阅读说明：本技术 一种可利用dna连接酶促进分子内环化的最适dna底物及应用 (Optimal DNA substrate capable of promoting intramolecular cyclization by using DNA ligase and application ) 是由 刘猛 燕毓 常洋洋 于 2021-09-27 设计创作，主要内容包括：本发明公开了一种可利用DNA连接酶促进分子内环化的最适DNA底物及应用,属于DNA合成技术领域。通过使用体外选择技术(SELEX),从一个随机序列的DNA库中,定向进化出针对T4DNA连接酶的最适DNA分子,能够采用结构安排,有利于分子内环化。本发明成功进化出一条最适底物,允许以极高的收率和选择性,在顺式和反式反应中大规模合成DNA环。此外,反式作用的模板有助于构建连锁DNA双环,可作为滚环扩增的自由模板。与传统的检测方法相比,本发明解决了传统成环过程中因没有明显熵差异不可避免得生成线性连接产物等问题,实现了环状核酸得高产率和高特异性制备。(The invention discloses an optimal DNA substrate capable of promoting intramolecular cyclization by using DNA ligase and application thereof, belonging to the technical field of DNA synthesis. By using in vitro selection technology (SELEX), the optimum DNA molecules for T4DNA ligase were evolved directionally from a pool of random-sequence DNA, enabling structural arrangements to be employed which favour intramolecular cyclisation. The present invention successfully evolved an optimal substrate that allows large scale synthesis of DNA circles in both cis and trans reactions with extremely high yield and selectivity. In addition, the trans-acting template facilitates the construction of linked DNA duplexes that can serve as free templates for rolling circle amplification. Compared with the traditional detection method, the invention solves the problems that linear connection products cannot be inevitably generated due to no obvious entropy difference in the traditional cyclization process, and the like, and realizes the preparation of the cyclic nucleic acid with high yield and high specificity.)

1. A DNA substrate capable of promoting intramolecular circulation using DNA ligase, wherein the DNA substrate comprises a nucleotide sequence set forth in SEQ ID No. 1.

2. The DNA substrate according to claim 1, characterized in that the secondary structure of the DNA substrate comprises a single-stranded region (SS1), three short double strands (P1, P2 and P3), a key hairpin loop (L1) and an inter-double stranded unpaired element (J2/3).

3. A single-loop ligation template prepared by trans-ligation of a random DNA library using DNA ligase, wherein the ligation template comprises the nucleotide sequence set forth in SEQ ID No. 25.

4. A ligation template for preparing an interlocked double loop by using a DNA ligase to perform a trans-ligation with a random DNA library, wherein the ligation template comprises a nucleotide sequence shown as SEQ ID No. 26.

5. The ligation template according to claim 3 or 4, wherein the random DNA library is any nucleotide sequence comprising the nucleotide sequence shown in SEQ ID No. 7.

6. The ligation template according to claim 3, wherein the ligation template is obtained by cleaving P1 and P2 of the nucleotide sequence shown in SEQ ID No.1 and extending the double strands of the binding arms at both ends by a certain nucleotide, respectively;

preferably, the nucleotide sequences of the double strands of the binding arms at both ends, which are respectively extended, are shown as SEQ ID NO.29 and SEQ ID NO.30, respectively.

7. The ligation template according to claim 4, wherein the ligation template is obtained by introducing additional nucleotides at both ends of the nucleotide sequence shown in SEQ ID No. 25;

preferably, extra nucleotides are introduced at both ends of the nucleotide sequence shown in SEQ ID NO.25 and are respectively shown in SEQ ID NO.31 and SEQ ID NO. 32.

8. Use of a DNA substrate according to any one of claims 1 to 7 for the intramolecular cyclization of DNA.

9. Use of a ligation template according to any of claims 3 to 7 in a DNA monocycle and/or an interlocking bicyclic ring.

10. Use of the ligation template according to claim 4 as a circular template for performing a rolling circle amplification reaction.

Technical Field

The invention belongs to the field of DNA synthesis, and particularly relates to an optimal DNA substrate capable of promoting intramolecular cyclization by using DNA ligase, two trans-ligation templates and application.

Background

Circular Nucleic Acids (CNAs) refer to naturally occurring or artificially generated nucleic acid molecules with a closed loop structure. Due to the unique characteristics of the molecular probe in the aspects of fluidity, stability, topological structure, function and the like, the molecular probe is widely applied to the fields of molecular cloning, disease treatment, medical diagnosis, biosensing, biochemistry and the like. CNAs are also replicated in a rolling circle manner by some polymerases, driving the popularity of Rolling Circle Amplification (RCA) and Rolling Circle Transcription (RCT) as nucleic acid amplification tools. Furthermore, CNAs can be readily coupled to functional nucleic acids (e.g., aptamers, dnases, ribozymes, and aptamer ribozymes) to create functional systems for bioassays and biomedical applications.

A common method for synthesizing circular single-stranded (ss) DNA is to use a ligase, including T4DNA ligase (T4DL), to seal the gap between the 3 '-hydroxyl and 5' -phosphate ends in ssDNA with the aid of paired DNA short strands. Another approach is to use other ligases (e.g., CircLigase) to catalyze end-joining of ssDNA with end-complementarity. However, since it is inevitable to simultaneously produce linear ligation products (LLP; ligation of two or more linear DNA molecules). This is because there is no significant entropy difference between the two ligation reactions, intramolecular cyclization and intermolecular ligation. Optimization of reaction conditions (e.g., Mg (II) concentration, temperature) and DNA sequence design (e.g., stem length) can improve yield and selectivity to some extent, but does not fundamentally preclude intermolecular ligation, often requiring longer reaction times (up to several hours). In addition, the base pairs near the linker ends must be carefully designed to reduce the negative effects of size, secondary structure and topology on cyclization. However, CNAs-based devices exhibit complex, controllable and complex functions on the nanometer scale and truly realize their potential, new methods providing high cyclization yields and selectivity are needed.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention obtains the optimal DNA substrate capable of specifically acting on the T4DNA ligase by utilizing an in vitro screening technology, prepares monocyclic DNA by adopting a structure more favorable for intramolecular cyclization rather than intermolecular ligation, provides a method for generating CNAs with high yield and high selectivity, and expands the practical application of the CNAs in the fields of chemical biology, diagnosis, calculation and biosensing.

In order to solve the technical problems, the invention provides an optimal DNA substrate with high affinity with T4DNA ligase, which comprises a nucleotide sequence shown as SEQ ID NO. 1.

The invention also provides a trans-ligation DNA template which can be used for circularization of a single-stranded DNA library and comprises a nucleotide sequence shown as SEQ ID NO. 25.

The invention also provides a trans-connecting DNA template, which can be used for preparing interlocking DNA dicyclo D2C (DNA 2 catenanes) and comprises the nucleotide sequence shown in SEQ ID NO. 26.

Further, in the above technical scheme, the secondary structure of the DNA substrate comprises a single-stranded region (SS1), three short double strands (P1, P2 and P3), a key hairpin loop (L1) and an inter-double strand unpaired element (J2/3), and the structure can perform high-efficiency intramolecular self-cyclization.

Further, in the above technical scheme, the random DNA library is any nucleotide sequence including a nucleotide sequence shown in SEQ ID No. 7.

Further, in the above technical solution, the ligation template cleaves P1 and P2 of the DNA substrate obtained by the above screening, and extends the double strands of the binding arms at both ends by a certain nucleotide, preferably by 12 nucleotides, respectively, which can greatly improve the yield of single-loop.

Preferably, the nucleotide sequences of the double strands of the binding arms at both ends, which are respectively extended, are shown as SEQ ID NO.29 and SEQ ID NO.30, respectively.

Furthermore, in the above technical scheme, extra nucleotides are introduced at both ends of the nucleotide sequence shown in SEQ ID No.25 for increasing the flexibility of the linking template and forming a circular linking template, which can greatly improve the yield of the interlocked double rings.

Preferably, extra nucleotides are introduced at both ends of the nucleotide sequence shown in SEQ ID NO.25 and are respectively shown in SEQ ID NO.31 and SEQ ID NO. 32.

Furthermore, in the above embodiment, the nucleic acid D2C can be used as a circular template to perform a rolling circle amplification reaction simultaneously.

The invention also provides the application of the DNA substrate in DNA intramolecular cyclization.

The invention also provides the application of the connecting template in DNA single ring and/or interlocking double ring.

The invention also provides the application of the connecting template as a circular template in carrying out rolling circle amplification reaction.

The ligation template allows high yield and high selectivity of intramolecular self-cyclization with only minimal linear ligation products.

The corresponding circular nucleic acid can serve as a template for RCA amplification at a rate of approximately 2.5 times the rate of the library (SEQ ID NO.6) used for its replication screening.

The DNA substrate or the connection template acts on T4DNA ligase specifically, and the dissociation constant K of the DNA substrate and the T4DNA ligase_d300 +/-21 nM, dissociation constant K from Taq DNA ligase_d1268. + -. 300 nM; as a control, the dissociation constant K of the nonspecific sequence (SEQ ID NO.21) to the two ligases described above_d550. + -.38 nM and 630. + -.32 nM, respectively.

The cross-matching 'bubble' area in the connection template weakens the limitation of the interlocking effect on phi29DP, and the connection template can be used as a circular template to carry out a rolling circle amplification reaction simultaneously.

Compared with the prior art, the invention has the beneficial effects that:

the invention obtains the most suitable DNA substrate with high affinity with T4DNA ligase by screening, and designs two trans-connecting templates for forming CNAs (CNAs) by utilizing the unique secondary structure thereof, wherein the two trans-connecting templates comprise a monocyclic ring and an interlocked bicyclic ring. The method can effectively avoid the linear connection product generated when the traditional method uses the auxiliary of pairing short sequences or other ligases (such as CircLigase) for connection, realizes the preparation of CNAs with high yield and high selectivity, and synthesizes DNA loops in cis-form and trans-form reactions on a large scale. In addition, the trans-acting template facilitates the construction of linked DNA duplexes that can serve as free templates for rolling circle amplification. When the method is used for preparing the library single ring, the yield and the selectivity can respectively reach 93 percent and 96 percent, and when the interlocking DNA double ring is prepared, the yield and the selectivity can respectively reach 90 percent and 92 percent (the connection reaction time is 10 min). Compared with the traditional detection method, the invention solves the problems that linear connection products cannot be inevitably generated due to no obvious entropy difference in the traditional cyclization process, and the like, and realizes the preparation of the cyclic nucleic acid with high yield and high specificity.

Drawings

FIG. 1 is a schematic diagram showing a screening strategy for an optimum substrate for T4DNA ligase in the present invention.

FIG. 2 is a diagram of the programmed secondary structure of the DNA substrate Dsub1 described in example 2.

FIG. 3 is a test chart of the intramolecular ligation characteristics of the DNA substrate Dsub1 prepared in example 3; in the figure, a is Dsub1 self-circularization (for T4DL), b is Dsub1 circularization assisted by template (for T4DL), and c is Dsub1 self-circularization (for TaqDL).

FIG. 4 is a schematic diagram of the structure optimization of the DNA substrate Dsub1 prepared in example 3.

FIG. 5 shows the P1 mutation of the DNA substrate Dsub1 prepared in example 3.

FIG. 6 shows the L1-P2 mutation of the DNA substrate Dsub1 prepared in example 3.

FIG. 7 shows SS1-J2/3 mutation of the DNA substrate Dsub1 prepared in example 3.

FIG. 8 shows Dsub1-I mutation in the DNA substrate prepared in example 3.

FIG. 9 is a kinetic analysis of the RCA of the DNA substrate prepared in example 3.

FIG. 10 is an affinity analysis of the DNA substrate Dsub1 prepared in example 3.

FIG. 11 is the library circularization profile of the trans-ligated template Dsub1.T1 obtained by example 4; in the figure, a is a schematic diagram of Dsub1.T1 mediated circularization ligation, and b is a time-dependent ligation result of DL 2.

FIG. 12 is the library circularization profile of the trans-ligated template Dsub1.T2 obtained by example 4; in the figure, a is a schematic diagram of Dsub1. T2-mediated circularization ligation, b is time-dependent ligation of D2C, and c is a diagram of RCA agarose gel electrophoresis of bicyclic D2C.

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the following will explain in detail the embodiments of the present invention with reference to examples, which include the screening of the most suitable DNA substrate, the trans-ligation of libraries and bicyclic rings, and the like.

Table 1: nucleic acid sequences for use in the invention

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

RCA buffer (10 ×) in the following examples: 330mM Tris acetate, 100mM magnesium acetate, 660mM potassium acetate, 1% Tween-20, 10mM DTT, pH 7.9.

T4DNA ligation buffer (10X) in the following examples: 400mM Tris-HCl, 100mM MgCl2, 100mM DTT, 5mM ATP, pH 7.8.

The specific implementation steps of the embodiment are as follows:

example 1 technical route for screening the most suitable DNA substrate with high affinity for T4DNA ligase using SELEX and high throughput sequencing techniques and designing two trans-ligation templates

(1) In vitro screening of the optimal substrate for specific recognition of T4DNA ligase; (2) characterization of the optimal DNA substrate Dsub1. Testing the intramolecular self-cyclization characteristic of the optimal substrate and the binding capacity of the optimal substrate and the ligase, and analyzing the key base mediating the high-efficiency intramolecular self-cyclization to optimize the structure of the optimal substrate; (3) based on the unique secondary structure of the optimal substrate, two trans-linked templates were designed and analyzed for their ability to form a single-loop library and to interlock double loops.

Example 2 in vitro screening of optimal DNA substrates for promoting intramolecular cyclization Using T4DNA ligase

The specific steps of screening the optimal substrate aiming at the T4DNA ligase (figure 1) comprise detailed processes such as protein ligation substrate screening, RCA amplification and digestion, and the specific operations are as follows:

(1) screening: mu.L of a working buffer containing 10. mu.M DNA library 1 and T4DNA ligase containing ATP was added to a 1.5mL sterile EP tube, denatured at 95 ℃ for 5min, cooled to room temperature, added with T4DNA ligase and incubated for 1h to give circular DNA molecules (CTA).

(2) RCA and digestion: performing standard ethanol precipitation on the circular DNA molecule (CTA) obtained in the step (1), purifying by 10% dPAGE, and performing Rolling Circle Amplification (RCA). The specific reaction conditions are as follows: mu.L of RCA buffer (containing CTA, 5. mu.L of 20. mu.M LT1, 5. mu.L of 2.5. mu.M dNTP) was heated at 90 ℃ for 5 minutes, and then cooled to RT for 10 minutes. Mu.l of Phi29DNA polymerase (Phi29DP, 10U/L) was then added and incubated for 5h at 30 ℃. Finally the mixture was heated at 65 ℃ for 10 minutes to inactivate the Phi29 DP. To the above mixture was added 10. mu.L of 100. mu.M LT1, 10. mu.L of 10 Xdigestion buffer, 25. mu.L of ddH₂O, heated at 90 ℃ for 5 minutes, cooled to RT and then 5. mu.L of EcoRV (final volume: 100. mu.L) is added. The reaction mixture was incubated overnight at 37 ℃ and inactivated for 10 minutes at 90 ℃. RCA monomer product was purified by standard ethanol precipitation, 10% dPAGE.

(3) The monomeric DNA obtained in step (2) was recovered and subjected to cyclization reaction using 50. mu.L of monomeric DNA, 2. mu.L of 100. mu.M LT2, 10. mu. L T4DNA ligase buffer and 33. mu.L of ddH₂O after mixing, the mixture was heated at 90 ℃ for 2 minutes and cooled at room temperature for 10 minutes, and 5. mu. L T4DL (5U/. mu.L) was added. After 1 hour of RT incubation, the resulting CTB molecules were purified by standard ethanol precipitation, 10% dPAGE.

(4) And (3) recovering the obtained CTB for the second RCA reaction. The reaction conditions were identical to the first RCA except LT1 was replaced with LT 2. The enzyme cleavage after RCA was replaced with LT1 instead of LT 2.

(5) And (4) repeating the steps (1) to (4) to the 14 th screening.

(6) MiSeq (Illumina) sequencing platform 200pmol of RCA amplification products from round 14 screening were subjected to deep sequencing.

The sequencing results were analyzed for the secondary mechanism that dominates the first sequence (by Mfold program (http:// Mfold. rna. albany. edu/.

The prediction results are shown in FIG. 2, and the secondary structure of the sequence comprises a single-stranded region (SS1), three short double strands (P1, P2 and P3), a key hairpin loop (L1) and an inter-double stranded unpaired element (J2/3). .

Table 2: the first 5 Dapt nucleic acid sequences with the highest frequency of occurrence obtained by deep sequencing in the invention

Example 3 characterization of the optimal DNA substrate Dsub1

(1) Assays for T4DNA ligase (T4DL) and Taq DNA ligase (TaqDL)

Intramolecular self-cyclization characteristics of substrate Dsub1

mu.L of 2. mu.M Dsub1 was denatured in 20. mu.L of 1 XT 4DNA ligase buffer at 90 ℃ for 5 minutes, after cooling RT for 10 minutes, 1U T4DL was added and the RT was incubated for 10,30,60,300, 600s, respectively. After heating the reaction mixture at 90 ℃ for 10 minutes, 10% dPAGE analysis was performed.

The intramolecular self-cyclization reaction against TaqDL is similar to the above reaction except for the following two points 1) the intramolecular cyclization reaction is performed in 1 × Taq DNA ligase buffer; 2) the reaction temperature was 45 ℃.

Figures 3a and 3b show that for T4DL, Dsub1 has 90% and 97% monocyclic product and selectivity, respectively, in 10 minutes; for TaqDL, Dsub1 produced 7% and 70% single-loop product and selectivity, respectively, within 10 minutes.

(2) Analysis of intramolecular cyclization characteristics of Dsub1 with the aid of DS1 against T4DL

mu.L of 2. mu.M Dsub1, and 6. mu.L of 2. mu.M DS1 were denatured in 20. mu.L of 1 XT 4DNA ligase buffer at 90 ℃ for 5 minutes, after cooling RT for 10 minutes, 1U T4DL was added and the RT was incubated for 10,30,60,300, 600s, respectively. After heating the reaction mixture at 90 ℃ for 10 minutes, 10% dPAGE analysis was performed.

Figure 3c shows monocyclic product and selectivity of 39% and 59%, respectively, within 10 minutes for T4DL, Dsub 1;

(3) base mutation analysis of the ligation substrate Dsub1

For the hypothetical P1, the order was changed and replaced with A-T base pairs for three base pairs G6-C17, A7-T16, C8-G15; for the hypothetical L1-P2: mutation of 5 nucleotides from G32 to T41 to AAAAA; g53, T55 and G54 in the order of presumed SS1-J2/3 to A; c47, T49 and C50 were mutated to a. The Dsub1-I mutation was made using a strong paired duplex between SS1 and J2/3.

For hypothetical P1 (fig. 5): base pair content is important; for hypothetical L1-P2 (FIG. 6): mutations at nucleotides G32-T41 resulted in a significant loss of circularization yield; for putative SS1-J2/3 (FIG. 7): the nucleotides at the junction should be perfectly matched, with 4 nucleotides (C47, T49, C50 and G53) being highly conserved; FIG. 8 shows that: when the Dsub1-I mutation was made using a strong double-stranded pairing between SS1 and J2/3, the yield decreased significantly.

(4) Kinetics of RCA reaction

1) DNA Synthesis reaction

1 μ L of 0.4 μ M Ring^CDsub1 or^CDL1 was mixed with 1. mu.L of 100. mu.M LT1, 5. mu.L of 10 × RCA buffer, 5. mu.L of 2.5mM dNTPs and 10U of Phi29DP in a total volume of 50. mu.L. The mixture was incubated at 30 ℃ for 10,30,60,300 and 600s and then heated at 90 ℃ for 10 minutes to inactivate Phi2 DP.

2) Digestion

mu.L of the above mixture was mixed with 5. mu.L of 100. mu.M LT2, 2. mu.L of 10 Xfast digestion buffer and 9. mu.L ddH₂O, heated at 90 ℃ for 5 minutes, cooled at RT for 10 minutes, and then 3. mu.L of EcoRV (15U/. mu.L) was added and reacted at 37 ℃ for 18 hours.

3) Analysis of digestion product

mu.L of the digestion product was mixed with 20. mu.L of 2 XDPAGE loading buffer. The mixture was then placed on a 10% dPAGE gel, stained with 1 × SYBRgold fluorochrome for 10 minutes at 4 ℃ and imaged.

4) Calculation of DNA product Length

Estimation of monomeric DNA bands in each digestion mixture Using Image Quant software (F)_72nt) And IC tape (F)_64nt) And using FR ═ F_72nt/F_64ntThe Fluorescence Ratio (FR) was calculated. From this, we can calculate the total amount of monomeric DNA N_72ntFR × 1pmol × 50, where 50 is the volume correction factor. The length of the product can be used as (N)_72ntX 72nt)/0.4pmol, where 72nt is the monomer length and 0.4pmol is^CDsub1 or^CAmount of DL 1.

FIG. 9 shows that by measuring the final RP length as a function of time, we calculated the phi29DP replication^CThe rate of Dsub1 was 1.12. + -. 0.03kb min^-1About its replication^CDL1 rate (0.44. + -. 0.032kb min)^-1) 2.5 times of the total weight of the powder.

(4) Affinity assay

The binding reaction was performed in 100. mu.L of DNA ligase buffer (40mM Tris-HCl,10mM MgCl)₂10mM DTT, pH 7.8) with 5nM 3' FAM-labeled Dsub1 in buffer and various concentrations of T4DL or TaqDL. After 30 minutes of RT incubation, fluorescence anisotropy values were measured with a microplate reader at an excitation wavelength of 485nm and an emission wavelength of 520 nm.

FIG. 10 shows that T4DL binds with significantly better affinity to Dsub1, K_d300. + -.21 nM, and the mutation Dsub1(^MMutation of the nucleotides Dsub1, L1 and J2/3 to A) with poor binding affinity, K_d1268. + -. 300 nM. As controls, Dsub1 and^MTaqDL from Dsub1 shows similar K_dValues (550. + -.38 nM and 630. + -.32 nM, respectively).

Example 4 construction of Trans-ligation templates for library monocyclic and interlocking bicyclic preparation

(1) Construction of Trans-ligation templates

Breaking the rings on P1 and P2 of the Dsub1 obtained by screening, and extending the sequences to be paired to 12 bases on the left and right respectively to obtainTo the trans-ligation template dsub1.t 1; introduction of additional nucleotides at both ends based on Dsub1.T1 for increasing flexibility of ligation template and for forming circular trans-ligation template: (^CDsub1.T2)。

(2) Dsub1.T1 mediated cyclization of library 2DL2

In a typical experiment, 3. mu.L of 1. mu.M Dsub1.T1 and 1. mu.L of 1. mu.M DL2 were incubated in 20. mu.L of 1 XT 4DNA ligase buffer (pH 7.8) at 90 ℃ for 5 minutes, 10 minutes after RT with 1U T4DL for 10s, 30s, 60s, 300s and 600s, respectively. After heating at 90 ℃ for 10min, the reaction mixture was analyzed by 15% dPAGE.

Figure 11 shows 93% and 96% for Dsub 110 min monocyclic product and selectivity, respectively, for T4 DL;

(3)^Cdsub1.T2 mediated formation of D2C

3 μ L of 1 μ M^CDsub1.T2 and 1. mu.L of 1. mu.M LsDNA were incubated in 20. mu.L of 1 XT 4DNA ligase buffer (pH 7.8) for 5 minutes at 90 ℃ and 10 minutes after RT with 1U T4DL for 10s, 30s, 60s, 300s and 600s, respectively. After heating at 90 ℃ for 10min, the reaction mixture was analyzed by 15% dPAGE.

Figure 12b shows Dsub 110 min monocyclic product and selectivity are 90% and 92% for T4DL, respectively.

(4) RCA reaction based on interlocking bicyclic DNA [2] catenanes (D2C)

In a typical experiment, 5. mu.L (10 × RCA buffer, 1. mu.L Phi29DP (10U/. mu.L) 5. mu.L of 2.5mM dNTP, 5. mu.L 3. mu. M D2C-i D2C-ii or D2C-iii, 1. mu.L 100. mu.M DP1, DP2 or both (total volume: 50. mu.L) were mixed, heated at 90 ℃ for 5 minutes, cooled at RT for 10 minutes, added to 1. mu.M L Phi29DP, reacted at 30 ℃ for 1 h. the amplification products thus produced were analyzed by 0.6% agarose gel electrophoresis.

Figure 12c shows Dsub 110 min monocyclic product and selectivity 93% and 96% for T4DL, respectively.

SEQUENCE LISTING

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<120> an optimal DNA substrate capable of promoting intramolecular cyclization by using DNA ligase and application thereof

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<170> PatentIn version 3.5

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atctccgata gtcatcgtcc ccagctttgt cgccagggag ttggggcatc taggtatccg 60

aatgtctcgg at 72

<210> 23

<211> 72

<212> DNA

<213> L1-P2.M1

<400> 23

atctcgacta gtcagtctcc ccagctttgt cgccagaaaa atggggcatc taggtatccg 60

aatgtctcgg at 72

<210> 24

<211> 72

<212> DNA

<213> L1-P2.M2

<400> 24

atctcgacta gtcagtctcc ccagctttgt caaaaaggag ttggggcatc taggtatccg 60

aatgtctcgg at 72

<210> 25

<211> 62

<212> DNA

<213> Dsub1.T1

<400> 25

ctgagactag tctccccagc tttgtcgcca gggagttggg gcatctagat atccgaatcg 60

cg 62

<210> 26

<211> 102

<212> DNA

<213> Dsub1.T2

<400> 26

atcatctgaa aaaaaaaaac tgagactagt ctccccagct ttgtcgccag ggagttgggg 60

catctagata tccgaatcgc gaaaaaaaaa actcgacctg at 102

<210> 27

<211> 49

<212> DNA

<213> LsDNA

<400> 27

atctcgacta gtctcagcct tgggatatct cacttatcgc gattcggat 49

<210> 28

<211> 64

<212> DNA

<213> IC

<400> 28

ggcgaagaca ggtgcttagt cgaaagatac ctgggggagt attgcggagg aaggttcaga 60

tatc 64

<210> 29

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ctgagactag tc 12

<210> 30

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

atccgaatcg cg 12

<210> 31

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

atcatctgaa aaaaaaaaa 19

<210> 32

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

aaaaaaaaaa ctcgacctga t 21