阅读说明：本技术 用于确定生物分子之间的相互作用水平的方法 (Method for determining the level of the interaction between biomolecule ) 是由 奥拉·索德伯格 于 2018-02-09 设计创作，主要内容包括：本发明涉及用于确定样品中的生物分子诸如蛋白质之间的相互作用水平的方法,其包括提供第一和第二携带信息(IC)的寡核苷酸,其中第一和第二IC寡核苷酸共价地或非共价地附接至具有与第一和第二生物分子结合的能力的第一和第二亲和试剂诸如抗体,其中第一和第二IC寡核苷酸各自包含与另一个寡核苷酸的一部分互补的至少一个单链链段,从而在第一和第二IC寡核苷酸中的至少一个中的至少一个单链链段与另一个寡核苷酸的其互补部分杂交后,能够在单细胞水平或单分子水平上测量样品中相互作用的第一和第二生物分子与非相互作用的第一和第二生物分子的相对比例。此外,本发明涉及包含实施本发明的方法必需的寡核苷酸和试剂的试剂盒。(The present invention relates to the methods for determining the interaction level between such as protein of the biomolecule in sample, it includes providing the first and second oligonucleotides for carrying information (IC), wherein the first and second IC oligonucleotides are covalently or non-covalently attached to the first and second affinity reagents such as antibody with the ability in conjunction with the first and second biomolecule, wherein the first and second IC oligonucleotides respectively contain a part of at least one complementary single-stranded segment with another oligonucleotides, to after the single-stranded segment of at least one of at least one of first and second IC oligonucleotides hybridizes with its complementary portion of another oligonucleotides, the phase of the first and second biomolecule and non-interacting first and second biomolecule that interact in sample can be measured on individual cell level or single molecules level Comparative example.In addition, the present invention relates to the kits comprising implementing oligonucleotides and reagent necessary to method of the invention.)

1. the method for determining the interaction level between the biomolecule such as protein in sample, the method includes The oligonucleotides of the oligonucleotides that first carries information (IC) and the second carrying information (IC) is provided, wherein the first IC widow core Thuja acid and the 2nd IC oligonucleotides, which are covalently or non-covalently attached to, to be had and the first biomolecule and the second biology point The first affinity reagent and the second affinity reagent for the ability that son combines, first affinity reagent and second affinity reagent are all Such as antibody, wherein the first IC oligonucleotides and the 2nd IC oligonucleotides respectively contain one with another oligonucleotides The single-stranded segment of at least one of partial complementarity, thus in the first IC oligonucleotides and the 2nd IC oligonucleotides extremely It, can be unicellular after at least one described single-stranded segment in one few hybridizes with its complementary portion of another oligonucleotides Measured on horizontal or single molecules level the first biomolecule to interact in the sample and the second biomolecule and it is non-mutually First biomolecule of effect and the relative scale of the second biomolecule.

2. according to the method described in claim 1, the wherein institute of the first IC oligonucleotides and the 2nd IC oligonucleotides (a) described first IC oligonucleotides and/or the 2nd IC oligonucleotides can be facilitated and receive information (IR) by stating single-stranded segment Oligonucleotides between interaction, (b) described first IC oligonucleotides and/or the 2nd IC oligonucleotides and activation it is few Interaction between nucleotide or (c) the direct phase between the first IC oligonucleotides and the 2nd IC oligonucleotides Interaction.

3. method according to claim 1 or 2 the described method comprises the following steps:

A. the DNA molecular of single-stranded reception information (IR) is provided, wherein the IR DNA molecular is cricoid or linear, carrying At least first cracking motif and the second cracking motif, wherein the cracking motif, which is chosen to cracking motif positions, to be become At double-strand to allow to crack；

B. the first DNA molecular for carrying information (IC) is provided, the described first DNA molecular for carrying information (IC) includes and carries institute The single-stranded segment of a part of complementation of the IR DNA molecular of the first cracking motif is stated, wherein the first IC DNA molecular Occur reflecting the amount of the first biomolecule in the sample；

C. the second DNA molecular for carrying information (IC) is provided, the described second DNA molecular for carrying information (IC) includes and carries institute The single-stranded segment of a part of complementation of the IR DNA molecular of the second cracking motif is stated, wherein the second IC DNA molecular Occur reflecting the amount of the second biomolecule in the sample；

D. the DNA molecular of step a- step c is mixed under conditions of allowing the combination of complementary single-stranded segment；

E. addition digestive ferment at the cracking motif positions for having changed into double-strand to create notch, to be formed

I. the first reporter label binding site, the first reporter label binding site will allow the first reporter label The combination of DNA molecular or sequence, the first reporter label dna molecule or sequence include and the first IC DNA molecular A part of complementary single-stranded segment, and/or

Ii. the second reporter label binding site, the second reporter label binding site will allow the second reporter label The combination of DNA molecular or sequence, the second reporter label dna molecule or sequence include and the 2nd IC DNA molecular A part of complementary single-stranded segment；

F. reporter label dna sequence is mixed by any one of following alternative solution:

I. the first reporter label dna molecule and the second reporter label dna molecule are added, wherein if existing Notch is created at the first cracking motif positions, then the first reporter label dna molecule is in the first reporter mark It is impregnated in the IR DNA molecular, and/or if is created at the second cracking motif positions at label binding site Notch, then the second reporter label dna molecule is impregnated in the IR at the second reporter label binding site In DNA molecular；Or

Ii. if notch is created at the first cracking motif positions, using archaeal dna polymerase and nucleotide by filling out The vacancy complementary with the first reporter label binding site on the first IC oligonucleotides is filled to report described first It accuses object label dna sequence to mix in the IR DNA molecular, and/or if is created at the second cracking motif positions Notch, then by fill the vacancy complementary with the second reporter label binding site on the 2nd IC oligonucleotides come The second reporter label dna sequence is mixed in the IR DNA molecular；And

Addition connects the ligase of the IR DNA molecular, to provide the IR DNA molecular re-created；

G. the IR DNA molecular re-created of optionally amplification step f；

H. the first reporter label and/or the second reporter label are monitored in the IR DNA molecular re-created In incorporation, as appearance to the first biomolecule described in the sample and/or second biomolecule and/or described The measurement of interaction between first biomolecule and/or second biomolecule.

4. according to the method described in claim 3, wherein cracking motif is selected from uracil or restriction site.

5. the method according to claim 3 or 4, wherein disappearing described in notch for being created at the cracking motif positions Change enzyme to be selected from

I. 3 ' the Nb.Bsr.DI of notch endonuclease for generating notch the 3 ' of double-strand restriction site,

Ii. 5 ' the Nt.BsmAI of notch endonuclease for generating notch the 5 ' of double-strand restriction site, or

Iii. the combination of uracil-DNA glycosylase (UDG) and EndoIV, uracil-DNA glycosylase (UDG) removal Uracil base at double stranded site, the EndoIV remove de- pyrimidine site.

6. the method according to any one of claim 3-5, wherein the monitoring passes through following progress:

A. the detection oligonucleotides of the first label and the detection oligonucleotides of the second label are provided, the detection of first label is few Nucleotide is complementary at least part of the first reporter label dna molecule, and the detection few nucleosides of second label It is sour complementary at least part of the second reporter label dna molecule, and

B. by the detection oligonucleotides of detection oligonucleotides that described first marks and second label be optionally amplified The IR DNA molecular hybridization re-created.

7. the method according to any one of claim 3-6, wherein the linear IR DNA molecular re-created is amplified, And it is separated thereafter through separation method, the separation method such as electrophoresis or chromatography, wherein having various sizes of separation to produce Object indicates the incorporation of different reporter labels.

8. the method according to any one of claim 3-7, wherein the identity of different reporter labels by be sequenced come Monitoring.

9. the method according to any one of claim 3-8, wherein having formed described the IR DNA re-created points In the sample of son such as by microscopy monitor described in the IR DNA molecular that re-creates, or wherein having been formed The IR DNA molecular re-created described in collecting in the sample of the IR DNA molecular re-created is stated, then such as by aobvious Micro- art carries out monomolecular sorting and analysis to the IR DNA molecular re-created.

10. the method according to any one of claim 3-9, the method is for removing abasic site and improving detection Efficiency, wherein the reporter tag molecule be added and the first reporter label and/or it is described second report After announcement object label has been incorporated into the IR, follow the steps below:

I. digestion template is hybridized with the IR DNA molecular, the region around remaining abasic site is made to become double-strand；

Ii. the abasic site is removed by suitable restriction Enzyme digestion double-stranded region, the restriction enzyme is such as EndoIV；

Iii. the area of digestion is filled with the polymerase such as T4 archaeal dna polymerase of the base such as thymidine of suitable addition missing The vacancy in domain,；And the IR DNA molecular is connected to provide the IR DNA re-created points with ligase such as T4 ligase Son.

11. method according to claim 1 or 2,

A. wherein the first IC oligonucleotides and the 2nd IC oligonucleotides include hairpin structure, fluorogen and quencher quilt It is placed in the hairpin structure, wherein the hairpin structure is designed to make each oligonucleotides only one fluorogen can be with Emit light, and wherein each fluorogen has unique signal；

B. wherein the hair clip in the first IC oligonucleotides and the 2nd IC oligonucleotides passes through any in following Kind is destroyed or goes to stablize:

I. the activation oligonucleotides complementary with the first IC oligonucleotides or the 2nd IC oligonucleotides is provided, thus with institute It states the first IC oligonucleotides or the 2nd IC oligonucleotides combines, or

Ii. a chain in the hair clip of degradation one or two IC oligonucleotides, to discharge the first IC few nucleosides The single-stranded segment of DNA in the sour and/or described 2nd IC oligonucleotides,

The first IC oligonucleotides and the 2nd IC oligonucleotides are interacted with each other, fluorogen and sudden is caused It goes out the repositioning of agent；

C. wherein conjugate is described to being used to inquire the proximity between two biomolecule in conjunction with the affinity reagent Conjugate is to the affinity reagent comprising being coupled with the first IC oligonucleotides or the 2nd IC oligonucleotides, wherein being used as The oligonucleotides hybridizes each other and causes glimmering when interacting between first biomolecule and second biomolecule The position of light blob and quencher change as a result, the first fluorophore signature mode will be in first biomolecule and described second It is shown when interacting between biomolecule, and the second fluorophore signature mode will be in first biomolecule and described second It is shown when lacking interaction between biomolecule.

12. method according to any of the preceding claims, wherein first biomolecule and second biology Molecule is protein or polypeptide.

13. method according to any of the preceding claims, wherein the sample is the life of one or more of cells The mixture of object sample or protein.

14. method according to any of the preceding claims resists wherein the first IC DNA molecular is conjugated to first Body molecule, the first antibody molecule are equal to first biomolecule or targeting first biomolecule, and described 2nd IC DNA molecular is conjugated to secondary antibody molecule, and the secondary antibody molecule is equal to second biomolecule or targeting Second biomolecule.

15. method according to any of the preceding claims, the method is for analyzing individual cells or individual molecule Protein-protein interaction.

16. for kit used in the method described in any one of claim 1-15, the kit includes

A. the DNA molecular of single-stranded reception information (IR), the DNA molecular of the single-stranded reception information (IR) carry the first cracking Motif and the second cracking motif；

B. first the DNA molecular of information (IC) and the DNA molecular of the second carrying information (IC) are carried, described first carries information (IC) DNA molecular and described second carries the first biomolecule and the second biology in the DNA molecular reflection sample of information (IC) The amount of molecule, wherein the first IC DNA molecular and the 2nd IC DNA molecular are conjugated to affinity reagent or are provided as having Need the chemical part being conjugated by kit user；

C. optionally enzyme, the enzyme are used to create notch at the cracking motif positions in the IR DNA molecular；

D. the first reporter label dna molecule and the second reporter label dna molecule；

E. the reagent of the IR DNA molecular re-created optionally for amplification；

F. the detection oligonucleotides of the detection oligonucleotides of the optionally first label and the second label；And

G. the DNA molecular and enzyme of abasic site are optionally removed.

17. for kit used in the method described in any one of claim 1-15, the kit includes

A. first the DNA molecular of information (IC) and the DNA molecular of the second carrying information (IC) are carried, described first carries information (IC) it includes hairpin structure that DNA molecular and described second, which carries the DNA molecular of information (IC), and fluorogen and quencher are placed In the hairpin structure, wherein the hairpin structure is designed to make each oligonucleotides only one fluorogen that can emit Light, and wherein each fluorogen has unique signal, to have the first biomolecule and the second biology in reflection sample The ability of the amount of molecule, wherein the first IC DNA molecular and the 2nd IC DNA molecular are conjugated to affinity reagent or are mentioned For for the chemical part to be conjugated by kit user；And

B. oligonucleotides is activated, the activation oligonucleotides and the first IC DNA molecular or the 2nd IC DNA molecular are mutual It mends, to have the ability in conjunction with the first IC DNA molecular or the 2nd IC DNA molecular.

Technical field

The present invention relates to the methods for determining the interaction level between such as protein of the biomolecule in sample. In addition, the present invention relates to the kits for using in the method for the invention.

Technical background

The method for determining protein-protein interaction level is required in the analysis to cell signaling activity 's.For many years, a variety of methods have been developed to promote the analysis.Several such methods are based on genetic constructs, wherein waiting Sortilin matter is merged with reporter molecule, and functional reporter will be reconstructed into interaction, i.e. Protein fragment complementation assay is surveyed Fixed (protein fragment complementation assay).The example of such method is yeast two-hybrid (Y2H) (Fields et al., 1989, Nature 340 (6230): 245-246), the double cross of mammal film (MaMTH) (Petschnigg Et al. 2014, Nat Methods 11 (5): 585-592) and biomolecule fluorescence complementary (BiFC) (Hu et al. 2002, Mol Cell 9(4):789-798).Another common method be usingResonance energy transfer (FRET) is to determine fluorescence The combination of fusion protein changes with associated transmitting.In order to determine the interaction between native protein, there are several bases In the method for the targeting protein of antibody, wherein antibody and functional group are conjugated to assign the separation or detection of protein complex, FRET or ortho position connection measurement (PLA) such as based on co-immunoprecipitation antibody.

Ortho position connection measurement (PLA) (such as WO2009/021031) is based on the few nucleosides being conjugated with referred to as adjacent to probe The antibody of acid, the neighbouring probe connect into ring molecule for the cyclisation oligonucleotides that two are then added and provide template.Only when When a pair of neighbouring probe combines adjacent epitope, it can just allow the creation of cyclic annular reporter molecule.Then on one of probe Oligonucleotides will cause rolling circle amplification (RCA).Single neighbouring probe by for cannot pass through RCA amplification linear molecule connection Template is provided.Therefore, it can be detected only proximate to event such as protein-protein interaction.

In WO2015/118029, the ortho position measurement with detection based on hybridization chain reaction is disclosed.

Summary of the invention

Although PLA and other known ortho position measurement be for detect the sensitive of protein-protein interaction and Selective method, but it is impossible for having much ratios actually to take part in interaction in the pond (pool) of determination protein 's.

The letter of the protein and both free proteins about interaction is provided in terms of two methods described herein Breath.It is a kind of design using DNA amplification oligonucleotides archaeal dna polymerase offer amplification of signal to detect unimolecule, the DNA few nucleosides The information whether to interact about protein has been received in acid, and another design is based only upon DNA hybridization to fluorogen Positioned with quencher so that whether they are dissociated or interacted with each other with different color reporter protein.

Therefore, in general, that the present invention relates to a kind of is mutual between such as protein of the biomolecule in sample for determining The method of exposure level, this method include providing the widow of the oligonucleotides that first carries information (IC) and the second carrying information (IC) Nucleotide, wherein the first IC oligonucleotides and the 2nd IC oligonucleotides, which are covalently or non-covalently attached to, to be had and the first biology The first affinity reagent and the second affinity reagent for the ability that molecule and the second biomolecule combine, first affinity reagent and second Affinity reagent such as antibody, wherein the first IC oligonucleotides and the 2nd IC oligonucleotides respectively contain and another oligonucleotides A part of at least one complementary single-stranded segment (stretch), thus in the first IC oligonucleotides and the 2nd IC oligonucleotides At least one at least one of after single-stranded segment hybridizes with its complementary portion of another oligonucleotides, can be unicellular The first biomolecule to interact in sample and the second biomolecule and non-interaction are measured on horizontal or single molecules level The first biomolecule and the second biomolecule relative scale.

" being covalently or non-covalently attached " means affinity reagent, such as antibody in the present context, by means of any kind of It is chemically combined (as long as can be analyzed in conjunction with strong to the interaction for being enough to allow between biomolecule) and is attached to IC few nucleosides Acid.

" affinity reagent " generally means that antibody or the reagent comprising antibody, although other kinds of molecule can be used, only " affinity reagent " to be somebody's turn to do with the ability in conjunction with biomolecule to be analyzed, and to achieve the object of the present invention." other The molecule of type " can be any kind of biomolecule for example with the ability in conjunction with biomolecule to be analyzed, all Such as nucleic acid molecules, polypeptide or any other type.

" IC probe " or " probe " mean the IC oligonucleotides in conjunction with affinity reagent.

" the proportional amount of measurement " of interaction and non-interacting biomolecule means to measure ties each other The relative quantity with uncombined biomolecule each other is closed, this is free-revving engine of the invention.

" individual cell level or single molecules level " means that biomolecule can be carried out to single cell or single molecule Between interaction and non-interacting measurement, i.e., reading characteristic is improved compared with art methods.

" the single-stranded segment " of first IC oligonucleotides and the 2nd IC oligonucleotides can facilitate (a) the first IC oligonucleotides And/or the 2nd IC oligonucleotides and receive information (IR) oligonucleotides between interaction, (b) the first IC oligonucleotides And/or the 2nd interaction between IC oligonucleotides and activation oligonucleotides or (c) the first IC oligonucleotides and the 2nd IC are few Direct interaction between nucleotide.Single-stranded segment, which must have, is enough to allow the mutual of single-stranded segment and another oligonucleotides Mend the length of partial hybridization.

" activation oligonucleotides " means such oligonucleotides, and IC widow's core can be caused after in conjunction with IC oligonucleotides The reconstruct of the intramolecular hybridization of thuja acid, and to manifest the oligonucleotides segment complementary with another IC oligonucleotides, promote At the hybridization between two different IC oligonucleotides (this will cause the repositioning of fluorogen and quencher).

In order to detect protein and both the non-interacting protein ponds of interaction, the present inventor Develop method in a first aspect, this method can when its reported on individual cell level or single molecules level it is non-interacting Visualize the interaction between a-protein and PROTEIN B while the amount of a-protein or non-interacting PROTEIN B. According to the method for the present invention in this respect, the information for carrying the identity about protein or biomolecule to be analyzed is provided Oligonucleotides (information belongings (IC)).(it can be ring-type to preformed single strand dna (information receives object (IR)) ) hybridize with these information belongings (IC), and in the region that the region i.e. IR molecule that they are double-strands hybridizes with IC molecule It is cut open.After cracking, it will be incorporated into IR molecule with the short oligonucleotide label of IC complementary element.It re-creates now IR molecule can be by RCA (if it is cricoid) or PCR (if it is linear) amplification, and the identity of the label mixed will It is visualized for example, by the hybridization of the detection oligonucleotides of fluorogen label, the incorporation of wherein one or two label will provide About the information of the interaction between protein or biomolecule to be analyzed, and free biomolecule or protein and The biomolecule of interaction or the relative quantity of protein.The method for molecule Boolean (MolBoolean) analysis mentions The unique tools for the relationship for determining the protein of free protein and interaction are supplied, which is logical to signal transduction The necessary condition of road activity mathematical modeling.

Therefore, in a first aspect, the method for the present invention includes the following steps:

A. the DNA molecular of single-stranded reception information (IR) is provided, wherein IR DNA molecular is cricoid or linear, the list The DNA molecular of the reception information (IR) of chain carries at least first cracking motif and the second cracking motif, wherein cracking motif is selected Be selected as so that crack motif positions must become double-strand to allow to crack；

B. provide first carry information (IC) DNA molecular, this first carry information (IC) DNA molecular include and carrying The single-stranded segment of a part of complementation of the IR DNA molecular of first cracking motif, wherein the appearance of the first IC DNA molecular reflects sample The amount of first biomolecule in product；

C. provide second carry information (IC) DNA molecular, this second carry information (IC) DNA molecular include and carrying The single-stranded segment of a part of complementation of the IR DNA molecular of second cracking motif, wherein the appearance of the 2nd IC DNA molecular reflects sample The amount of second biomolecule in product；

D. the DNA molecular of step a- step c is mixed under conditions of allowing the combination of complementary single-stranded segment；

E. addition digestive ferment is to create notch (nick) at the cracking motif positions for having changed into double-strand, to be formed

I. the first reporter label binding site, the first reporter label binding site will allow the first reporter label The combination of DNA molecular or sequence, the first reporter label dna molecule or sequence include a part with the first IC DNA molecular Complementary segment, and/or

Ii. the second reporter label binding site, the second reporter label binding site will allow the second reporter mark The combination of DNA molecular or sequence is signed, the second reporter label dna molecule or sequence include one with the 2nd IC DNA molecular Divide complementary segment；

F. reporter label dna sequence is mixed by any one of following alternative solution:

I. the first reporter label dna molecule and the second reporter label dna molecule are added, wherein if first It cracks and creates notch at motif positions, then the first reporter label dna molecule is incorporated at the first reporter label binding site Enter in IR DNA molecular, and/or if creates notch, the second reporter label dna at the second cracking motif positions Molecule is impregnated in IR DNA molecular at the second reporter label binding site；Or

Ii. if notch is created at the first cracking motif positions, using archaeal dna polymerase and nucleotide by filling out The vacancy complementary with the first reporter label binding site on the first IC oligonucleotides is filled by the first reporter label dna sequence It mixes in IR DNA molecular, and/or if creating notch at the second cracking motif positions, passes through filling and the 2nd IC Second reporter label dna sequence is mixed IR DNA by the vacancy of the second reporter label binding site complementation on oligonucleotides In molecule；And

The ligase of addition connection IR DNA molecular, to provide the IR DNA molecular re-created；

G. the IR DNA molecular of optionally amplification step f re-created；

H. the first reporter label and/or the second reporter label mixing in the IR DNA molecular re-created are monitored Enter, as to the appearance of the first biomolecule and/or the second biomolecule in sample and/or the first biomolecule and/or second The measurement of interaction between biomolecule.

IR DNA molecular can be linear or cricoid.Importantly, IR DNA molecular has when itself and IC DNA points The ability that son is cleaved when combining, and it is needed and IC DNA molecular partial complementarity.

IC DNA molecular must with IR DNA molecular partial complementarity, and including other DNA base, the DNA base Template is provided for insertion of the complementary series into the IR DNA molecular of cracking.

The amount of biomolecule " reflection " mean IC DNA molecular relative quantity or appearance and its then with IR DNA molecular Hybridization/combination, be to occur or relative quantity to the opposite of the biomolecule of the biomolecule and interaction dissociated in sample Measurement.Therefore, the relative quantity of the first IC DNA molecular and the 2nd IC DNA molecular is the first biology point to dissociate in sample respectively The amount of the first biomolecule and the second biomolecule of son and the second biomolecule and interaction or the relative measurement of appearance.

" condition for allowing the combination of complementary single-stranded segment " means the such condition for commonly known as allowing stingent hybridization. Those skilled in the art will know and/or will easily find the suitable condition for being used for practical hybridization reaction.

In the context of the present invention, " cracking motif " mean motif that restriction enzyme of DNA molecular etc. can identify or Short sequence.Then restriction enzyme of identification cracking motif etc. can be with site (i.e. " cracking motif positions ") knot of cracking motif It closes, and under certain conditions, cracks a chain of double chain DNA molecule or the chain " creation notch " to double chain DNA molecule, So that the binding site of short reporter label dna molecule, i.e. " reporter label binding site " are created.

Many possible cracking motifs can be used, as long as they only allow to crack when motif has changed into double-strand.It is excellent The cracking motif of choosing is selected from uracil or restriction site.

In addition, the digestive ferment for creating notch at cracking motif positions is preferably chosen from

I. notch endonuclease (nicking endonuclease), such as Nb.Bsr.DI or Nt.BsmAI, the notch Endonuclease generates notch to the specific chain in double-strand restriction site, or

Ii. the combination of uracil-DNA glycosylase (UDG) and EndoIV, the uracil-DNA glycosylase (UDG) are gone Except the uracil base at double stranded site, which removes de- pyrimidine site.

Other enzymes or enzyme combination are also entirely possible, such as restriction enzyme, the enzyme used in DNA reparation are such as MutY, or the restriction enzyme such as transcriptional activation increment effector nuclease (Talen) of engineering, as long as selected enzyme or enzyme Combination allows to crack a DNA chain at double stranded site.

In one embodiment, the first IC DNA molecular, which is conjugated to, is equal to the first biology of the first biomolecule or targeting The first antibody molecule of molecule, and the 2nd IC DNA molecular is conjugated to and is equal to the second biology point of the second biomolecule or targeting The secondary antibody molecule of son.

Reporter label dna molecule is a part of complementary short dna molecule with IC DNA molecular.

As the optinal plan of reporter label dna molecule is provided, reporter label dna sequence is polymerize by using DNA Enzyme is created with adding nucleotide (respectively referring to step f (i) and step f (ii)).

It therefore, is using archaeal dna polymerase to add nucleotide (with IC by the optional method that sequence information is transferred to IR molecule Nucleotide be template), the vacancy that will be formed and to IR-IC hybrid notch sealing (seal).

Ligase can be for example selected from DNA ligase or any other substitute.

The amplification step of this method for example by the RCA (rolling circle amplification) for cyclic annular IR DNA molecular or can be used for line The PCR (polymerase chain reaction) of property IR DNA molecular or any other common method carry out.Those skilled in the art will Easily know suitable amplification method.

The monitoring step of method of the invention can pass through following progress:

A., the detection oligonucleotides of first label and the detection oligonucleotides of the second label, the detection of first label are provided Oligonucleotides is complementary at least part for the first reporter label dna molecule having been incorporated into IR, and second mark The detection oligonucleotides of note is complementary at least part for the second reporter label dna molecule having been incorporated into IR, and And

B. by the detection oligonucleotides that the detection oligonucleotides of the first label and second mark and the weight being optionally amplified Newly created IR DNA molecular hybridization,

Wherein the marker of the first detection oligonucleotides and the second detection oligonucleotides, which is selected from, has different reading wavelength Fluorogen, the fluorogen (Fig. 3), the enzyme that are combined with quencher (such as the horseradish mistake of colored precipitate thing can be converted a substrate into Oxide enzyme and alkaline phosphatase) and molecule with different quality (quality tab can be recorded by mass spectrograph).Marker is worked as It so can differently select, as long as they include the specific feature that can be measured and recorded.

In one embodiment, the linear IR DNA molecular re-created is amplified, and thereafter through separation side Method separation, the separation method such as electrophoresis, especially gel electrophoresis, or chromatography, wherein having various sizes of separation product to refer to Show the incorporation of different reporter labels.

In another embodiment, the identity of different reporter labels is monitored by being sequenced.

In another embodiment again, such as by aobvious in the sample for having formed the IR DNA molecular re-created Micro- art monitors the IR DNA molecular re-created, or wherein receives in the sample for having formed the IR DNA molecular re-created Collect the IR DNA molecular re-created, monomolecular sorting and analysis are such as then carried out to it by microscopy.

In a further embodiment, oligonucleotides is conjugated to antibody, and wherein oligonucleotides is carried about every kind of antibody The information (information belongings (IC)) of identity.Preformed single stranded DNA ring (information receives object (IR)) is miscellaneous with these conjugates It hands over, and is the region of double-strand at them, i.e., the region that DNA circle hybridizes with antibody-oligonucleotide acid conjugate is cut.It is cracking Afterwards, the short oligonucleotide label complementary with antibody-oligonucleotide acid conjugate will be incorporated into ring, and hereafter the ring is connected.It is existing The detection few nucleosides that the identity for the label that will be expanded, and mixed by RCA in the ring re-created will be marked by fluorogen The hybridization of acid visualizes.If RCA product is generated from the ring for wherein only mixing a label, RCA product will be only at one Wavelength is fluorescence, but if being mixed with two labels, RCA product will be marked by two different fluorogens.

In another embodiment again, abasic site is removed to improve detection efficiency.This can be in reporter label Molecule has been added and the first reporter label and/or the second reporter label have been incorporated into rear progress in IR, and And it follows the steps below:

I. digestion template is hybridized with IR DNA molecular, the region around remaining abasic site is made to become double-strand；

Ii. double-stranded region is digested by suitable restriction enzyme such as EndoIV；

Iii. digestion is filled with the polymerase such as T4 archaeal dna polymerase of the base such as thymidine of suitable addition missing Region vacancy；And IR DNA molecular is connected with ligase such as T4 ligase, to provide the IR DNA re-created points Son.

It removes abasic site and connects ring molecule in this way, will improve for after the incorporation of reporter label The detection efficiency of IR DNA molecular with remaining abasic (depurination or de- pyrimidine) site." detection of raising is imitated Rate " means that more amplified productions for the IR DNA molecular for wherein having been removed abasic site will be generated.

" digestion template " is usually also to have the length for being enough to hybridize with IR DNA molecular after removing abasic site Oligonucleotides.In general, length is about 20-30 nucleotide, but it can change, as long as digestion template and IR DNA points Son hybridizes under conditions of allowing and hybridizing.

The embodiment is particularly useful for the case where motif wherein in IR DNA molecular for cracking is uracil, Wherein thymidine is added in the filling process of vacancy.

After carrying out these steps, the IR DNA molecular re-created that can be optionally amplified is obtained, is hereafter monitored The incorporation of first reporter label and/or the second reporter label.

In second aspect, the present invention relates to a kind of methods, in which:

A. the first IC oligonucleotides and the 2nd IC oligonucleotides include hairpin structure, and fluorogen and quencher are placed in this In hairpin structure, wherein hairpin structure is designed to make each oligonucleotides only one fluorogen that can emit light, and its In each fluorogen have unique signal；

B. the hair clip in the first IC oligonucleotides and the 2nd IC oligonucleotides is destroyed or is gone by any one of following Stablize:

I. the activation oligonucleotides complementary with the first IC oligonucleotides or the 2nd IC oligonucleotides is provided, thus with the first IC Oligonucleotides or the 2nd IC oligonucleotides combine, or

Ii. degrade one or two IC oligonucleotides hair clip in a chain, thus release the first IC oligonucleotides with/ Or the single-stranded segment of the 2nd DNA in IC oligonucleotides,

First IC oligonucleotides and the 2nd IC oligonucleotides are interacted with each other, fluorogen and quencher are caused Repositioning；And

C. conjugate comprising the affinity reagent being coupled with the first IC oligonucleotides or the 2nd IC oligonucleotides is to being used for The proximity between two biomolecule in conjunction with affinity reagent is inquired, wherein as in the first biomolecule and the second biology Between molecule interact when oligonucleotides hybridize each other cause the position of fluorogen and quencher to be changed as a result, the first fluorescence Group's signal mode will be shown when will interact between the first biomolecule and the second biomolecule, and the second fluorophore signature Mode will be shown when will lack interaction between the first biomolecule and the second biomolecule.

" hairpin structure " means a part of oligonucleotides, and wherein the two of sequence segment is complimentary to one another and to each other Hybridization, leaves the segment of non-hybridized sequence therebetween, so that molecule forms hair clip spline structure in the part.

" hair clip of IC oligonucleotides " quilt " destroying or go to stablize " means that oligonucleotides forms being partially destroyed for hair clip, makes Obtain some other structures that hair clip disappears and forms oligonucleotides.

" chain in degradation hair clip " means that at least part of nucleotide sequence structure of hairpin portion disappears.

The single-stranded segment of " release " IC oligonucleotides means complementary portion of the segment being released no longer with such as hairpin structure Divide and combine, and can at least partly hybridize instead with another oligonucleotides or with a part of same oligonucleotides.

" interaction between each other of IC oligonucleotides " means that the single-stranded segment of some part of oligonucleotides hybridizes each other, causes " repositioning " of fluorogen and quencher in IC oligonucleotides and/or " position change ".

" conjugate " means the combination of affinity reagent and IC oligonucleotides, and wherein affinity reagent and IC oligonucleotides be covalently Or it noncovalently combines.Correspondingly, " conjugate to " means respectively two IC oligonucleotides in conjunction with affinity reagent, wherein parent It can be interacted with each other with reagent.

" inquiry proximity " means to monitor the proximity and/or interaction between affinity reagent.

" fluorophore signature mode " means that fluorophore signature (such as one or two fluorophore signature of different wave length) is It is readable, and the signal mode is unique, and IC few nucleosides for certain interaction/structures between IC oligonucleotides Another interaction/structure between acid has another " fluorophore signature mode ".

This aspect is based on two or more oligonucleotides with fluorogen and quencher modification, the fluorogen and quenching Agent is arranged to that each oligonucleotides only one fluorogen is allowed to emit light.These oligonucleotides can be activated, therefore A pair of of antibody of such oligonucleotides is conjugated to close to when combining, oligonucleotides can hybridize each other.Therefore it relocates glimmering Light blob and quencher so that the fluorogen that can previously emit light is quenched now, and manifests and can emit the new of light Fluorogen (is reported protein-protein interaction).

Therefore, it in terms of the two shown, provides a method, this method solve the technologies of the prior art Challenge, and by providing the information of the protein combined about free protein and compound on individual cell level, it provides The reading characteristic improved compared with art methods.For detecting the prior art side of protein-protein interaction Method, such as PLA or Y2H give the information about interaction level, but do not provide about non-interacting protein Horizontal information.Accordingly, it is determined that record interaction level difference whether be the protein by interacting difference Caused by expression, or whether interaction is impossible by the posttranslational modification regulation of such as protein.Pass through inspection The information of both ratios of protein complex that rope is made of about the expression of protein and participation these protein, makes The variation visualization of dissociation constant in unicellular will be it is possible, the variation of dissociation constant reflection is drawn by such as posttranslational modification The conformation change of the protein risen.Method of the invention is beneficial to research and understanding to cell signaling activity, and Thus provide the novel research tool with extensive clinical application.

The first biomolecule and the second biomolecule of method of the invention can be such as protein or polypeptide, although also The biomolecule of other biological molecule such as DNA, RNA, carbohydrate, lipid, antibody or any other type can be monitored.

In general, sample is biological sample, the mixture of such as one or more of cells or protein.Side of the invention Method can be used for many applications.In preferred embodiments, this method be used to analyze for example unicellular, histotomy, Protein-protein interaction in body fluid or protein extract.

In a third aspect, it the present invention relates to for kit used in the method in the first aspect of the present invention, is somebody's turn to do Kit includes

A. the DNA molecular of the DNA molecular of single-stranded reception information (IR), the single-stranded reception information (IR) carries first Crack motif and the second cracking motif；

B. first the DNA molecular of information (IC) and the DNA molecular of the second carrying information (IC) are carried, described first carries letter It ceases the DNA molecular of (IC) and second and carries the first biomolecule and the second biology point in the DNA molecular reflection sample of information (IC) The amount of son, wherein the first IC DNA molecular and the 2nd IC DNA molecular are conjugated to affinity reagent or are provided with to by reagent Box user uses the chemical part for conjugation；

C. optionally enzyme, the enzyme are used to create notch at the cracking motif positions in IR DNA molecular；

D. the first reporter label dna molecule and the second reporter label dna molecule；And

E. the reagent of the IR DNA molecular re-created optionally for amplification；

F. the detection oligonucleotides of the detection oligonucleotides of the optionally first label and the second label；And

G. the DNA molecular and enzyme of abasic site are optionally removed.

" DNA molecular and enzyme of removal abasic site " means digestion template for example as defined above, and as above Suitable restriction enzyme, polymerase and/or the ligase discussed in the embodiment for being related to removing abasic site.

" being used for the chemical part being conjugated " means to prepare IC DNA molecular with chemical part, and the chemical part is in later period rank It can be used to be conjugated with affinity reagent in section.Such " chemical part " can be for example selected from biotin, Streptavidin, parent With element, NHS- ester, maleimide, hydrazone, aldehyde, alkynes, azide, mercaptan, amine or any other suitable chemical part.Skill Art personnel will be appreciated by other possible parts.

In fourth aspect, the present invention relates to for kit used in the method in the second aspect of the present invention, it is somebody's turn to do Kit includes

A. first the DNA molecular of information (IC) and the DNA molecular of the second carrying information (IC) are carried, described first carries letter The DNA molecular of the DNA molecular and the second carrying information (IC) that cease (IC) includes hairpin structure, and fluorogen and quencher are placed in In the hairpin structure, wherein hairpin structure is designed to make each oligonucleotides only one fluorogen that can emit light, and Wherein each fluorogen has unique signal, to have the amount of the first biomolecule and the second biomolecule in reflection sample Ability such as antibody or be provided as having wherein the first IC DNA molecular and the 2nd IC DNA molecular are conjugated to affinity reagent Need the chemical part being conjugated by kit user；And

B. oligonucleotides is activated, the activation oligonucleotides is complementary with the first IC DNA molecular or the 2nd IC DNA molecular, from And there is the ability in conjunction with the first IC DNA molecular or the 2nd IC DNA molecular.

Brief description

Fig. 1: the schematic diagram of oligonucleotides design.IR ring respectively with two IC probes (most upper row), an IC probe (rear two Row) hybridize, IC probe forms (left figure) by the IC oligonucleotides for being conjugated to antibody (indicating with A and B).IR is digested by enzymatic treatment Ring, wherein IR ring hybridizes with IC probe, and tagged oligonucleotides invade IC probe (middle).Reconnect IR ring, and label Sequence is impregnated in (right figure).

Fig. 2: the schematic diagram of the different designs of the enzymatic digestion for IR ring indicates uracil base and restriction site Position.

Fig. 3: it interacts using for detecting beta-catenin, CAM 120/80 and beta-catenin-CAM 120/80 MolBoolean method, to quantifying for the +/- SD of average of the signal from the individual image of three width.Image, which is shown, to be directed to Beta-catenin, CAM 120/80 and beta-catenin-labeled cell of CAM 120/80 interaction.It marks in the picture The boundary of nucleus is gone out.

Fig. 4: the design of three kinds of Alexa dyestuffs and the Invader-MolBoolean of two kinds of Black hole quenchers is used Schematic diagram.(A) when neighbouring probe (antibody (referring to table) for being conjugated to IC oligonucleotides 1 or IC oligonucleotides 2) combines individually Antigen when, a fluorogen of each probe will emit light: Alexa555 (A555) and Alexa647 (A647), and Alexa488 (A488) would be quenched because it is located near quencher (BHQ1).(B) when addition activates sub- oligonucleotides When (activator oligonucleotide), it will invade IC oligonucleotides 1.If neighbouring probe be it is adjacent, open IC oligonucleotides 1 will hybridize with IC oligonucleotides 2, to change the position of fluorogen and quencher.Present A555 will be located at Near BHQ1, and A647 is located near quencher (BHQ3), both fluorogens would be quenched, and A488 is now and quencher BHQ1 is separated and will be emitted light.

Fig. 5: the schematic diagram of vacancy filling process.Firstly, digestion template hybridizes across AP site, ring is allowed to be disappeared by EndoIV Change.Hereafter, the notch (arrow) of creation can be filled by that will mix the T4 archaeal dna polymerase of thymidine.Ligase will make incision Ring sealing so that it is amplified in the next step of the program.

Fig. 6: the protein detection in situ with vacancy filling design.By probe at four kinds under different conditions in HaCat It is incubated on cell: with both anti-CAM 120/80 antibody and anti-beta-catenin antibody, there is anti-CAM 120/80 antibody, tool There is anti-beta-catenin antibody or does not have any Primary antibodies (background).All conditions shown are by subtracting background condition To normalize.Error bar represents the standard error (SEM) of average value.

Detailed description of the invention

The present inventor has developed mutual between such as protein of the biomolecule in sample for determining The conventional method of exposure level, this method include providing the oligonucleotides and second that first carries information (IC) to carry information (IC) Oligonucleotides, wherein the first IC oligonucleotides and the 2nd IC oligonucleotides be covalently or non-covalently attached to have and first The first affinity reagent and the second affinity reagent for the ability that biomolecule and the second biomolecule combine, first affinity reagent and Second affinity reagent such as antibody, wherein the first IC oligonucleotides and the 2nd IC oligonucleotides respectively contain and another few nucleosides At least one single-stranded segment of a part of complementation of acid, thus in the first IC oligonucleotides and the 2nd IC oligonucleotides at least After at least one of one single-stranded segment hybridizes with its complementary portion of another oligonucleotides, can in individual cell level or The first biomolecule and the second biomolecule and non-interacting first to interact in sample is measured on single molecules level The relative scale of biomolecule and the second biomolecule.

This method will be illustrated by two kinds of selectable methods in the present specification；A kind of selectable method is few using IC Hairpin structure and the combination of fluorogen/quencher technology in nucleotide, hairpin structure are designed to make each IC few nucleosides Sour only one fluorogen can emit light, and another selectable method is other than IC oligonucleotides, using carrying extremely IR (receiving information) oligonucleotides of few two cracking motif positions, the cracking motif positions must become double-strand so as to Allow to crack.By using any one of these methods, can be measured in sample on individual cell level or single molecules level The proportional amount of measured value of the biomolecule of interaction and non-interacting biomolecule.

Therefore, for the one aspect in the various aspects of method of the invention, the present inventor is devised by single-stranded The oligonucleotides system of ring-shaped DNA molecule (information receives object (IR)) composition, (information receives object to the single stranded circle DNA molecular (IR)) motif for cracking, such as uracil or restriction site are carried.Oligonucleotides system is created so that cracking will It is required that site is double-strand, which only will be double-strand when combining complementary oligonucleotide.These complementary oligonucleotide (information Belongings (IC)) it is designed to make them to be made of hairpin structure, which is will be complementary with IR ring single-stranded The segment of DNA.Notch of the creation in IR ring is beneficial to then adding few with IC probe a part of complementary short label Nucleotide invades the hair clip of IC oligonucleotides and is connected in IR ring.Selectively, archaeal dna polymerase can be used, with from IC The template of probe carries out vacancy filling.In order to seal and re-create ring-shaped DNA molecule for vacancy, DNA ligase is added.IC The unique label and hair clip of oligonucleotides can be used to for information to be transferred to the IR ring for having been combined IC oligonucleotides.When IR ring When in conjunction with two different IC oligonucleotides, two kinds of corresponding labels will be mixed.Such as in one embodiment, by the way that IC is few Nucleotide is conjugated to antibody, and the present inventor has created a kind of method, and wherein IR ring can be used to monitor two kinds of protein Whether interact, i.e., whether two labels are incorporated into IR ring.The protein for being intended to research for people selects antibody.It is right In noninteracting protein, only one label will be incorporated into IR ring (Fig. 1).

It tests for cutting ring and integrating the distinct methods of label.IR ring can be cut by notch endonuclease, should Notch endonuclease only cracks a chain in its double-strand target.Selectively, pass through the help in enzyme combination UNG and EndoIV Lower removal urine pyrimidine alkali base can create notch.For notch endonuclease, the present inventor tests two different enzymes: will The Nb.BsrDI (3 ' Nb.BsrDI) cut in 3 ' sides of hair clip the and Nt.BsmAI (5 ' Nt.BsmAI) cut in 5 ' lateral incisions. The present inventor is also surveyed by mixing uracil base relative to 3 ' sides of hair clip or 5 ' sides (3 ' uracils and 5 ' uracils) Try lysis efficiency.(Fig. 2).With the help of Nupack.org, the present inventor designs and analyzes all oligonucleotide sequences To ensure correct secondary structure and hybridization (table 1).

Then, the IR ring of the label comprising incorporation re-created can be by rolling circle amplification (RCA) from one of IC probe Cause to expand.RCA product includes the repetition of the IR ring of hundreds of complementations.Then the identity of RCA product can by with wherein The hybridization of the detection oligonucleotides of the label of the partial complementarity of the RCA product of label has been mixed to decode.Depending on that will use Which kind of reads, and the label for detecting oligonucleotides can be for example different fluorogens, enzyme or the molecule with different quality.Dan Biao The RCA product of note will only mix a label as a result, and the RCA product of double labelling will be the result for mixing two labels.

As a result it can seem that example how is shown in FIG. 3.Here, IC oligonucleotides is conjugated the present inventor's use It is designed to 5 ' uracils of anti-mouse antibody and anti-rabbit antibody.These IC probes with targeting CAM 120/80 mouse antibodies and It is tested in the MCF10 cell line of the rabbit antibody label of targeting beta-catenin.Answering comprising beta-catenin and CAM 120/80 The RCA product that object detects generation with both Cy3 and FITC is closed, and non-interacting protein is provided with Cy3 or FITC The RCA product of label.The different identity of RCA product is determined using software CellProfiler, and different types of RCA product It is pseudo-colours so that interaction and individual protein (beta-catenin between beta-catenin and CAM 120/80 And CAM 120/80) visualization.

The selectable method for extracting information is using the proximity between IR ring or linear IR molecule inquiry IC probe.In After connecting sequence label, the IR molecule re-created can be separated by PCR amplification and by gel electrophoresis.Size difference will Indicate the incorporation of different labels.Expand single IR molecule and the identity by decoding different labels to the sequencing of amplicon It will be possible.

MolBoolean analysis can be in cell, in the mixture of protein, (protein be in batch mixed object In (bulk mixture), or for example separated by gel electrophoresis) in carry out.If readout platform supports Single Molecule Detection, can Directly to inquire that (such as by microscopy, as shown in Figure 3) is created again in the sample for forming the IR molecule re-created The IR molecule built, or the IR molecule re-created can be collected and then can individually unimolecule be sorted and be divided Analysis.

In some cases, remaining abasic site (AP site) can interfere amplification and/or detection effect in IR molecule Rate.As the means for improving detection efficiency and in order to reduce indigested abasic site (AP of cyclic annular IR molecule Point) prevent signal detection risk, the present inventor has modified the design (Fig. 5) with alternate design version.In order to remove dealkalize Base site is added to two steps after the step of mixing label.In first other step, template and DNA circle are digested Hybridization makes the region around AP site become double-strand.Hereafter abasic site is removed by EndoIV digestion.In next step In rapid, vacancy filling is carried out with T4 archaeal dna polymerase to add the thymidine of missing, and makes notch by connecting with T4 ligase Closure.

Vacancy filling design assesses (figure using previously described CAM 120/80 and beta-catenin interaction measurement 6).Detect the amount of CAM 120/80 and beta-catenin interaction and considerably higher total CAM 120/80, total E- calcium The amount of mucoprotein is equal to there is only the total amounts of the CAM 120/80 when antibody.Similarly, in two conditions using the antibody Under, the total amount of beta-catenin is maintained at comparable level.

For other aspects of method of the invention, inventor developed a kind of selectable methods to monitor free egg Both white matter and the protein of interaction.In the method, the IC oligonucleotides connecting with antibody can be designed to Hairpin structure.By the way that fluorogen and quencher are attached to such hair clip, only when fluorogen and quencher separate a spacing From when, will allow Fluorophore emission light.The positioning of fluorogen and quencher is beneficial to allow each such IC oligonucleotides Only one Fluorophore emission light.Once IC oligonucleotides under the guidance of antibody in connection in conjunction with their target, one Kind hairpin structure will be by activating the intrusion of sub- oligonucleotides be gone to stablize.This will change the conformation of IC oligonucleotides, and The segment for the DNA being previously concealed in stem, the segment reverse complemental of the segment of the DNA and the 2nd IC oligonucleotides will be manifested. Only when the first IC oligonucleotides and the 2nd IC oligonucleotides that go stable/activation are bound to adjacent target molecule, go to stablize / the first IC oligonucleotides of activation will be with the 2nd IC oligonucleotide hybridization, this will be such that fluorogen and quencher relocates, and make Previous transmission light fluorogen be quenched now and a fluorogen being quenched in one of IC oligonucleotides with it is sudden Agent of going out separation.Only when a pair of of IC oligonucleotides hybridizes each other, which will emit light.Therefore, free protein and The fluorescence of the protein of interaction can be recorded simultaneously in different wavelength.Referred to herein as Invader- This method of MolBoolean describes in Fig. 4.With the help of Nupack.org, the present inventor designs and analyzes all Oligonucleotide sequence is to ensure correct secondary structure and hybridization (table 1).

The present invention is described referring now to following non-limiting embodiment.

Embodiment