapted to facilitate the binding of ligase and lyase if desired.

The auxiliary strand may comprise nucleotides, nucleotide analogs/derivatives and/or non-nucleotides.

Preferably, mismatches to the support strand within the sequence region of the helper strand should be avoided, GC-and AT-rich regions should be avoided, and regions of secondary structure, such as hairpins or bulges, should be avoided.

The length of the helper strand may be 10 bases or more. Optionally, the length of the helper strand may be 15 bases or more, preferably 30 bases or more. However, the length of the helper strand may vary, provided that the helper strand is capable of facilitating cleavage and/or ligation.

The auxiliary strand must hybridize to the corresponding region of the support strand. It is not necessary that the entire helper strand hybridises to the corresponding region of the support strand if the helper strand can facilitate binding of ligase in the ligation step and/or binding of lyase in the cleavage step. Thus, mismatches between the respective regions of the helper and support strands can be tolerated. The auxiliary chain may be longer than the corresponding region of the supporting chain. The support strand may extend beyond the region corresponding to the auxiliary strand in a direction away from the primer strand. The helper strand may be linked to the corresponding region of the support strand, e.g., by a hairpin.

The auxiliary strand may be hybridized to the support strand such that the terminal nucleotide of the auxiliary strand at the nick site occupies the next sequential nucleotide position in the synthetic strand relative to the terminal nucleotide of the primer strand portion at the nick site. Thus, in this configuration, there is no nucleotide position nick between the helper strand and the primer strand. However, due to the presence of single strand breaks or gaps, the helper strand and primer strand will be physically separated.

The nucleotides in the auxiliary strand that pair with the universal nucleotides can be any suitable nucleotides. Preferably, pairing that could distort the helical structure of the molecule should be avoided. Preferably, cytosine serves as a partner of a universal nucleotide. In a particularly preferred embodiment, the universal nucleotide is inosine or an analogue, variant or derivative thereof and the partner nucleotide of the universal nucleotide in the helper strand is cytosine.

Removal of auxiliary chains

In any of the synthetic methods of the invention described herein, the helper strand provided by the polynucleotide linker molecule may be removed from the scaffold polynucleotide prior to the step of combining the second nucleotides of the predetermined sequence.

The auxiliary strand portion of the synthetic strand may be removed from the scaffold polynucleotide by any suitable method, including but not limited to: (i) heating the scaffold polynucleotide to a temperature of about 80 ℃ to about 95 ℃ and separating the auxiliary strand portion from the scaffold polynucleotide, (ii) treating the scaffold polynucleotide with a urea solution (e.g., 8M urea) and separating the auxiliary strand portion from the scaffold polynucleotide, (iii) treating the scaffold polynucleotide with formamide or a formamide solution (e.g., 100% formamide) and separating the auxiliary strand portion from the scaffold polynucleotide, or (iv) contacting the scaffold polynucleotide with a single-stranded polynucleotide molecule comprising a nucleotide sequence region complementary to the sequence of the auxiliary strand portion, thereby competitively inhibiting hybridization of the auxiliary strand portion to the scaffold polynucleotide.

In a method of removing the helper strand portion from the scaffold polynucleotide after the step of ligating the double stranded polynucleotide linker molecule to the cleaved scaffold polynucleotide and before the step of cleaving the scaffold polynucleotide, the cleaving step will comprise cleaving the support strand in the absence of the double stranded region provided by the helper strand. Any suitable enzyme may be selected to perform such a cleavage step, for example selected from any suitable enzyme disclosed herein.

Primer chain

The primer strand portion should be suitable to allow an enzyme (e.g., a polymerase or an enzyme with terminal transferase activity) to prime synthesis, i.e., catalyze the addition of a new nucleotide at the end of the primer strand portion.

The primer strand may include a sequence region (e.g., as shown by the dashed lines in the structures depicted in each of fig. 1 to 5) that can be used to prime synthesis of a new polynucleotide. The primer strand may be composed of a region of sequence that can act to prime synthesis of a new polynucleotide, and thus the entirety of the primer strand may be a sequence that can act to prime synthesis of a new polynucleotide as described herein.

There is no particular requirement for the length, sequence and structural parameters of the primer strand, provided that the primer strand is suitable for priming new polynucleotide synthesis.

The primer strand may comprise nucleotides, nucleotide analogs/derivatives and/or non-nucleotides.

The skilled person can readily construct primer strands capable of priming new polynucleotide syntheses. Thus, within the sequence region of the primer strand that can serve to prime a new polynucleotide, mismatches with the support strand should be avoided, GC-and AT-rich regions should be avoided, and in addition regions of secondary structure, such as hairpins or bulges, should be avoided.

One skilled in the art can select the length of the sequence region of the guide strand that can be used to prime the synthesis of a new polynucleotide, as desired and with the polymerase used. The region may be 7 bases or more, 8 bases or more, 9 bases or more, or 10 bases or more in length. Optionally, the region is 15 bases or more, preferably 30 bases or more in length.

The primer strand must hybridize to the corresponding region of the support strand. If the primer strand is capable of priming new polynucleotide synthesis, it is not necessary that the entire primer strand hybridises to the corresponding region of the support strand. Thus, mismatches between the corresponding regions of the primer strand and the support strand can be tolerated to some extent. Preferably, the region of the primer strand sequence that can be used to prime synthesis of a new polynucleotide should include nucleobases complementary to the corresponding nucleobases in the support strand.

The primer strand may be linked to the corresponding region of the support strand, for example, by a hairpin.

Support chain

In the methods of the invention, including but not limited to the synthetic methods versions 1-2 of the invention as described above, the scaffold polynucleotide is provided with a support strand. The support strand hybridizes to the synthetic strand. There is no particular requirement for the length, sequence and structural parameters of the support strand, so long as the support strand is compatible with the primer strand portion described herein and, if present, the auxiliary strand portion of the synthetic strand, as described above.

Synthesis of polynucleotides

Polynucleotides having a predetermined sequence synthesized according to the methods described herein are double-stranded. The synthesized polynucleotide is generally double-stranded, and wherein the first strand is linked to the second strand by hybridization. As long as the entire first strand is linked to the second strand by hybridization, both mismatched and non-hybridized regions can be tolerated.

Hybridization can be defined by moderately stringent or stringent hybridization conditions. Moderately stringent hybridization conditions are performed using a pre-wash solution (or other similar hybridization solution, e.g., a solution containing about 50% formamide at 42 ℃ C.) at 55 ℃ in a hybridization buffer containing 5 Xsodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0mM EDTA (pH 8.0), about 50% formamide, and 60 ℃ in 0.5XSSC, 0.1% SDS. Stringent hybridization conditions are performed in 6XSSC at 45 ℃ followed by one or more washes in 0.1XSSC, 0.2% SDS at 68 ℃.

A double-stranded polynucleotide having a predefined sequence synthesized according to the methods described herein can be retained as a double-stranded polynucleotide. Alternatively, the two strands of a double-stranded polynucleotide may be separated to provide a single-stranded polynucleotide having a predefined sequence. Conditions that allow separation of the two strands of a double-stranded polynucleotide (melting) are well known in the art (e.g., Sambrook et al, 2001, Molecular Cloning: a Laboratory manual,3rd edition, Cold Spring harbor Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley-lntervention, New York (1995)).

Double-stranded polynucleotides having a predefined sequence synthesized according to the methods described herein can be amplified post-synthesis. Any region of the double-stranded polynucleotide can be amplified. All or any region of the double stranded polynucleotide may be amplified together with all or any region of the scaffold polynucleotide. Conditions that allow amplification of double-stranded polynucleotides are well known in the art (e.g., Sambrook et al, 2001, Molecular Cloning: a Laboratory manual,3rd edition, Cold Spring harbor Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley-lnterscience, New York (1995)). Thus, any of the synthetic methods described herein may further comprise an amplification step wherein a double stranded polynucleotide having a predefined sequence or any region thereof is amplified as described above. Amplification may be performed by any suitable method, such as Polymerase Chain Reaction (PCR), polymerase helix reaction (PSR), loop-mediated isothermal amplification (LAMP), Nucleic Acid Sequence Based Amplification (NASBA), autonomous sequence replication (3SR), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA), Ligase Chain Reaction (LCR), Helicase Dependent Amplification (HDA), reticular branched amplification method (RAM), and the like. Preferably, the amplification is performed by Polymerase Chain Reaction (PCR).

Double-stranded or single-stranded polynucleotides having a predefined sequence synthesized according to the methods described herein can be of any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 nucleotides or nucleotide pairs in length. For example, a polynucleotide may be about 10 to about 100 nucleotides or nucleotide pairs, about 10 to about 200 nucleotides or nucleotide pairs, about 10 to about 300 nucleotides or nucleotide pairs, about 10 to about 400 nucleotides or nucleotide pairs and about 10 to about 500 nucleotides or nucleotide pairs in length. The polynucleotide may be up to about 1000 or more nucleotides or nucleotide pairs, up to about 5000 or more nucleotides or nucleotide pairs in length or up to about 100000 or more nucleotides or nucleotide pairs in length.

RNA synthesis

The methods described for DNA synthesis may be applied to the synthesis of RNA. In one modification, the synthesis steps described for the synthesis method versions of inventions 1 and 2 and their variants may be modified. Thus, as described above, in each of synthesis method versions 1 and 2 and variants thereof, the support strand of the scaffold polynucleotide is a DNA strand. The primer strand portion of the synthetic strand of the scaffold polynucleotide is an RNA strand. The helper strand, if present, may be an RNA strand. The helper strand, if present, may be a DNA strand.

Nucleotides can be incorporated from ribonucleoside-5' -O-triphosphates (NTPs), which can be modified to include a reversible terminator group, as described above. Preferably, 3 '-O-modified ribonucleoside-5' -O-triphosphates are used. The modified nucleotides are incorporated by the action of RNA polymerase.

Thus, the description of the synthetic method versions of the invention 1-5 can be applied mutatis mutandis to RNA synthesis, but as described applies.

FIGS. 31 and 32 depict reaction schemes for RNA synthesis that are modifications of the DNA synthesis method versions 1 and 2 of the examples shown in FIGS. 6 and 7, respectively. The method versions of the invention 1 and 2 and their variants can be modified in the same way as shown in fig. 1 to 5, respectively.

The above descriptions of the support strand, primer strand, helper strand, polynucleotide linker molecule and universal nucleotides may be mutatis mutandis in any suitable RNA synthesis method, but may be modified as described above. The cleavage steps and cleavage positions as described above may be applied mutatis mutandis, since the support strand comprising the universal nucleotide is a DNA strand. In a preferred embodiment, SplintR DNA ligase is used for the ligation step.

Solid phase synthesis

Synthetic polynucleotides produced according to the synthetic methods of the invention can preferably be synthesized using solid phase or reversible solid phase techniques. Various such techniques are known in the art and may be used. The scaffold polynucleotide may be immobilized onto a surface, such as a surface of a flat surface, e.g., glass, gel-based material, or a microparticle, e.g., a bead or functionalized quantum dot, before synthesis of a new double stranded polynucleotide of a predetermined sequence is initiated. The material comprising the surface may itself be bonded to the substrate. For example, the scaffold polynucleotide may be immobilized on a gel-based material, such as polyacrylamide, and wherein the gel-based material is bound to a support substrate, such as glass.

The polynucleotide may be immobilized or tethered, directly or indirectly, to a surface. For example, they may be attached directly to the surface by chemical bonding. They may be tethered indirectly to a surface through an intermediate surface, such as the surface of a particle or bead, for example in SPRI or in an electrowetting system, as described below. The synthesis cycle can then be initiated and completed while the scaffold polynucleotide incorporating the newly synthesized polynucleotide is immobilized.

In such methods, the double stranded scaffold polynucleotide can be immobilized to a surface prior to pooling the first nucleotide of the predetermined sequence. Thus, such immobilized double stranded scaffold polynucleotides can act as anchors to attach double stranded polynucleotides of a predefined sequence to a surface during and after synthesis.

Only one strand of such a double stranded anchor/scaffold polynucleotide may be immobilized on the surface at the same end of the molecule. Alternatively, the two strands of the double stranded anchor/scaffold polynucleotide may each be immobilized on the surface at the same end of the molecule. A double stranded anchor/scaffold polynucleotide can be provided in which each strand is ligated at adjacent ends, e.g., by a hairpin loop at the end opposite the new synthesis start site, and the ligated ends can be immobilized on a surface (e.g., as schematically depicted in fig. 11).

In methods involving scaffold polynucleotides, the scaffold polynucleotides can be attached to a surface prior to incorporation of the first nucleotide in a predetermined sequence, as described herein. Thus, synthetic strands including a primer strand portion and a support strand portion hybridized therewith can be attached to the surface, respectively, as shown in FIGS. 11(a) and (c). The synthetic strand comprising the primer strand portion and the support strand portion hybridized thereto may be ligated at adjacent ends, for example, by hairpin loops, for example, at opposite ends of the new synthesis initiation site, and the ligated ends may be tethered to the surface, as shown in FIGS. 11(b) and (d). One or the other synthetic strand comprising the primer strand portion and the support strand portion hybridized therewith may be attached to the surface as shown in FIGS. 11(e) to (h), respectively. Preferably, the synthetic strand comprising the primer strand portion and the support strand portion hybridized thereto is attached to a surface.

Solid phase synthesis on flat surfaces

Prior to starting the synthesis of a new double stranded polynucleotide of a predefined sequence, the synthetic anchor/scaffold polynucleotide can be synthesized by methods known in the art, including those described herein, and tethered to a surface.

Preformed polynucleotides can be tethered to a surface by methods commonly used to create nucleic acid microarrays attached to planar surfaces. For example, an anchor/scaffold polynucleotide can be produced and then spotted or printed onto a flat surface. The anchor/scaffold polynucleotide can be deposited on the surface using contact printing techniques. For example, a solid or hollow tip or needle may be dipped into a solution comprising a preformed scaffold polynucleotide and contacted with a flat surface. Alternatively, the oligonucleotides may be adsorbed onto the microstamp and then transferred to a flat surface by physical contact. Non-contact printing techniques include thermal or piezoelectric printing, in which sub-nanoliter-sized droplets comprising pre-formed scaffold polynucleotides can be ejected from a printing tip using methods similar to those used in inkjet and bubble jet printing.

Single-stranded oligonucleotides can be synthesized directly on a planar surface, for example using the so-called "on-chip" method for generating microarrays. Such single stranded oligonucleotides may then serve as attachment sites to immobilize the preformed anchor/scaffold polynucleotide.

On-chip techniques for generating single-stranded oligonucleotides include photolithography, which involves the use of UV light directed through a photolithographic mask to selectively activate protected nucleotides, allowing subsequent incorporation of new protected nucleotides. The cycle of UV-mediated deprotection and predetermined nucleotide coupling allows for the in situ generation of oligonucleotides with the desired sequence. As an alternative to using photolithographic masks, oligonucleotides having the desired sequence can be produced on a planar surface by sequentially depositing nucleobases using inkjet printing techniques and using cycles of coupling, oxidation and deprotection (for review, see Kosuri and Church, Nature Methods,2014,11, 499-507).

In any of the synthetic methods described herein, including methods involving reversible immobilization as described below, the surface may be made of any suitable material. Typically, the surface may comprise silicon, glass or a polymeric material. The surface may comprise a gel surface, for example a polyacrylamide surface, for example about 2% polyacrylamide, optionally derivatized with N- (5-bromoacetylpentyl) acrylamide (BRAPA), preferably a polyacrylamide surface coupled to a solid support such as glass.

Reversible fixation

Synthetic polynucleotides having a predefined sequence may be synthesized according to the present invention using binding surfaces and structures (e.g., microparticles and beads) that facilitate reversible immobilization. Solid Phase Reversible Immobilization (SPRI) methods or modified methods are known in the art and may be used (see, e.g., DeAngelis M.M. et al (1995) Solid-Phase Reversible Immobilization for the Isolation of PCR Products, Nucleic Acids Research, 23 (22): 4742) 4743).

The surface may be provided in the form of particles, such as paramagnetic beads. The paramagnetic beads may be concentrated under the influence of a magnetic field. For example, the paramagnetic surface may have chemical groups, such as carboxyl groups, which under appropriate attachment conditions will act as binding moieties for nucleic acids, as described in more detail below. Nucleic acids can be eluted from these surfaces under appropriate elution conditions. The surfaces of the particles and beads may be provided with UV sensitive polycarbonate. The nucleic acid may be bound to the activated surface in the presence of a suitable immobilization buffer.

The microparticles and beads can be allowed to move freely in the reaction solution and then reversibly immobilized, for example by holding the beads in microwells or wells etched into the surface. The beads may be positioned as part of an array, for example, by using a unique nucleic acid "barcode" attached to the beads or by using color coding.

Thus, an anchor/scaffold polynucleotide according to the invention can be synthesized and then reversibly immobilized onto such a binding surface before starting to synthesize a new double-stranded polynucleotide of a predefined sequence. Polynucleotides synthesized by the methods of the invention can be synthesized while reversibly immobilized on such binding surfaces.

Microfluidic technology and system

The surface may be part of an electrowetting on dielectric system (EWOD). The EWOD system provides a dielectric coated surface that facilitates microfluidic manipulation of very small liquid volumes in Droplet form (see, e.g., Chou, W-L. et al (2015) Recent Advances in Applications of Droplet Microfluidics, Micromachines,6: 1249-. Drop volumes can be programmably created, moved, partitioned, and combined on a chip by electrowetting techniques. Thus, electrowetting systems provide an alternative way to reversibly immobilize polynucleotides during and after synthesis.

Polynucleotides having a predetermined sequence can be synthesized in a solid phase by the methods described herein, wherein the polynucleotides are immobilized on an EWOD surface, and the steps required in each cycle are facilitated by electrowetting techniques. For example, in a method involving scaffold polynucleotides and requiring pooling, cleavage, ligation and deprotection steps, the reagents required for each step, as well as any required washing steps to remove used and unwanted reagents, may be provided in the form of droplets that are transported under the influence of an electric field by electrowetting techniques.

Other microfluidic platforms are available that can be used in the synthesis methods of the invention. For example, emulsion-based microdroplet techniques commonly used for nucleic acid manipulation may be used. In such systems, droplets are formed in an emulsion created by mixing two immiscible fluids (typically water and oil). Emulsion droplets can be programmably created, moved, segmented, and combined in a microfluidic network. Hydrogel systems may also be provided. In any of the synthesis methods described herein, the droplets may be operated in any suitable compatible system, such as the EWOD system described above and other microfluidic systems, such as microfluidic systems including structures based on components comprising elastomeric materials.

The droplets can be of any suitable size, provided that they are compatible with the synthetic methods herein. The droplet size will vary depending on the particular system employed and the architecture associated with the system. The size can be adjusted appropriately. In any of the synthesis methods described herein, the droplet diameter can be in the range of about 150nm to about 5 mm. Droplet diameters below 1 μm may be verified by methods known in the art, for example by techniques involving capillary spray methods, e.g. Et al (Nature Physics,2007,3, pp 737-742)

Sequencing of the intermediate or final synthesis product.

Synthetic or assembled intermediates or final polynucleotide synthesis products can be sequenced for quality control checks to determine whether the desired polynucleotide or polynucleotides have been correctly synthesized or assembled. One or more polynucleotides of interest can be removed from the solid phase synthesis platform and sequenced by any of a variety of known commercially available sequencing techniques, for example using the MinION sold by Oxford nanopore technologies, Inc^TMNanopore sequencing by the device. Sequencing can be performed on the solid phase platform itself, eliminating the need to transfer the polynucleotide to a separate synthesis apparatus. Sequencing may conveniently be performed on the same electrowetting device, e.g. an EWOD device for synthesis, whereby the synthesis device comprises one or more pairs of measurement electrodes.A droplet comprising the polynucleotide of interest can be contacted with one of the electrodes of the electrode pair, the droplet forming a droplet interface bilayer, wherein the second droplet is contacted with the second electrode of the electrode pair, wherein the droplet bilayer interface comprises a nanopore in an amphiphilic membrane. For example, a polynucleotide may be translocated into a nanopore under the control of an enzyme, and the ionic current through the nanopore may be measured at a potential difference between a pair of electrodes as the polynucleotide passes through the nanopore. Ion current measurements over time can be recorded and used to determine polynucleotide sequence. Prior to sequencing, the polynucleotides may be subjected to one or more sample preparation steps to optimise them for sequencing, for example patent application No. pct/GB 2015/050140. Examples of enzymes, amphiphilic membranes and nanopores that may be suitably used are disclosed in patent applications nos. PCT/GB2013/052767 and PCT/GB 2014/052736. Reagents required for preparing samples of polynucleotides, nanopores, amphiphilic membranes, etc. may be provided to the EWOD device through the sample inlet. The sample inlet may be connected to the reagent chamber.

Surface attachment chemistry

Although oligonucleotides are usually chemically linked, they can also be attached to a surface by indirect means, e.g., by affinity interaction. For example, oligonucleotides can be functionalized with biotin and bound to a surface coated with avidin or streptavidin.

To immobilize polynucleotides to surfaces (e.g., flat surfaces), microparticles, beads, and the like, various surface attachment methods and chemicals can be used. The surface may be functionalized or derivatized to facilitate attachment. Such functionalization is known in the art. For example, the surface may be functionalized with: a polyhistidine tag (hexahistidine tag, 6 XHis-tag, His6 tag or) Ni-NTA, streptavidin, biotin, oligonucleotides, polynucleotides (such as DNA, RNA, PNA, GNA, TNA or LNA), carboxyl groups, quaternary amine groups, thiol groups, azide groups, alkyne groups, DIBO, lipids, FLAG-tags (F)LAG octapeptide), polynucleotide binding protein, peptide, protein, antibody, or antibody fragment. The surface may be functionalized with molecules or groups that specifically bind to the anchor/scaffold polynucleotide.

Some examples of chemicals suitable for attaching polynucleotides to a surface are shown in fig. 11i and 11 j.

In any of the methods described herein, the scaffold polynucleotides comprising synthetic strands comprising a portion of the primer strand and a portion of the support strand hybridized thereto may be tethered to a common surface by one or more covalent bonds. One or more covalent bonds may be formed between functional groups on the common surface and functional groups on the scaffold molecules. The functional groups on the scaffold molecules may be, for example, amine groups, thiol groups, phosphorothioate groups, or thioamide groups. The functional group on the common surface may be a bromoacetyl group, optionally wherein the bromoacetyl group is provided on a polyacrylamide surface derivatized with N- (5-bromoacetylpentyl) acrylamide (BRAPA).

In any of the methods of the invention, the scaffold polynucleotide may be attached to the surface directly or indirectly through a linker. Any suitable linker of which the properties are biocompatible and hydrophilic may be used.

The linker may be a linear linker or a branched linker.

The linker may comprise a hydrocarbon chain. The hydrocarbon chain can include 2 to about 2000 or more carbon atoms. The hydrocarbon chain may include alkylene groups, such as C2 to about 2000 or more alkylene groups. The hydrocarbon chain may have the general formula- (CH)₂)_n-, where n is 2 to about 2000 or higher. The hydrocarbon chain may optionally be substituted with one or more ester groups (i.e., -C (O) -O-) or one or more amide groups (i.e., -C (O) -N (H) -).

Any linker selected from the group comprising: PEG, polyacrylamide, poly (2-hydroxyethyl methacrylate), poly-2-methyl-2-oxazoline (PMOXA), zwitterionic polymers such as poly (carboxybetaine methacrylate) (pcbmma), poly [ N- (3-sulfopropyl) -N-methacryloyloxyethyl-N, N-dimethylammonium betaine ] (PSBMA), sugar polymers, and polypeptides.

The linker may comprise polyethylene glycol (PEG) having the general formula:

-(CH ₂-CH ₂-O) n-, wherein n is 1 to about 600 or more.

The linker may comprise a linker having the general formula- [ (CH)₂-CH₂-O)_n-PO₂ ^--O]_m-wherein n is 1 to about 600 or more and m may be 1-200 or more.

Any of the above linkers can be attached to a scaffold molecule as described herein at one end of the linker and attached to a first functional group at the other end of the linker, wherein the first functional group can provide covalent attachment to a surface. The first functional group can be, for example, an amine group, a thiol group, a phosphorothioate group, or a thioamide group, as further described herein. The surface may be functionalized with additional functional groups to provide covalent bonds with the first functional group. The additional functional group can be, for example, a 2-bromoacetamido group as further described herein. Optionally, bromoacetyl groups are provided on the surface of polyacrylamide derivatized with N- (5-bromoacetamidopentyl) acrylamide (BRAPA). Further functional groups on the surface may be bromoacetyl groups, optionally wherein the bromoacetyl groups are provided on a polyacrylamide surface derivatized with N- (5-bromoacetylpentyl) acrylamide (BRAPA), and where appropriate the first functional group may be, for example, an amine group, a thiol group, a phosphorothioate group, or a thioamide group. The surface to which the polynucleotides are attached may comprise a gel. The surface comprises a polyacrylamide surface, for example about 2% polyacrylamide, preferably the polyacrylamide surface is coupled to a solid support such as glass.

In any of the methods of the invention, the scaffold polynucleotide may optionally be linked to the linker by incorporating branched nucleotides in the scaffold polynucleotide. Any suitable branched nucleotide may be used with any suitable compatible linker.

Prior to beginning the synthesis cycle of the invention, a scaffold polynucleotide may be synthesized in which one or more branched nucleotides are incorporated into the scaffold polynucleotide. The exact position at which one or more branched nucleotides are incorporated into the scaffold polynucleotide and thus to which a linker may be attached may vary and may be selected as desired. The position may be, for example, at the end of the support strand and/or the synthesis strand or, for example, in a loop region connecting the support strand to the synthesis strand in embodiments comprising hairpin loops.

During synthesis of the scaffold polynucleotide, one or more branched nucleotides may be incorporated into the scaffold polynucleotide, wherein the blocking group blocks the reactive group of the branched moiety. The blocking group may then be removed (deblocked) prior to coupling to the branched portion of the linker, or the first unit (molecule) of the linker if the linker comprises multiple units.

During synthesis of the scaffold polynucleotide, one or more branched nucleotides may be incorporated into the scaffold polynucleotide having groups suitable for subsequent "click chemistry" reactions to couple to the branching portion of the linker, or to the first unit if the linker comprises multiple units. An example of such a group is ethynyl.

Some non-limiting exemplary branched nucleotides are shown below.

The linker may optionally include one or more spacer molecules (units), such as the SP9 spacer, wherein the first spacer unit is attached to a branched nucleotide.

The linker may comprise one or more further spacer groups attached to the first spacer group. For example, the linker may comprise a plurality of, for example, Sp9 spacer groups. A first spacer group is attached to the branched portion and then one or more additional spacer groups are added in sequence to extend a spacer chain comprising a plurality of spacer units attached to phosphate groups therebetween.

Shown below are some non-limiting examples of spacer units (Sp3, Sp9, and Sp13), which may include a first spacer unit attached to a branched nucleotide, or an additional spacer unit attached to an existing spacer unit already attached to a branched nucleotide.

The linker may comprise one or more ethylene glycol units.

The linker may comprise an oligonucleotide, wherein the plurality of units are nucleotides.

In the structures described above, the term 5 "is used to distinguish the 5 'end of the nucleotide linked to the branching moiety, where 5' has the ordinary meaning in the art. 5 "means a position on the nucleotide where the linker can be extended. The position of 5 "may vary. The 5 "position is typically a position in the nucleobase of a nucleotide. The 5 "position in the nucleobase can vary depending on the nature of the desired branching moiety, as shown in the above structure.

Microarray

Any of the polynucleotide synthesis methods described herein can be used to make polynucleotide microarrays (Trevino, v. et al, mol.med.200713, pp 527-541). Thus, the anchor or scaffold polynucleotide may be attached to a plurality of individually addressable reaction sites on the surface, and polynucleotides having a predefined sequence may be synthesized in situ on the microarray.

After synthesis, a polynucleotide of a predetermined sequence can be provided with a unique sequence in each reaction region. The anchor or scaffold polynucleotide may be provided with barcode sequences to facilitate identification.

In addition to methods of synthesizing polynucleotides of predefined sequences, microarray fabrication can be performed using techniques commonly used in the art, including the techniques described herein. For example, anchor or scaffold polynucleotides can be tethered to a surface using known surface attachment methods and chemistry, including those described herein.

After synthesis of the polynucleotide of the predetermined sequence, a final cleavage step may be provided to remove any unwanted polynucleotide sequences from the unbound ends.

The polynucleotide of the predefined sequence may be provided in a double stranded form at the reaction site. Alternatively, after synthesis, the double stranded polynucleotides may be separated and one strand removed, leaving the single stranded polynucleotide at the reaction site. Selective tethering of the strands may be provided to facilitate the process. For example, in methods involving scaffold polynucleotides, the synthetic strands may be tethered to a surface and the support strands may not be tethered, or vice versa. The synthetic strand may have a non-cleavable linker and the support strand may have a cleavable linker, or vice versa. The isolation of the chains can be carried out by conventional methods, such as heat treatment.

Assembly of synthetic polynucleotides

A polynucleotide having a predefined sequence synthesized by the methods described herein, and optionally amplified by the methods described herein, can be ligated to one or more other such polynucleotides to produce a larger synthetic polynucleotide.

Ligation of multiple polynucleotides can be achieved by techniques well known in the art. A first polynucleotide and one or more additional polynucleotides synthesized by the methods described herein can be cleaved to produce compatible ends, and the polynucleotides then ligated together by ligation. Cleavage may be achieved by any suitable method. In general, restriction enzyme cleavage sites may be created in the polynucleotide, and a cleavage step then performed using the restriction enzyme, thereby releasing the synthesized polynucleotide from any anchor/scaffold polynucleotide. The cleavage site may be designed as part of the anchor/scaffold polynucleotide. Alternatively, the cleavage site may be generated within the newly synthesized polynucleotide as part of a predetermined nucleotide sequence.

The assembly of the polynucleotide is preferably performed using a solid phase method. For example, after synthesis, the first polynucleotide may be subjected to a single cleavage at a suitable position away from the surface fixation site. Thus, the first polynucleotide will remain immobilized on the surface and a single cleavage will produce an end that is compatible for ligation with another polynucleotide. The additional polynucleotide may be cleaved at two suitable positions to create compatible ends at each end for ligation of other polynucleotides, while releasing the additional polynucleotide from the surface immobilization. The additional polynucleotide may be compatibly ligated with the first polynucleotide, resulting in a larger immobilized polynucleotide having a predefined sequence and having ends that are compatibly ligated with another additional polynucleotide. Thus, iterative cycles of ligation of preselected cleaved synthetic polynucleotides can produce longer synthetic polynucleotide molecules. The order of ligation of the additional polynucleotides will be determined by the desired predefined sequence.

Thus, the assembly method of the present invention may allow for the generation of synthetic polynucleotide molecules of around one or more Mb in length.

The assembly and/or synthesis methods of the present invention can be performed using equipment known in the art. Available techniques and devices allow very small volumes of reagents to be selectively moved, dispensed and combined with other volumes in different locations of the array, typically in the form of droplets, and electrowetting techniques, such as electrowetting on dielectric (EWOD), can be used, as described above. Suitable electrowetting techniques and systems capable of manipulating liquid droplets that can be used in the present invention are disclosed, for example, in US8653832, US8828336, US20140197028 and US 20140202863.

Cleavage from the solid phase may be achieved by providing a cleavable linker in one or both of the primer strand portion and the support strand portion to which it hybridizes. The cleavable linker may be, for example, a UV cleavable linker.

An example of a cleavage process involving enzymatic cleavage is shown in fig. 29. The schematic shows scaffold polynucleotides attached to a surface (shown by black diamond structures) and comprising polynucleotides of predefined sequences. The scaffold polynucleotide includes top and bottom hairpins. In each case, the top hairpin can be cleaved using a cleavage step of a universal nucleotide to define a cleavage site. The bottom hairpin can be removed by restriction endonucleases via sites designed into the scaffold polynucleotide or engineered into a newly synthesized polynucleotide of predefined sequence.

Thus, as described above, a polynucleotide having a predefined sequence can be synthesized while immobilized on an electrowetting surface. The synthesized polynucleotide can be cleaved from the electrowetting surface and moved in the form of droplets under the influence of an electric field. The droplets may be combined at specific reaction sites on the surface where they can deliver the cleaved synthetic polynucleotides for ligation with other cleaved synthetic polynucleotides. The polynucleotides may then be ligated (e.g., by ligation). Using these techniques, populations of different polynucleotides can be synthesized and linked in sequence according to the desired predefined sequence. Using this system, a fully automated polynucleotide synthesis and assembly system can be designed. The system can be programmed to receive a desired sequence, supply reagents, perform a synthesis cycle, and then assemble a desired polynucleotide according to a desired predefined sequence.

System and kit

The present invention also provides a polynucleotide synthesis system for carrying out any of the synthesis methods described and defined herein and any subsequent amplification and assembly steps described and defined herein.

Typically, the synthesis cycle reaction will be carried out by incorporating a predetermined sequence of nucleotides into a scaffold polynucleotide molecule that is tethered to the surface in the manner described and defined herein. The surface may be any suitable surface as described and defined herein.

In one embodiment, the reaction of combining the nucleotides of the predetermined sequence into the scaffold polynucleotide molecule involves performing any synthetic method on the scaffold polynucleotide within the reaction region.

The reaction region is any region of a suitable substrate to which the scaffold polynucleotide molecules are attached and in which reagents for performing the synthetic process can be delivered.

In one embodiment, the reaction region may be a single region of the surface comprising a single scaffold polynucleotide molecule, wherein the single scaffold polynucleotide molecule may be addressed with the agent.

In another embodiment, the reaction region may be a single region of the surface comprising a plurality of scaffold polynucleotide molecules, wherein the scaffold polynucleotide molecules cannot be individually addressed with reagents that are isolated from each other. Thus, in such embodiments, a plurality of scaffold polynucleotide molecules in a reaction region are exposed to the same reagents and conditions, and thus synthetic polynucleotide molecules having the same or substantially the same nucleotide sequence may be produced.

In one embodiment, a synthesis system for performing any of the synthesis methods described and defined herein may comprise a plurality of reaction regions, wherein each reaction region comprises one or more attached scaffold polynucleotide molecules, and wherein each reaction region may be individually addressed with a reagent in isolation from each other reaction region. Such systems may be configured, for example, in the form of an array, for example, wherein the reaction regions are formed on a substrate, typically a planar substrate.

Systems having a substrate comprising a single reaction region or comprising multiple reaction regions may be included, for example, within an EWOD system or a microfluidic system, and the system is configured to deliver reagents to the reaction sites. EWOD and microfluidic systems are described in more detail herein. For example, the EWOD system can be configured to deliver reagents to a reaction site under electrical control. Microfluidic systems, e.g., including microfabricated structures, e.g., formed of elastomers or similar materials, may be configured to deliver reagents to reaction sites under fluid pressure and/or suction control or by mechanical means. The agent may be delivered by any suitable means, for example by carbon nanotubes which act as a delivery conduit for the agent. Any suitable system may be envisaged.

EWOD, microfluidic, and other systems may be configured to deliver any other desired reagents to the reaction site, such as enzymes for cleaving the synthesized double-stranded polynucleotide from the scaffold polynucleotide after synthesis, and/or reagents for cleaving the linker to release the entire scaffold polynucleotide from the substrate and/or reagents for amplifying the polynucleotide molecule after synthesis or any region or portion thereof, and/or reagents for assembling larger polynucleotide molecules from smaller polynucleotide molecules synthesized according to the synthesis methods of the invention.

The invention also provides kits for carrying out any of the synthetic methods described and defined herein. The kit may contain any desired combination of reagents for performing any of the synthesis and/or assembly methods of the invention described and defined herein. For example, a kit may comprise any one or more volumes of reaction reagents comprising a scaffold polynucleotide, a volume of reaction reagents corresponding to any one or more steps of a synthesis cycle as described and defined herein, a volume of reaction reagents comprising nucleotides comprising a reversible blocking group or a reversible terminator group, a volume of reaction reagents for amplifying one or more polynucleotide molecules or any region or portion thereof after synthesis, a volume of reaction reagents for assembling larger polynucleotide molecules from smaller polynucleotide molecules synthesized according to the synthesis methods of the invention, a volume of reaction reagents for cleaving a synthesized double stranded polynucleotide from a scaffold polynucleotide after synthesis, and a volume of reaction reagents for cleaving one or more linkers to release an intact scaffold polynucleotide from a substrate.

Exemplary method

Described herein are exemplary, non-limiting methods of synthesizing polynucleotide or oligonucleotide molecules according to the invention, including the appended claims.

In the following two exemplary methods of synthesizing polynucleotide or oligonucleotide molecules according to the invention and variants thereof, reference to synthesis method versions 1 and 2 will be explained according to the reaction schemes set out in fig. 1 to 5, respectively, rather than according to the descriptions in the reaction schemes or example sections set out in any of fig. 6 to 10. In the examples section below, the reaction schemes listed in any of figures 6 to 10 and the description thereof provide illustrative support for the process of the invention based on a modified reaction scheme compared to the process of the invention.

In each of the exemplary methods described below, the structures described in each step may be referenced by reference numerals to a particular figure as appropriate. Accordingly, reference numerals in the following text correspond to those in fig. 1 to 5. However, such references are not intended to be limited to the structures shown in the drawings, and the description of the relevant structures corresponds to all the descriptions as provided herein, including but not limited to those of the specific illustrations.

Two non-limiting exemplary methods of the invention, referred to herein as synthetic method versions of invention 1 and 2 (see, e.g., fig. 1 and 2, respectively), are described below. In step (1) of each of these exemplary methods, a scaffold polynucleotide (see the structure shown in step 1 of fig. 1 and 2) is provided (101, 201) comprising a synthetic strand (see the strand labeled "b" in the structure depicted in step 1 of each of fig. 1 and 2) hybridized to a complementary support strand (see the strand labeled "a" in the structure depicted in step 1 of each of fig. 1 and 2).

The scaffold polynucleotide is double stranded and provides a support structure to modify the region of the synthetic polynucleotide as it is synthesized de novo. The scaffold polynucleotide comprises a synthetic strand comprising a primer strand portion (see the dotted portion of the strand labeled "b" in the structure depicted in step 1 of each of fig. 1 and 2) and an auxiliary strand portion (see the dashed portion of the strand labeled "b" in the structure depicted in step 1 of each of fig. 1 and 2). The primer strand portion and the auxiliary strand portion of the synthetic strand are hybridized with the complementary support strand.

The ends of the scaffold polynucleotide comprising the primer strand portions comprise blunt ends, i.e., no overhanging nucleotides in either strand.

In step (2) of the method, a ligation step (102, 202) is performed in which the polynucleotide linker molecule is ligated to the double stranded scaffold polynucleotide. The polynucleotide linker molecule comprises a first nucleotide of a predetermined nucleotide sequence. The polynucleotide linker molecule comprises a support strand and an auxiliary strand hybridized to the support strand. The polynucleotide linker molecule comprises blunt-ended complementary linkers, i.e., no overhanging nucleotides in either strand. The blunt end complementary ligation ends are complementary to the blunt ends of the double stranded scaffold polynucleotide. The support strand of the polynucleotide linker molecule comprises a first nucleotide of a predetermined nucleotide sequence at the complementary linker end. The first nucleotide of the predefined nucleotide sequence is the terminal nucleotide of the support strand of the polynucleotide linker molecule at the complementary linker end. The first nucleotide of the predetermined nucleotide sequence is a ligatable nucleotide and is ligated to the terminal nucleotide of the support strand of the scaffold polynucleotide. Following ligation, the first nucleotide of the predetermined nucleotide sequence is incorporated into the double-stranded scaffold polynucleotide by ligation to the support strand of the double-stranded scaffold polynucleotide at the blunt end of the double-stranded scaffold polynucleotide.

In each of these two method versions and variants thereof, the support strand of the polynucleotide linker molecule also comprises a universal nucleotide (labeled "Un" in the structures shown in fig. 1 and 2) at the complementary linker end, which will facilitate cleavage during the cleavage step. The role of the universal nucleotide will be apparent from the detailed description of each method below.

The terminal nucleotide of the auxiliary strand of the polynucleotide linker molecule at the complementary linker end is provided such that the auxiliary strand cannot be ligated to the primer strand portion of the synthetic strand, i.e. it is provided as a non-ligatable nucleotide. This is usually achieved by providing the terminal nucleotide of the auxiliary strand without a phosphate group, i.e. it is provided in nucleoside form. Alternatively, a 5 '-protected nucleoside, a nucleoside having a non-linkable group at the 5' position, such as a 5 '-deoxynucleoside or a 5' -aminonucleoside, or any other suitable non-linkable nucleotide or nucleoside may be used.

Thus, when the support strand of the polynucleotide linker molecule is ligated to the support strand of the double stranded scaffold polynucleotide, a single stranded break or "nick" is provided in the synthetic strand between the primer strand portion and the auxiliary strand of the synthetic strand.

Upon ligation of the polynucleotide linker molecule to the double stranded scaffold polynucleotide, a double stranded scaffold polynucleotide is formed comprising the newly incorporated first nucleotide, the universal nucleotide and the "nick" for facilitating cleavage in the cleavage step.

Since the first nucleotide of the predetermined sequence of the cycle is ligated to the terminal nucleotide of the support strand of the double-stranded scaffold polynucleotide, the terminal nucleotide of the support strand of the double-stranded scaffold polynucleotide must be provided with an attached phosphate group or other ligatable group prior to the ligation step in order for the terminal nucleotide of the support strand of the double-stranded scaffold polynucleotide to serve as a substrate for the ligase.

As described in more detail herein, in certain exemplary methods relating to versions 1 and 2 and variants thereof described herein, the auxiliary strand may be removed prior to the step of incorporating the second nucleotide of the predetermined sequence into the synthesis cycle, for example by denaturation and release from the support strand to which it was previously hybridized.

In the context of the first cycle of synthesis, the term "first nucleotide of the predetermined sequence" is not necessarily understood to mean the first nucleotide of the predetermined sequence. The methods described herein involve synthesis of a double-stranded polynucleotide having a predetermined sequence, and may be presynthesized in the scaffold polynucleotide to provide a portion of the predetermined sequence prior to the initiation of a first synthesis cycle. In this context, the term "a" first nucleotide of the predefined sequence may mean "any" nucleotide of the predefined sequence.

The end of the primer strand portion of the synthetic strand provides a primer site that is a site for attaching a second nucleotide of the predetermined sequence that is attached/incorporated into the synthetic strand by an enzyme that has the ability to extend an oligonucleotide or a polynucleotide molecule having a single nucleotide. Such enzymes are typically nucleotidyl transferases or polymerases. Any suitable enzyme may be used as further defined herein and/or known to the skilled person. Thus, the enzyme will act to extend the terminal nucleotide of the primer strand portion. Thus, this terminal nucleotide will generally define the 3' end of the primer strand portion, e.g., allowing extension by a polymerase or transferase that catalyzes extension in the 5' to 3' direction. The opposite end of the synthetic strand, including the primer strand portion, will thus generally define the 5' end of the synthetic strand, and the terminal nucleotide of the support strand adjacent to the 5' end of the synthetic strand will thus generally define the 3' end of the support strand.

The terminal nucleotide of the auxiliary strand portion of the synthetic strand at the site of single strand break will generally define the 5 'end of the auxiliary strand portion, and thus the opposite end of the auxiliary strand portion of the synthetic strand will generally define the 3' end of the synthetic strand.

In the step of merging the second nucleotides (step 3; 103, 203), the second nucleotides are provided with a reversible terminator group (depicted as small triangles of merged nucleotides in step 3 of each of fig. 1 and 2), which prevents further extension by the enzyme. Therefore, only single nucleotides are pooled in step (3).

Nucleotides comprising any suitable reversible terminator group may be used. Preferred nucleotides having a reversible terminator group are 3 '-O-allyl-dNTPs and/or 3' -O-azidomethyl-dNTPs or other groups further described herein.

It will be apparent from the description of the various methods defined herein that the term "second nucleotide of the predetermined sequence" is not to be understood as referring to the next nucleotide from the first nucleotide in the linear sequence in one strand comprising the predetermined sequence, but only to "a" further nucleotide of the predetermined sequence in the context of the entire synthetic double stranded polynucleotide. In the case of the specific and non-limiting method versions 1 and 2 of the invention and certain variants thereof defined herein, each "first nucleotide" in one cycle will be sequentially linked in the same order to the "first nucleotide" nucleic acid strand of the previous cycle, such that each cycle extends the first strand sequentially by one nucleotide. In each cycle, a "second nucleotide" will pair with a "first nucleotide", and each "second nucleotide" in one cycle will be introduced sequentially in the same nucleic acid strand adjacent to the "second nucleotide" of the previous cycle, such that the extended second strand is arranged one nucleotide in each cycle sequence. Thus, when a synthesis cycle is complete, the synthesized double-stranded polynucleotide molecule will comprise a predetermined sequence of one strand defined by the linked first nucleotides of each cycle, and a predetermined sequence of the opposite strand defined by the bound second nucleotides of each cycle. The sequence of both strands must be predefined and determined by the identity of the first and second nucleotides selected by the user during each synthesis cycle. In method versions 1 and 2 described herein, assuming that the first and second nucleotides of each cycle form a nucleotide pair, the final synthesized strand will be fully complementary if the user selects that the second nucleotide of each cycle is naturally complementary to the first nucleotide of each cycle. If the user selects that certain cycles of the second nucleotide are not complementary to those cycles of the respective first nucleotides, the final synthesized strand will not be fully complementary. In either case, however, the final synthesized strand comprises a sequence that is predetermined throughout the synthesized double-stranded polynucleotide.

After the step of combining the second nucleotides (step 3), the scaffold polynucleotide is then cleaved (steps 4, 104, 204). Cleavage causes the polynucleotide linker molecule to be released from the scaffold polynucleotide and the first nucleotide of the cycle remains on the support strand of the cleaved scaffold polynucleotide and pairs with the second nucleotide of the cycle that links the cleaved scaffold polynucleotide to the synthetic strand of the scaffold. Cleavage will cause the auxiliary strand, if present, to be released and hybridise to the support strand immediately prior to cleavage, releasing the support strand comprising the universal nucleotide. Cleavage thus leaves in situ the cleaved double stranded scaffold polynucleotide comprising at the cleavage site the cleaved end of the cleaved support strand and the end of the primer strand portion of the synthetic strand comprising the pre-cleavage nick site, wherein the cleaved double stranded scaffold polynucleotide comprises the first nucleotide of the cycle as the terminal nucleotide of the cleaved end of the support strand, paired with the second nucleotide of the cycle as the terminal nucleotide of the primer strand portion of the synthetic strand.

In step (5) of the method, a deprotection step is performed to remove the reversible terminator from the combined second nucleotide of the predetermined nucleotide sequence (105, 205). The deprotection step may optionally be performed before the cleavage step (step 4), in which case the deprotection step is defined as step (4) (steps 4; 104, 204 of fig. 1 and 2) and the cleavage step is defined as step (5) (steps 5; 105, 205 of fig. 1 and 2).

An iterative loop of synthesis including the same steps described above is performed to produce a synthesized polynucleotide.

Specific methods are described in more detail below.

Synthesis method version 1

Referring to FIG. 1, in a first specific non-limiting exemplary form of the synthetic method of the invention, a double stranded scaffold polynucleotide is provided (step 1; 101 of FIG. 1). The double stranded scaffold polynucleotide comprises a support strand and a synthetic strand hybridized thereto. The synthetic strand comprises a primer strand portion. The double stranded scaffold polynucleotide has at least one blunt end, wherein the at least one blunt end comprises an end of a primer strand portion and an end of a support strand hybridized thereto. The terminal nucleotide of the support strand is capable of serving as a substrate for a ligase and preferably comprises a phosphate group or other linkable group.

In step (2) of the method, a polynucleotide linker molecule (see the structure shown in the upper right of FIG. 1) is attached to the scaffold polynucleotide. Ligation incorporates the first nucleotide of the predetermined sequence into the support strand of the scaffold polynucleotide (step 2; 102 of FIG. 1).

In step (3) of the method, a second nucleotide of the predetermined nucleotide sequence is added to the end of the primer strand portion of the synthesized strand under the action of an enzyme having the ability to extend the oligonucleotide or polynucleotide molecule using a single nucleotide. Such enzymes are typically nucleotidyl transferases or polymerases (step 3; 103 of FIG. 1). The second nucleotide has a reversible terminator group, which prevents further extension by the enzyme. Therefore, only single nucleotides are pooled in step (3).

The scaffold polynucleotide is then cleaved at each synthesis cycle at a cleavage site defined by the sequence comprising the universal nucleotide in the support strand (step 4; 104). In method version 1, the cleaving includes cleaving the support strand in a direction close to the primer strand portion/away from the auxiliary strand immediately after the universal nucleotide, that is, cleaving the support strand between a position occupied by the universal nucleotide and a next nucleotide position in the support strand in the direction close to the primer strand portion/away from the auxiliary strand. Cleavage of the scaffold polynucleotide (step 4) results in release of the polynucleotide linker molecule from the scaffold polynucleotide and retention of the first nucleotide of the cycle on the first strand of the cleaved scaffold polynucleotide and pairing with the second nucleotide of the cycle, the oligonucleotide being ligated to the primer strand portion of the synthetic strand. Cleavage of the scaffold polynucleotide (step 4) results in loss of the helper strand of the scaffold polynucleotide (if present and hybridized to the support strand immediately prior to cleavage), and loss of the universal nucleotide from the scaffold polynucleotide. Cleavage leaves in situ the cleaved double stranded scaffold polynucleotide comprising at the cleavage site the cleaved end of the cleaved support strand and the end of the primer strand portion of the synthesized strand comprising the pre-cleavage nick site. Cleavage results in a double-stranded scaffold polynucleotide that is cleaved at the blunt end of the cleavage site, with no overhang in either strand, and the first and second nucleotides are nucleotide pairs.

In step (5) of the method, a deprotection step is performed to remove the terminator group from the newly incorporated nucleotides. The deprotection step may alternatively be performed before the cleavage step, in which case the deprotection step is defined as step (4) and the cleavage step as step (5), as shown in fig. 1 (104 and 105, respectively).

Step 1-providing a scaffold polynucleotide

In exemplary version 1 of the synthesis method of the invention, a double stranded scaffold polynucleotide is provided in step (1) (101). Double-stranded scaffold polynucleotides are provided that comprise a synthetic strand and a support strand hybridized thereto, wherein the synthetic strand comprises a primer strand portion. The terminal nucleotide of the support strand is paired with the terminal nucleotide of the primer strand portion, thereby forming a blunt end. The terminal nucleotide of the support strand is capable of acting as a substrate for a ligase and comprises a ligatable group, preferably a phosphate group.

Step 2-ligating the polynucleotide linker molecule to the scaffold polynucleotide and incorporating the first nucleotide of the predetermined sequence

In step (2) of the method, a double stranded polynucleotide linker molecule is ligated (102) to the scaffold polynucleotide by the action of a ligase in a blunt end ligation reaction.

The polynucleotide linker molecule comprises a support strand and an auxiliary strand hybridized thereto. The polynucleotide linker molecule further comprises a complementary linker comprising the universal nucleotide and the first nucleotide of the predetermined sequence in the support strand.

The complementary linking ends of the polynucleotide linker molecules are configured such that the terminal nucleotide of the support strand is the first nucleotide of the predetermined sequence that is incorporated into the scaffold polynucleotide in any given synthesis cycle. The terminal nucleotide of the support strand is paired with the terminal nucleotide of the helper strand. The terminal nucleotide of the support strand, i.e. the first nucleotide of the predetermined sequence of the loop, occupies nucleotide position n in the support strand. Position n refers to the nucleotide position relative to the second nucleotide of the predetermined sequence of the cycle after combining the second nucleotides, whereby the first and second nucleotides form a nucleotide pair. In FIG. 1, the first nucleotide of the predetermined sequence is depicted as adenosine, the second nucleotide of the predetermined sequence in step (3) is depicted as thymine, and the terminal nucleotide of the auxiliary strand is depicted as thymine. Adenosine and thymine are for illustration only. The first and second nucleotides and the terminal nucleotide of the helper strand may be any suitable nucleotide selected by the user. The nucleotide depicted as X can also be any suitable nucleotide selected by the user.

Typically, the terminal nucleotide of the support strand at the complementary linker will define the 3' end of the support strand of the complementary linker of the polynucleotide linker molecule.

The terminal nucleotide of the auxiliary strand of the polynucleotide linker molecule is configured such that it cannot be linked to another polynucleotide in the polynucleotide strand (the position purely labeled "T" is used to illustrate the structure at the top right of the figure) 1). This nucleotide is referred to as the non-ligatable terminal nucleotide. Typically, the terminal nucleotide will lack a phosphate group, i.e., it will be a nucleoside. Typically, the terminal nucleotide of this helper strand will define the 5' end of the helper strand, as described above.

In the complementary linking end of the polynucleotide linker molecule, the universal nucleotide in the support strand is positioned such that it is the penultimate nucleotide of the support strand, pairs with the penultimate nucleotide of the auxiliary strand, and occupies nucleotide position n +1 in the support strand. Position n +1 refers to the next nucleotide position in the support strand distal to the complementary linker relative to position n.

The complementary linking end is configured such that it will compatibly link with the blunt end of the scaffold polynucleotide when subjected to suitable linking conditions. After the support strand of the polynucleotide linker molecule is ligated to the scaffold polynucleotide, the first nucleotide is incorporated into the scaffold polynucleotide. Since the terminal nucleotide of the auxiliary strand of the polynucleotide linker molecule is a non-ligatable nucleotide, ligation of the ligase into the auxiliary strand and the primer strand portion of the strand will be prevented, thereby creating a single strand break or "gap" between the auxiliary strand and the primer strand portion of the synthetic strand.

Ligation of the polynucleotide linker molecule to the scaffold polynucleotide extends the length of the support strand of the double stranded scaffold polynucleotide of step (1), and wherein the first nucleotide of the predetermined nucleotide sequence is incorporated into the support strand of the scaffold polynucleotide.

The linking of the support chains may be performed in any suitable manner. Ligation may generally, and preferably is, performed by an enzyme having ligase activity. For example, ligation may be performed using T3DNA ligase or T4DNA ligase or functional variants or equivalents thereof or other enzymes described herein. The use of such an enzyme will cause single strand breaks to be maintained in the synthesized strand because the terminal nucleotide of the helper strand is provided such that it cannot serve as a substrate for the ligase, for example due to the absence of a terminal phosphate group or the presence of an unlinkable blocking group.

After ligation of the polynucleotide linker molecule to the scaffold polynucleotide, the first nucleotide of the predetermined nucleotide sequence is referred to as occupying nucleotide position n, the universal nucleotide is referred to as occupying nucleotide position n +1, and the terminal nucleotide of the support strand of the scaffold polynucleotide that was proximal to the primer strand portion prior to ligation is referred to as occupying nucleotide position n-1. Nucleotide position n-1 refers to the next nucleotide position in the support strand relative to position n in the direction of the proximal end of the primer strand portion/distal end of the auxiliary strand.

Step 3-merging second nucleotides of the predetermined sequence

In step (3) of the method, after the polynucleotide linker molecule is ligated to the scaffold polynucleotide, the second nucleotide of the predetermined sequence is then incorporated into the synthesized strand by extending the primer strand portion.

Extension of the primer strand portion can be achieved by the action of any suitable enzyme that has the ability to extend an oligonucleotide or polynucleotide molecule having a single nucleotide. Such enzymes are typically nucleotidyl transferases or polymerases. Any suitable enzyme may be used as further defined herein or known to those skilled in the art.

If the helper strand is present in the scaffold polynucleotide immediately prior to primer strand partial extension, particularly if a polymerase is used, the enzyme may act to "invade" the helper strand and displace the helper strand terminal nucleotides. The incorporated second nucleotide will then occupy the position previously occupied by the displaced terminal nucleotide of the helper strand (see figure 1 for the structure shown in the middle lower part). In certain embodiments, the helper strand may be removed prior to the extension/pooling step (3), in which case the enzyme may access and extend the terminal nucleotide of the primer strand portion of the synthesized strand without displacing the helper strand or a portion thereof.

The second nucleotide of the predetermined sequence incorporated in step (3) comprises a reversible terminator group that prevents further extension by the enzyme, or any other similar function that prevents further extension by the enzyme. Any suitable reversible terminator group or function as further defined herein or known to those skilled in the art may be used.

Upon incorporation into the primer strand portion of the scaffold polynucleotide, the second nucleotide of the predetermined sequence of the cycle is paired with the first nucleotide of the predetermined sequence of the cycle to form a nucleotide pair of the cycle. The nucleotide pair may be any suitable nucleotide pair as further defined herein.

Step 4-lysis

In step (4) of the method, the scaffold polynucleotide is cleaved at the cleavage site (104). The cleavage site is defined by a sequence comprising the universal nucleotide in the support strand. Cleavage causes a double strand break in the scaffold polynucleotide. Cleavage of the scaffold polynucleotide (step 4) causes loss of the helper strand (if present and hybridized to the support strand immediately prior to cleavage) and loss of the support strand comprising the universal nucleotide.

Cleavage of the scaffold polynucleotide thereby releases the polynucleotide linker molecule from the scaffold polynucleotide, but causes the first nucleotide of the cycle to remain on the first strand of the cleaved scaffold polynucleotide and pair with the second nucleotide of the cycle. Cleavage of the scaffold polynucleotide leaves in situ the cleaved blunt-ended double-stranded scaffold polynucleotide comprising a first nucleotide of a predetermined sequence at the end of the support strand and a second nucleotide of a predetermined sequence at the end of the primer strand portion of the synthetic strand, wherein the first and second nucleotides form a nucleotide pair.

In this exemplary method, the synthetic strand already has a single strand break or "nick", and thus only cleavage of the support strand is required to provide a double strand break in the scaffold polynucleotide. Furthermore, as previously described, in this exemplary method version, the cleavage results in a blunt-end cleaved double-stranded scaffold polynucleotide, with no overhang in either the synthetic strand or the support strand, and the universal nucleotide is located at position n +1 cleavage step in the support strand prior to the support strand. To obtain such blunt-end cleaved double-stranded scaffold polynucleotides when the universal nucleotide is located at the n +1 position in the support strand, the support strand is cleaved at a specific position relative to the universal nucleotide. When the support strand of the scaffold polynucleotide is cleaved between nucleotide positions n +1 and n, the polynucleotide linker molecule is released from the scaffold polynucleotide (see the structure shown in the upper left of figure 1), except that the cycle remains in the scaffold polynucleotide attached to the support strand of the cleaved scaffold polynucleotide.

The phosphate group should continue to be attached to the terminal nucleotide of the support strand of the cleaved scaffold polynucleotide at the cleavage site. This ensures that the support strand of the cleaved scaffold polynucleotide can be ligated to the support strand of the polynucleotide linker molecule in the ligation step of the next synthesis cycle. The cleavage is performed such that the terminal nucleotides of the support strands of the cleaved scaffold polynucleotide retain a ligatable group, preferably a terminal phosphate group, and thus no phosphorylation step is required.

Thus, in method version 1, the universal nucleotide is located at position n +1 in the support strand in steps (2), (3) and (4), and the support strand is cleaved between nucleotide positions n +1 and n in step (4).

Preferably, the support strand is cleaved by cleavage of the phosphodiester bond between nucleotide positions n +1 and n (the first phosphodiester bond of the support strand is in a direction away from the helper strand/towards the primer strand, relative to the position of the universal nucleotide).

The support strand may be cleaved by cleaving one of the ester bonds of the phosphodiester bond between nucleotide positions n +1 and n.

Preferably, the support strand is cleaved by cleavage of the first ester bond relative to nucleotide position n + 1. This will have the effect of retaining the terminal phosphate group on the support strand of the cleaved scaffold polynucleotide at the cleavage site.

When the universal nucleotide occupies position n +1, cleavage of the support strand between nucleotide positions n +1 and n can be achieved using any suitable mechanism.

As described above, cleavage of the support strand between nucleotide positions n +1 and n can be carried out by the action of an enzyme.

As described above, the cleavage of the support strand between nucleotide positions n +1 and n can be carried out as a two-step process.

The first cleavage step may comprise removing the universal nucleotide from the support strand, thereby forming an abasic site at position n +1, and the second cleavage step may comprise cleaving the support strand between positions n +1 and n at the abasic site.

One mechanism for cleaving the support strand at the cleavage site defined by the sequence comprising the universal nucleotide in the manner described above is described in example 2. The cleavage mechanism described in example 2 is exemplary, and other mechanisms may be employed so long as the above-described blunt-end cleaved double-stranded scaffold polynucleotide is achieved.

In the first cleavage step of the two-step cleavage process, the universal nucleotide is removed from the support strand while preserving the integrity of the sugar-phosphate backbone. This can be achieved by the action of an enzyme that specifically cleaves a single universal nucleotide from a double-stranded polynucleotide. In an exemplary cleavage method, the universal nucleotide is inosine, and inosine is cleaved from the support strand by the action of an enzyme to form an abasic site. In an exemplary cleavage method, the enzyme is a 3-methyladenine DNA glycosylase, in particular human alkyl adenine DNA glycosylase (hAAG). Other enzymes, molecules, or chemicals may be used, provided that abasic sites are formed. The nucleotide excising enzyme may be an enzyme that catalyzes the release of uracil from a polynucleotide, such as uracil-DNA glycosylase (UDG).

In the second cleavage step of the two-step cleavage process, the support strand is cleaved at the abasic site by single strand cleavage. In an exemplary method, the support chain is cleaved by the action of a chemical that is a base such as NaOH. Alternatively, organic chemicals such as N, N' -dimethylethylenediamine may be used. Alternatively, an enzyme having no base site lyase activity, such as AP endonuclease 1, endonuclease III (Nth), or endonuclease VIII, may be used. Other enzymes, molecules or chemicals may be used provided that the support strand is cleaved at the base-free sites as described above.

Thus, in embodiments in which the universal nucleotide is at position n +1 of the support strand in steps (1) and (2) and the support strand is cleaved between positions n +1 and n, the first cleavage step may be performed with a nucleotide excising enzyme. An example of such an enzyme is 3-methyladenine DNA glycosylase, such as human alkyl adenine DNA glycosylase (hAAG). The second cleavage step may be performed with a chemical that is a base, such as NaOH. The second step can be carried out with an organic chemical having abasic site cleavage activity, such as N, N' -dimethylethylenediamine. The second step may be carried out with an enzyme having abasic site cleaving enzyme activity such as endonuclease VIII or endonuclease III.

As described above, the cleavage of the support strand between nucleotide positions n +1 and n can also be carried out as a one-step cleavage process. Examples of enzymes that can be used in any such process include endonuclease III, endonuclease VIII. Other enzymes that may be used in any such method include enzymes that cleave 8-oxoguanosine, such as formamidopyrimidine DNA glycosylase (Fpg) and 8-oxoguanine DNA glycosylase (hOGG 1).

Step 5-deprotection

In this exemplary method form and all forms of the invention, the reversible terminator group must be removed from the first nucleotide in order to allow the next nucleotide to be incorporated into the next synthesis cycle (deprotection step; 105). This may be performed at various stages of the first cycle. After the cleavage step (4), it may be carried out as step (5) of the process. However, the deprotection step may be performed after the introduction of the second nucleotide in step (3) and before the cleavage step (4), in which case the deprotection step is defined as step (4) and the cleavage step is defined as step (5), as shown in fig. 1 (104 and 105, respectively). Whichever protecting group step is performed, the enzyme and residual unbound second nucleotide should first be removed after step (3) to prevent multiple pooling of the second nucleotide in the same synthesis cycle.

The reversible terminator group may be removed from the first nucleotide by any suitable means known to those skilled in the art. For example, removal can be performed by using a chemical, such as tris (carboxyethyl) phosphine (TCEP).

Further circulation

After completion of the first synthesis cycle, the second and further synthesis cycles may be performed using the same method steps.

The cleavage products of steps (4) and (5) of the previous cycle are provided (in step 6) as double stranded scaffold polynucleotides for the next synthesis cycle.

In step (6) of the next and every other synthesis cycle, another double-stranded polynucleotide linker molecule is ligated to the cleavage products of steps (4) and (5) of the previous cycle. The polynucleotide linker molecule may be constructed in the same manner as described above for step (2) of the previous cycle, except that the polynucleotide linker molecule comprises the first nucleotide of the further synthesis cycle. The polynucleotide linker molecule can be linked to the cleavage products of steps (4) and (5) of the previous cycle in the same manner as step (2) described above.

In step (7) of the next and each further synthesis cycle, the end of the primer strand portion of the synthetic strand of the double stranded scaffold polynucleotide is further extended by the introduction of a second nucleotide of the further synthesis cycle. Action of nucleotidyl transferase, polymerase or other enzyme. The second nucleotide of the cycle can be further synthesized in the same manner as in the above step (3).

In step (8) of the next and every further cycle of synthesis, the ligated scaffold polynucleotide is cleaved at the cleavage site. Cleavage causes a double strand break in the scaffold polynucleotide. Cleavage of the scaffold polynucleotide (step 8) causes loss of the helper strand (if present and hybridized to the support strand immediately prior to cleavage) and loss of the support strand comprising the universal nucleotide. Cleavage of the scaffold polynucleotide thereby releases the additional polynucleotide linker molecule from the scaffold polynucleotide, but causes retention of the first nucleotide of the additional cycle on the support strand of the cleaved scaffold polynucleotide and pairing with the second nucleotide of the additional cycle. Cleavage of the scaffold polynucleotide leaves in situ the cleaved blunt-ended double-stranded scaffold polynucleotide comprising another cycle of the first nucleotide at the end of the support strand of the scaffold polynucleotide and another cycle of the "end" of the primer strand portion of the synthetic strand of the second nucleotide scaffold polynucleotide at the end. The cleavage in step (8) may be carried out in the same manner as in step (4) described above.

In step (9), the reversible termination group is removed from the second nucleotide of another cycle (deprotection step; 109). This may be performed at various stages, as described above for the first cycle. After the cleavage step (8), it may be carried out as step (9) of the process. Alternatively, the deprotection step may be performed at any step after the combination step (7) and before the cleavage step (8), in which case the deprotection step is defined as step (8) and the cleavage step as step (9), as shown in fig. 1 (109 and 110, respectively). Deprotection by removal of the reversible terminating group in the next and subsequent cycles can be performed as described above for the first synthesis cycle.

The synthesis cycle is repeated as many times as necessary as described above to synthesize a double-stranded polynucleotide having a predetermined nucleotide sequence.

Synthesis method version 2

Referring to FIG. 2, in a second specific non-limiting exemplary form of the synthetic method of the invention, a double stranded scaffold polynucleotide is provided (step 1; 201 of FIG. 2). The double stranded scaffold polynucleotide comprises a support strand and a synthetic strand hybridized thereto. The synthetic strand comprises a primer strand portion. The double stranded scaffold polynucleotide has at least one blunt end, wherein the at least one blunt end comprises an end of a primer strand portion and an end of a support strand hybridized thereto. The terminal nucleotide of the support strand is capable of serving as a substrate for a ligase and preferably comprises a phosphate group or other linkable group.

In step (2) of the method, a polynucleotide linker molecule (see the structure shown in the upper right of FIG. 2) is attached to the scaffold polynucleotide. Ligation incorporates the first nucleotide of the predetermined sequence into the support strand of the scaffold polynucleotide (step 2; 202 of FIG. 2).

In step (3) of the method, a second nucleotide of the predetermined nucleotide sequence is added to the end of the primer strand portion of the synthesized strand under the action of an enzyme having the ability to extend the oligonucleotide or polynucleotide molecule using a single nucleotide. This enzyme is typically a nucleotidyl transferase or polymerase (step 3; 203 of FIG. 2). The second nucleotide has a reversible terminator group, which prevents further extension by the enzyme. Therefore, only single nucleotides are pooled in step (3).

The scaffold polynucleotide is then cleaved at each synthesis cycle at a cleavage site defined by the sequence comprising the universal nucleotide in the support strand (step 4; 204). In method version 2, the cleaving includes cleaving the support strand immediately after the nucleotide at the nucleotide position next to the universal nucleotide in the direction next to the primer strand portion/away from the auxiliary strand. Cleavage of the scaffold polynucleotide (step 4) results in release of the polynucleotide linker molecule from the scaffold polynucleotide and retention of the first nucleotide of the cycle on the first strand of the cleaved scaffold polynucleotide and pairing with the second nucleotide of the cycle, the oligonucleotide being ligated to the primer strand portion of the synthetic strand. Cleavage of the scaffold polynucleotide (step 4) results in loss of the helper strand of the scaffold polynucleotide (if present and hybridized to the support strand immediately prior to cleavage), and loss of the universal nucleotide from the scaffold polynucleotide. Cleavage leaves in situ the cleaved double stranded scaffold polynucleotide comprising at the cleavage site the cleaved end of the cleaved support strand and the end of the primer strand portion of the synthesized strand comprising the pre-cleavage nick site. Cleavage results in a double stranded scaffold polynucleotide that is cleaved at the blunt end of the cleavage site, with no overhang in either strand, and the first and second nucleotides are nucleotide pairs.

In step (5) of the method, a deprotection step is performed to remove the terminator group from the newly incorporated nucleotides. The deprotection step may also be performed before the cleavage step, in which case the deprotection step is defined as step (4) and the cleavage step as step (5), as shown in fig. 2 (204 and 205, respectively).

Step 1-providing a scaffold polynucleotide

In exemplary version 2 of the synthesis method of the invention, a double stranded scaffold polynucleotide (201) is provided in step (1). Double-stranded scaffold polynucleotides are provided that comprise a synthetic strand and a support strand hybridized thereto, wherein the synthetic strand comprises a primer strand portion. The terminal nucleotide of the support strand is paired with the terminal nucleotide of the support strand, thereby forming a blunt end. The terminal nucleotide of the support strand is capable of acting as a substrate for a ligase and comprises a ligatable group, preferably a phosphate group.

Step 2-ligating the polynucleotide linker molecule to the scaffold polynucleotide and incorporating the first nucleotide of the predetermined sequence

In step (2) of the method, a double stranded polynucleotide linker molecule is ligated (202) to the scaffold polynucleotide by the action of a ligase in a blunt end ligation reaction.

The complementary linking ends of the polynucleotide linker molecules are configured such that the terminal nucleotide of the support strand is the first nucleotide of the predetermined sequence that is incorporated into the scaffold polynucleotide in any given synthesis cycle. The terminal nucleotide of the support strand is paired with the terminal nucleotide of the helper strand. The terminal nucleotide of the support strand, i.e. the first nucleotide of the predetermined sequence of the loop, occupies nucleotide position n in the support strand. Position n refers to the nucleotide position relative to the second nucleotide of the predetermined sequence of the cycle after combining the second nucleotides, whereby the first and second nucleotides form a nucleotide pair. In FIG. 2, the first nucleotide of the predetermined sequence is depicted as adenosine, the second nucleotide of the predetermined sequence in step (3) is depicted as thymine, and the terminal nucleotide of the auxiliary strand is depicted as thymine. Adenosine and thymine are for illustration only. The first and second nucleotides of the helper strand, as well as the terminal nucleotide, can be any suitable nucleotide. The nucleotide depicted as X can also be any suitable nucleotide selected by the user.

Typically, the terminal nucleotide of the support strand at the complementary linker will define the 3' end of the support strand of the complementary linker of the polynucleotide linker molecule.

The terminal nucleotide of the auxiliary strand of the polynucleotide linker molecule is configured such that it cannot be linked to another polynucleotide in the polynucleotide strand (the position purely labeled "T" is used to illustrate the structure at the top right of the figure) 2). This nucleotide is referred to as the non-ligatable terminal nucleotide. Typically, the terminal nucleotide will lack a phosphate group, i.e., it will be a nucleoside. Typically, the terminal nucleotide of this helper strand will define the 5' end of the helper strand, as described above.

In the complementary linker end of the polynucleotide linker molecule, the universal nucleotide in the support strand is positioned such that it occupies nucleotide position n +2 in the support strand and pairs with the partner nucleotide in the auxiliary strand. Position n +2 refers to the second nucleotide position at position n in the support strand in the direction distal to the complementary linker.

The linking of the support chains may be performed in any suitable manner. Ligation may generally, and preferably is, performed by an enzyme having ligase activity. For example, ligation may be performed using T3 DNA ligase or T4 DNA ligase or a functional variant or equivalent thereof or other enzymes described further herein. The use of such an enzyme will cause single strand breaks to be maintained in the synthesized strand because the terminal nucleotide of the helper strand is provided such that it cannot serve as a substrate for the ligase, for example due to the absence of a terminal phosphate group or the presence of an unlinkable blocking group.

After ligation of the polynucleotide linker molecule to the scaffold polynucleotide, the first nucleotide of the predetermined nucleotide sequence is referred to as occupying nucleotide position n, the universal nucleotide is referred to as occupying nucleotide position n +2, and the terminal nucleotide of the support strand of the scaffold polynucleotide that was proximal to the primer strand portion prior to ligation is referred to as occupying nucleotide position n-1. Nucleotide position n-1 refers to the next nucleotide position in the support strand relative to position n in the direction of the proximal end of the primer strand portion/distal end of the auxiliary strand.

Step 3-merging second nucleotides of the predetermined sequence

If the helper strand is present in the scaffold polynucleotide immediately prior to primer strand partial extension, particularly if a polymerase is used, the enzyme may act to "invade" the helper strand and displace the helper strand terminal nucleotides. The merged second nucleotide will then occupy the position previously occupied by the displaced terminal nucleotide of the helper strand (see figure 2 for the structure shown in the middle lower part). In certain embodiments, the helper strand may be removed prior to the extension/pooling step (3), in which case the enzyme may access and extend the terminal nucleotide of the primer strand portion of the synthesized strand without displacing the helper strand or a portion thereof.

Step 4-lysis

In step (4) of the method, the ligated scaffold polynucleotides are cleaved at the cleavage site (204). The cleavage site is defined by a sequence comprising the universal nucleotide in the support strand. Cleavage causes a double strand break in the scaffold polynucleotide. Cleavage of the scaffold polynucleotide (step 4) causes loss of the helper strand (if present and hybridized to the support strand immediately prior to cleavage) and loss of the support strand comprising the universal nucleotide.

In this exemplary method, the synthetic strand already has a single strand break or "nick", and thus only cleavage of the support strand is required to provide a double strand break in the scaffold polynucleotide. Furthermore, as previously described in this exemplary method version, cleavage results in a blunt-end cleaved double-stranded scaffold polynucleotide that does not protrude in either the synthetic strand or the support strand, and the universal nucleotide is located at the n +2 position cleavage step in the support strand prior to the support strand. To obtain such blunt-end cleaved double-stranded scaffold polynucleotides when the universal nucleotide is located at the n +2 position in the support strand, the support strand is cleaved at a specific position relative to the universal nucleotide. When the support strand of the scaffold polynucleotide is cleaved between nucleotide positions n +1 and n, the polynucleotide linker molecule is released from the scaffold polynucleotide (see the structure shown in the upper left of figure 1), except that the cycle remains in the scaffold polynucleotide attached to the support strand of the cleaved scaffold polynucleotide.

Thus, in method version 1, the universal nucleotide occupies position n +2 in the support strand in steps (2), (3) and (4), and the support strand is cleaved between nucleotide positions n +1 and n in step (4).

The support strand may be cleaved by cleaving one of the ester bonds of the phosphodiester bond between nucleotide positions n +1 and n.

When the universal nucleotide occupies position n +2, cleavage of the support strand between nucleotide positions n +1 and n can be achieved using any suitable mechanism.

As described above, cleavage of the support strand between nucleotide positions n +1 and n can be carried out by the action of an enzyme.

As described above, when the universal nucleotide occupies position n +2 in the support strand, cleavage of the support strand between nucleotide positions n +1 and n can be performed by the action of an enzyme, such as endonuclease V.

In a similar manner, one mechanism for cleaving the support strand between nucleotide positions n +1 and n at a cleavage site defined by a sequence comprising a universal nucleotide occupying position n +2 in the support strand is described in example 3. The mechanisms described are exemplary and other mechanisms may be employed provided that the cleavage arrangement described above is implemented.

In this exemplary mechanism, an endonuclease is employed. In an exemplary method, the enzyme is endonuclease V. Other enzymes, molecules or chemicals may be used as long as the support strand is cleaved between nucleotide positions n +1 and n when the universal nucleotide is at position n +2 in the support strand.

Step 5-deprotection

In this exemplary method form and all forms of the invention, the reversible terminator group must be removed from the first nucleotide in order to allow the next nucleotide to be incorporated into the next synthesis cycle (deprotection step; 205). This may be performed at various stages of the first cycle. After the cleavage step (4), it may be carried out as step (5) of the process. However, the deprotection step may be performed after the introduction of the second nucleotide in step (3) and before the cleavage step (4), in which case the deprotection step is defined as step (4) and the cleavage step is defined as step (5), as shown in fig. 2 (204 and 205, respectively). Whichever protecting group step is performed, the enzyme and residual unbound second nucleotide should first be removed after step (3) to prevent multiple pooling of the second nucleotide in the same synthesis cycle.

Further circulation

After completion of the first synthesis cycle, the second and further synthesis cycles may be performed using the same method steps.

The cleavage products of steps (4) and (5) of the previous cycle are provided (in step 6) as double stranded scaffold polynucleotides for the next synthesis cycle.

In step (9), the reversible termination group is removed from the second nucleotide of another cycle (deprotection step; 209). This may be performed at various stages, as described above for the first cycle. After the cleavage step (8), it may be carried out as step (9) of the process. Alternatively, the deprotection step may be performed at any step after the combination step (7) and before the cleavage step (8), in which case the deprotection step is defined as step (8) and the cleavage step as step (9), as shown in fig. 2 (209 and 210, respectively). Deprotection by removal of the reversible terminating group in the next and subsequent cycles can be performed as described above for the first synthesis cycle.

The synthesis cycle is repeated as many times as necessary as described above to synthesize a double-stranded polynucleotide having a predetermined nucleotide sequence.

Synthetic methods variants of version 2

In addition to the above method, a variation of synthesis method version 2 is provided, wherein the method is performed in the same manner as synthesis method version 2 described above, except for the following variations.

In the first cycle of the ligation step (step 2), the complementary linkers of the polynucleotide linker molecules are configured such that the universal nucleotide is at nucleotide position n +2+ x on the support strand and pairs with the partner nucleotides. Removing the auxiliary strand at the 2+ x position from the terminal nucleotide of the auxiliary strand at the complementary ligation end; wherein nucleotide position n +2 is the second nucleotide position in the support strand at nucleotide position n in the distal direction relative to the complementary linking end.

In the first cycle of the cleavage step (step 4), the universal nucleotide occupies instead nucleotide position n +2+ x in the support strand of the scaffold polynucleotide, wherein nucleotide position n +2 is the second nucleotide position in the support strand relative to nucleotide position n in the direction proximal to the helper strand/distal to the primer strand portion; and cleaving the helper strand of the scaffold polynucleotide between positions n +1 and n.

In the second cycle of the ligation step (step 6) and all other cycles of the ligation step, the complementary linkers of the polynucleotide linker molecules are configured such that the universal nucleotides occupy nucleotide positions n +2+ x in the support strand and pair with partner nucleotides that remove position 2+ x from the terminal nucleotides of the auxiliary strand at the complementary linkers; wherein nucleotide position n +2 is the second nucleotide position in the support strand at nucleotide position n in the distal direction relative to the complementary linking end.

Finally, in the second cycle of the cleavage step (step 8) and all other cycles of the cleavage step, the universal nucleotide occupies instead nucleotide position n +2+ x in the support strand of the scaffold polynucleotide, wherein nucleotide position n +2 is the second nucleotide position in the support strand proximal to the helper strand/distal to the primer strand portion relative to nucleotide strand position n; the support strand of the scaffold polynucleotide is cleaved between positions n +1 and n.

In all of these variant methods, x is an integer of 1 to 10 or more, and wherein x is the same integer in steps (2), (4), (6) and (8).

As with the synthesis method of version 2, in the variant method based on version 2, it is noted that in any given cycle, after ligation and in the merging and cleavage steps, the nucleotide position occupied by the first nucleotide of that cycle in the support strand is defined as nucleotide position n, and that after completion of a given synthesis cycle, the position occupied by the first nucleotide of that cycle in the support strand of the cleaved scaffold polynucleotide is defined as nucleotide position n-1 in the next synthesis cycle.

Thus, in these particular variants of synthesis method version 2, the position of the cleavage site relative to nucleotide position n remains constant, and the position of the universal nucleotide relative to nucleotide position n increases the number of nucleotide positions determined by the number selected for x by shifting the position of the universal nucleotide in a direction closer to the helper strand/further away from the primer strand portion.

A schematic of these variant processes is provided in fig. 3, where the deprotection step is shown as step (4) and the cleavage step is shown as step (5). As described above, the order in which these steps are performed may be switched.

Synthetic methods variants of versions 1 and 2, each cycle comprising more than two nucleotides

The present invention further provides additional variant methods in which more than two nucleotides are combined per cycle, including other additional variant methods based on the specific versions 1 and 2 and variants thereof described above. Any of these other variant methods may be performed as described above, except that the following modifications may be made.

In step (2), the polynucleotide linker molecule has a complementary linker comprising the first nucleotide of the predetermined sequence of the first cycle and further comprising one or more additional nucleotides of the predetermined sequence of the first cycle. The polynucleotide linker molecule is then attached to the scaffold polynucleotide. The first nucleotide of the predetermined sequence of the first cycle is the terminal nucleotide of the support strand of the complementary linker. The complementary linkers are preferably configured such that the first and other nucleotides of the first cyclic predetermined sequence comprise a linear nucleotide sequence, wherein each nucleotide in the sequence occupies the next nucleotide position in the support strand in a direction away from the complementary linkers.

In step (3), the ends of the primer strand portion of the synthesized strand of the double-stranded scaffold polynucleotide are extended by the action of a nucleotidyl transferase or polymerase by pooling the second nucleotide of the predetermined sequence of the first cycle. An enzyme, wherein the ends of the primer strand portions are further extended by incorporation of one or more further nucleotides of the predetermined sequence of the first cycle by the action of a nucleotidyl transferase or polymerase, wherein each of the second and further nucleotides of the first cycle comprises a reversible terminator group which prevents further extension by the enzyme, and wherein, after each further extension, the reversible terminator group is removed from the nucleotide in a deprotection step (step 4) before merging the next nucleotide.

The complementary linkers of the polynucleotide linker molecules are configured such that in step (4) prior to cleavage, the universal nucleotide occupies a position in the support strand that is after the nucleotide position of the first and other nucleotides in the direction distal to the complementary linkers.

In step (4) after the cleavage, the first, second and further nucleotides of the predetermined sequence of the first cycle are retained in the cleaved scaffold polynucleotide.

In step (6), the polynucleotide linker molecule has a complementary linker comprising the first nucleotide of the further cycle of the predetermined sequence and further comprising one or more further nucleotides of the further cycle of the predetermined sequence. The polynucleotide linker molecule is then attached to the scaffold polynucleotide as described in step (2). As in step (2), the first nucleotide of the predetermined sequence of the other cycle is the terminal nucleotide of the support strand of the complementary linker. The complementary linkers are preferably structured such that the first and further nucleotides of the further cyclic predetermined sequence comprise a linear nucleotide sequence, wherein each nucleotide in the sequence occupies the next nucleotide position in the support strand in the direction of the distal end of the complementary linker.

In step (6), the ends of the primer strand portion of the synthetic strand of the double-stranded scaffold polynucleotide are extended by incorporating a further cycle of a second nucleotide of the predetermined sequence by the action of a nucleotidyl transferase or polymerase. An enzyme, wherein the ends of the primer strand portions are further extended by incorporation of one or more further nucleotides of the predetermined sequence in further cycles by the action of a nucleotidyl transferase or polymerase, wherein the other nucleotides of the second and said further cycles comprise a reversible terminator group which prevents further extension by the enzyme, and wherein, after each further extension, the reversible terminator group is removed from the nucleotide before merging of the next nucleotide.

In step (8) after the cleaving, additional cycles of the first, second and additional nucleotides of the predetermined sequence are retained in the cleaved scaffold polynucleotide.

In these variant methods, cleavage is always performed following steps (5) and (9) after the final deprotection step (steps 4 and 8), except that the reversible termination group of the last additional nucleotide incorporated in any given cycle may alternatively be removed from the last additional nucleotide after the step of cleaving the linked scaffold polynucleotide at the cleavage site.

In these variant methods, in any given synthesis cycle, prior to cleavage, in the support strand, the position occupied by the first nucleotide of the predetermined sequence may be referred to as position n, the position occupied by the first additional nucleotide of the predetermined sequence in the support strand may be referred to as position n +1, and the position occupied by the universal nucleotide may be referred to as position n +2+ x, where x is an integer from zero to 10 or more, where x is zero if the support strand contains only one additional nucleotide; if the support strand contains only two additional nucleotides, x is one, and so on.

A schematic representation of these variant processes is provided in fig. 4, where the deprotection step is shown as step (4) and the cleavage step is shown as step (5). As described above, the order in which these steps can be performed can be varied relative to the last nucleotide to be combined.

In any of these variant methods, the structure of the complementary linking end of the polynucleotide linker molecule is such that in steps (4) and (8) prior to cleavage, the universal nucleotide occupies a position in the support strand that is the next nucleotide position in the support strand after the nucleotide positions of the first and other nucleotides in a direction away from the complementary linking end, and the support strand is cleaved between the position occupied by the last nucleotide and the position occupied by the universal nucleotide.

In any of these variant methods, the complementary linkers of the polynucleotide linker molecules are alternately configured such that in steps (4) and (8) prior to cleavage, the universal nucleotide occupies a position in the support strand that is the next nucleotide position in the support strand after the nucleotide positions of the first and other nucleotides in a direction away from the complementary linkers, and alternatively, the support strand is cleaved between the position occupied by the last additional nucleotide and the position occupied by the next nucleotide in the support strand.

It will be appreciated that in respect of the position of the universal nucleotide relative to nucleotide position n, conventional modifications to the configuration of the complementary linkers of the polynucleotide linker molecules may be required in addition to each of the first and second nucleotides in order to modify the incorporation of the other nucleotides in the first and other cycles of synthesis. In all of the methods of the invention described and defined herein, nucleotide position n is always a nucleotide position in the support strand that is or will be opposite to the second nucleotide of the predetermined sequence at any given cycle before or after pooling. Thus, the skilled person is readily able to routinely modify the selection of positions of the universal nucleotides and cleavage sites relative to nucleotide position n to adapt any method version and variants thereof to accommodate incorporation of additional nucleotides for the first and additional cycles of synthesis.

Examples of the invention

The following examples provide support for methods for synthesizing polynucleotides or oligonucleotides according to the invention and exemplary constructs for use in the methods. The examples do not limit the invention.

In addition to example 13, the following example describes a synthetic method according to a reaction scheme that is related to, but not within the scope of, the synthetic method of the present invention.

The examples demonstrate the ability to perform a synthetic reaction comprising the steps of: adding nucleotides of a predetermined sequence to the synthesis strand of the scaffold polynucleotide, cleaving the scaffold polynucleotide at a cleavage site defined by the universal nucleotides and ligating polynucleotide linker molecules comprising partner nucleotides for the added nucleotides of the predetermined sequence, and new universal nucleotides for creating a cleavage site for the next synthesis cycle. The method of the present invention combines these steps in a modified manner. Thus, in addition to example 13, the following example provides illustrative support for the process of the invention as defined herein. Example 13 provides the data for incorporation of 3' -O-modified-dNTPs by terminator X DNA polymerase in combination with a method according to the invention, e.g., a synthetic method version of invention 1 and 2 and variants thereof (FIGS. 1 to 5).

In the following examples, and in corresponding fig. 12-50, references to, for example, synthetic methods "versions 1, 2, and 3" or "version 1, 2, or 3" are explained in accordance with fig. 6, 7, and 8 and not in accordance with the reaction schemes set forth in any of fig. 1-5 or the description herein. Example 13 and FIG. 51 will be explained according to the synthesis method of the present invention. In particular, the synthesis processes according to the invention 1 and 2, as well as variants and associated reaction schemes thereof, are shown in figures 1-5, more particularly including step 3 of these processes.

Example 1: there is no synthesis of the auxiliary chain.

This example describes the synthesis of a polynucleotide using 4 steps: the 3' -O-modified dNTPs are cleaved, ligated and deprotected on the partially double-stranded DNA combined, and the first step is performed opposite to the universal nucleotide (in this case, inosine).

Step 1: merging

The first step describes the controlled addition of 3' -O-protected mononucleotides to oligonucleotides by enzymatic incorporation of DNA polymerase (FIG. 12 a).

Materials and methods

Material

3' -O-modified dNTPs were synthesized internally according to the protocol described in the PhD paper: jian Wu Molecular Engineering of Novel Nucleotide analogs for DNA Sequencing by Synthesis, Columbia University, 2008. Protocols for synthesis are also described in patent application publications: william Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for polydeoxnitrile Synthesis, Molecular Assemblies US2016/0108382A 1.

2. The oligonucleotides were designed internally and obtained from Sigma-Aldrich (FIG. 12 h). Stock solutions were prepared at a concentration of 100. mu.M.

3. Terminator IX DNA polymerase, engineered by New England BioLabs, was used with enhanced ability to incorporate 3-O-modified dNTPs. However, any DNA polymerase that can incorporate modified dNTPs can be used.

Two types of reversible terminators were tested:

method of producing a composite material

1. Mu.l of 10XBuffer (20mM Tris-HCl, 10mM (NH)₄)₂SO₄、10mM KCl、2mM MgSO₄、0.1％X-100, pH 8.8, New England BioLabs) was mixed with 12.25. mu.l sterile deionized water (ELGA VEOLIA) in a 1.5ml Eppendorf tube.

2. Mu.l of 10. mu.M primer (synthetic strand) (5pmol, 1 eq.) (SEQ ID NO:1, FIG. 12h) and 0.75. mu.l of 10. mu.M template (support strand) (6pmol, 1.5 eq.) (SEQ ID NO:2, FIG. 12h) were added to the reaction mixture.

3. 3' -O-modified-dTTP (100. mu.M in 2. mu.l) and MnCl were added₂(40 mM in 1. mu.l).

4. Then 1.5. mu.l of Therminator IX DNA polymerase (15U, New England Biolabs) was added. However, any DNA polymerase that can incorporate modified dNTPs can be used.

5. The reaction was incubated at 65 ℃ for 20 minutes.

6. The reaction was stopped by adding TBE-urea sample buffer (Novex).

7. The reactions were separated on polyacrylamide gel (15%) with TBE buffer and visualized by a ChemiDoc MP imaging system (BioRad).

Gel electrophoresis and DNA visualization:

1. mu.l of the reaction mixture was added to 5. mu.l of TBE-urea sample buffer (Novex) in a sterile 1.5ml Eppendorf tube and heated to 95 ℃ using a heating ThermoMixer (Eppendorf) for 5 minutes.

2. Mu.l of the sample was then loaded into wells of 1.0mmx10 wells (Invitrogen) of a 15% TBE-urea gel containing preheated 1XTBE buffer Thermo Scientific (89mM Tris,89mM boric acid and 2mM EDTA).

3. Fixing an X-cell spare lock module (Novex) in place and performing electrophoresis under the following conditions; 260V, 90Amp, 40 min at room temperature.

4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS. Visualization and analysis were performed on the Image lab 2.0 platform.

Results

Custom engineered Therminator IX DNA polymerase from New England BioLabs is a highly efficient DNA polymerase capable of integrating 3' -O-modified dNTPs such as inosine as opposed to universal nucleotides (FIG. 12 b-c).

Efficient incorporation of inosine occurred at a temperature of 65 deg.C relative to the other (FIGS. 12 d-e).

Binding of 3' -O-modified dTTP to inosine requires the presence of Mn ²⁺Ions (FIG. 12 f-g). Successfully convert intoIn FIGS. 12c, e, g and h are marked in bold.

Conclusion

3-O-modified dTTP in contrast to inosine in the pool custom engineered terminator IX DNA polymerase from New England BioLabs was used at Mn²⁺In the presence of ions and at a temperature of 65 ℃ with particularly high efficiency.

Step 2: cracking

The second step describes the two-step cleavage of the polynucleotide with hAAG/Endo VIII or hAAG/chemical base (FIG. 13 a).

Materials and methods

Material

1. The oligonucleotide used in example 1 was designed internally and synthesized by Sigma Aldrich (see sequence listing of fig. 13 (e)).

2. The oligonucleotides were diluted to a stock concentration of 100 μ M using sterile distilled water (ELGA VEOLIA).

Method of producing a composite material

The oligonucleotides were subjected to cleavage reaction using the following procedure:

1. 41. mu.l of sterile distilled water (ELGA VEOLIA) were transferred to a 1.5ml Eppendorf tube with a pipette (Gilson).

2. Then 5. mu.l of 10XReaction buffer NEB (20mM Tris-HCl, 10mM (NH)₄)₂SO₄、10mM KCl、2mM MgSO₄、0.1％X-100, pH 8.8) were added to the same Eppendorf tube.

3. 1 each oligonucleotide (FIG. 13 e); the template (SEQ ID NO:3) or any fluorescently labeled long oligo, primer with T (SEQ ID NO:4) and control (SEQ ID NO:5), were added to the same tube at 5 pmol.

4. Mu.l of human alkyl adenine DNA glycosylase (hAAG) NEB (10 units/. mu.l) was added to the same tube.

5. The reaction mixture was then gently mixed by resuspension with a pipette, centrifuged at 13,000rpm for 5 seconds and incubated at 37 ℃ for 1 hour.

6. Typically after the incubation time has elapsed, the reaction is terminated by enzymatic heat inactivation (i.e., 20 minutes at 65 ℃).

Purification was performed under ambient conditions. The sample mixture was purified using the protocol outlined below:

1. 500 μ l of buffer PNI QIAGEN (5M guanidinium chloride) was added to the sample and gently mixed by resuspension with a pipette.

2. The mixture was transferred to a QIAquick spin column (QIAGEN) and centrifuged at 6000rpm for 1 minute.

3. After centrifugation, the flow-through was discarded, 750. mu.l of buffer PE QIAGEN (10mM Tris-HCl pH 7.5 and 80% ethanol) was added to the spin column, and centrifuged at 6000rpm for 1 minute.

4. The flow through was discarded and the spin column was centrifuged at 13000rpm for an additional 1 minute to remove residual PE buffer.

5. The spin columns were then placed in sterile 1.5ml Eppendorf tubes.

6. For DNA elution, 50. mu.l of buffer EB QIAGEN (10mM Tris.CL, pH 8.5) was added to the center of the column membrane and left to stand at room temperature for 1 minute.

7. The tubes were then centrifuged at 13000rpm for 1 minute. The eluted DNA concentration was measured and stored at-20 ℃ for later use.

The purified DNA concentration was determined using the following protocol:

1. mu.l of sterile distilled water (ELGA VEOLIA) was added to the base balance NanoDrop one (Thermo Scientific).

2. After equilibration, the water was gently wiped off with lint-free lens cleaning paper (Whatman).

3. NanoDrop one was masked (blank) by adding 2. mu.l of buffer EB QIAGEN (10mM Tris.CL, pH 8.5). Step 2 is then repeated after masking.

4. The DNA concentration was measured by adding 2. mu.l of the sample to the base and selecting the measurement icon on the touch screen.

Cleavage of the resulting abasic sites was performed using the following procedure:

1. mu.l (10-100 ng/. mu.l) of DNA was added to a sterile 1.5ml Eppendorf tube.

2. Mu.l (0.2M) NaOH or 1.5. mu.l Endo VIII NEB (10 units/. mu.l) and 5. mu.l 10 Xreaction buffer NEB (10mM Tris-HCl, 75mM NaCl, 1mM EDTA, pH [email protected] ℃) were added to the same tube and gently mixed by resuspension and centrifuged at 13000rpm for 5 seconds.

3. The resulting mixture was incubated at room temperature for 5 minutes to allow the NaOH to treat the sample while the Endo VIII reaction mixture was incubated at 37 ℃ for 1 hour.

4. After the incubation time has elapsed, the reaction mixture is purified using steps 1-7 of the purification scheme as outlined above.

Gel electrophoresis and DNA visualization:

1. Mu.l of DNA and TBE-urea sample buffer (Novex) were added to a sterile 1.5ml Eppendorf tube and heated to 95 ℃ using a heating block (Eppendorf) for 2 minutes.

2. The DNA mix was then loaded into wells of 1.0mmx10 wells (Invitrogen) of a 15% TBE-urea gel containing preheated 1XTBE buffer Thermo Scientific (89mM Tris,89mM boric acid and 2mM EDTA).

3. Fixing an X-cell spare lock module (Novex) in place and performing electrophoresis under the following conditions; 260V, 90Amp, 40 min at room temperature.

4. Detection and visualization of DNA in the gel was performed using ChemiDoc MP (BioRad) using Cy3 LEDS. Visualization and analysis were performed on the Image lab 2.0 platform.

Results and conclusions

Cleavage reactions without the auxiliary strand showed a low percentage yield of cleaved DNA to uncleaved DNA of about 7%: 93% (FIG. 13 b-d).

The cleavage results show that in this particular example, and based on the specific reagents used, low yields of cleaved DNA were obtained in the absence of the auxiliary strand compared to the positive control. Furthermore, the use of chemical bases for cleavage of abasic sites is less time consuming than EndoVIII cleavage.

And step 3: connection of

The third step describes the ligation of polynucleotides with DNA ligase in the absence of the helper strand. A schematic diagram is shown in fig. 14.

Materials and methods

Material

1. The oligonucleotides used in example 1 were designed internally and synthesized by Sigma Aldrich (see sequence listing of fig. 14 c).

2. The oligonucleotides were diluted to a stock concentration of 100 μ M using sterile distilled water (ELGA VEOLIA).

Method of producing a composite material

The ligation of the oligonucleotides was performed using the following procedure:

1. mu.l of sterile distilled water (ELGA VEOLIA) were transferred to a 1.5ml Eppendorf tube with a pipette (Gilson).

2. Then 10. mu.L of 2 Xquick ligation reaction buffer NEB (132mM Tris-HCl, 20mM MgCl)₂2mM dithiothreitol, 2mM ATP, 15% polyethylene glycol (PEG6000) and pH 7.6 at 25 ℃ were added to the same Eppendorf tube.

3. Add 1. mu.l of each oligonucleotide (FIG. 14 c); TAMRA or any fluorescently labeled phosphate strand (SEQ ID NO: 7), primers with T (SEQ ID NO: 8) and inosine strand (SEQ ID NO: 9) were added to the same tube at 5pmol each.

4. Mu.l of Quick T4 DNA ligase NEB (400 units/. mu.l) was added to the same tube.

5. The reaction mixture was then gently mixed by resuspending with a pipette, centrifuged at 13,000rpm for 5 seconds and incubated at room temperature for 20 minutes.

6. Typically after the incubation time has elapsed, the reaction is stopped by adding TBE-urea sample buffer (Novex).

7. The reaction mixture was purified using the protocol outlined in purification steps 1-7 as described above.

The purified DNA concentration was determined using the following protocol:

1. mu.l of sterile distilled water (ELGAVEOLIA) was added to the base balance NanoDrop one (Thermo Scientific).

2. After equilibration, the water was gently wiped off with lint-free lens cleaning paper (Whatman).

3. NanoDrop one was masked (blank) by adding 2. mu.l of buffer EB QIAGEN (10mM Tris.CL, pH 8.5) and then step 2 was repeated after masking.

4. The DNA concentration was measured by adding 2. mu.l of the sample to the base and selecting the measurement icon on the touch screen.

5. The purified DNA was run on a polyacrylamide gel and visualized according to the procedure in steps 5-8 above. No changes in conditions or reagents were introduced.

Results and conclusions

In this particular example, and based on the particular reagents used, ligation of oligonucleotides with DNA ligase (in this particular case rapid T4 DNA ligase) at room temperature (24 ℃) in the absence of the helper strand resulted in a reduced amount of ligation product (fig. 14 b).

Example 2 version 1 chemistry using the helper chain.

This example describes the synthesis of a polynucleotide using 4 steps: merging

Cleaving the 3' -O-modified dNTP from the nick site, ligation and deprotection, wherein the first step is performed opposite to the universal nucleotide, which in this particular case is inosine. The method uses auxiliary strands, which increase the efficiency of the ligation and cleavage steps.

Step 1: merging

The first step describes the controlled addition of 3' -O-protected mononucleotides to oligonucleotides by enzymatic incorporation of DNA polymerase (FIG. 15 a).

Materials and methods

Material

1. The 3' -O-modified dntps were synthesized internally according to the scheme described below: doctor graduation paper Jian Wu: molecular Engineering of Novel Nucleotide analogs for DNA Sequencing by Synthesis. Columbia University,2008. the protocol for synthesis is also described in the patent application publication: william Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for polydeoxnitrile Synthesis, Molecular Assemblies US2016/0108382A 1.

2. Oligonucleotides were designed internally and obtained from Sigma-Aldrich. Stock solutions were prepared at a concentration of 100. mu.M. The oligonucleotides are shown in FIG. 15 b.

3. Terminator IX DNA polymerase, engineered by New England BioLabs, was used with enhanced ability to incorporate 3-O-modified dNTPs.

Two types of reversible terminators were tested:

method of producing a composite material

1.2μl 10XBuffer (20mM Tris-HCl, 10mM (NH)₄)₂SO₄，10mM KCl，2mM MgSO₄、0.1％X-100, pH 8.8, new england laboratory) was mixed with 10.25 μ l sterile deionized water (ELGA veoli) in a 1.5ml Eppendorf tube.

2. Mu.l of 10. mu.M primer (5pmol, 1 eq.) (SEQ ID NO:10, Table in FIG. 15 (b)), 0.75. mu.l of 10. mu.M template (6pmol, 1.5 eq.) (SEQ ID NO:11, Table in FIG. 15 (b)), 2. mu.l of 10. mu.M helper strand (SEQ ID NO:12, Table in FIG. 15 (b)) were added to the reaction mixture.

3. 3' -O-modified-dTTP (100. mu.M in 2. mu.l) and MnCl were added₂(40 mM in 1. mu.l).

4. Then 1.5. mu.l of Therminator IX DNA polymerase (15U, New England Biolabs) was added.

5. The reaction was incubated at 65 ℃ for 20 minutes.

6. The reaction was stopped by adding TBE-urea sample buffer (Novex).

7. The reactions were separated on polyacrylamide gel (15%) with TBE buffer and visualized by a ChemiDoc MP imaging system (BioRad).

Gel electrophoresis and DNA visualization:

1. mu.l of the reaction mixture was added to 5. mu.l of TBE-urea sample buffer (Novex) in a sterile 1.5ml Eppendorf tube and heated to 95 ℃ using a heating ThermoMixer (Eppendorf) for 5 minutes.

2. Mu.l of the sample was then loaded into wells of 1.0mmx10 wells (Invitrogen) of a 15% TBE-urea gel containing preheated 1XTBE buffer Thermo Scientific (89mM Tris,89mM boric acid and 2mM EDTA).

3. Fixing an X-cell spare lock module (Novex) in place and performing electrophoresis under the following conditions; 260V, 90Amp, 40 min at room temperature.

4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS. Visualization and analysis were performed on the Image lab 2.0 platform.

The merging step can be studied according to the above scheme.

Step 2: cracking

The second step describes two-step cleavage of the polynucleotide with hAAG/Endo VIII or hAAG/chemical base (x2) (FIG. 16 a).

Materials and methods

Material

1. The oligonucleotides used in example 2 were designed internally and synthesized by Sigma Aldrich (see sequence of figure 16 f).

2. The oligonucleotides were diluted to a stock concentration of 100 μ M using sterile distilled water (ELGA VEOLIA).

Method of producing a composite material

The cleavage reaction of the oligonucleotides was performed using the following procedure:

1. 41. mu.l of sterile distilled water (ELGA VEOLIA) were transferred to a 1.5ml Eppendorf tube with a pipette (Gilson).

2. Then 5. mu.l of 10XReaction buffer NEB (20mM Tris-HCl, 10mM (NH)₄)₂SO₄、10mM KCl、2mM MgSO₄，0.1％X-100, pH 8.8) were added to the same Eppendorf tube.

3. Mu.l of each oligonucleotide (FIG. 16 f); the template (SEQ ID NO:13) or any fluorescently labeled long oligo strand, primer with T (SEQ ID NO:14), control (SEQ ID NO:15) and auxiliary strand (SEQ ID NO:16), were added to the same tube at 5 pmol.

4. Mu.l of human alkyl adenine DNA glycosylase (hAAG) NEB (10 units/. mu.l) was added to the same tube.

5. In the reaction using the surrogate base, 1. mu.l of human alkyl adenine DNA glycosylase (hAAG) NEB (100 units/. mu.l) was added.

6. The reaction mixture was then gently mixed by resuspension with a pipette, centrifuged at 13,000rpm for 5 seconds and incubated at 37 ℃ for 1 hour.

7. Typically after the incubation time has elapsed, the reaction is terminated by enzymatic heat inactivation (i.e., 20 minutes at 65 ℃).

Purification was performed under ambient conditions. The sample mixture was purified using the protocol outlined below:

1. 500 μ l of buffer PNI QIAGEN (5M guanidinium chloride) was added to the sample and gently mixed by resuspension with a pipette.

2. The mixture was transferred to a QIAquick spin column (QIAGEN) and centrifuged at 6000rpm for 1 minute.

3. After centrifugation, the flow-through was discarded, and 750. mu.l of a buffer PE QIAGEN (10mM Tris-HCl pH7.5 and 80% ethanol) was added to the spin column and centrifuged at 6000rpm for 1 minute.