WO2022155986A1 - 一种基于共价连接的已知分子与蛋白质相互作用检测系统及其鉴定或验证方法 - Google Patents

一种基于共价连接的已知分子与蛋白质相互作用检测系统及其鉴定或验证方法 Download PDF

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WO2022155986A1
WO2022155986A1 PCT/CN2021/073899 CN2021073899W WO2022155986A1 WO 2022155986 A1 WO2022155986 A1 WO 2022155986A1 CN 2021073899 W CN2021073899 W CN 2021073899W WO 2022155986 A1 WO2022155986 A1 WO 2022155986A1
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protein
molecule
pup
dna
proteins
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French (fr)
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陶生策
江何伟
郑云萧
陈红
王雪宁
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上海交通大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/36—Post-translational modifications [PTMs] in chemical analysis of biological material addition of addition of other proteins or peptides, e.g. SUMOylation, ubiquitination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • the invention belongs to the technical field of molecular biology, and relates to a known molecule-protein interaction detection system, in particular to a known molecule-protein interaction detection system based on covalent connection and an identification or verification method thereof.
  • Protein is the executor of life activities. More than 80% of proteins function by interacting with other molecules in a wide range of life processes including embryonic development, cell communication, receptor-ligand binding, signaling, and more. Disordered, uncontrolled protein-molecular interactions may trigger cancer, neurodegenerative diseases, etc. (kesin et al., 2016. Chem. Rev., 116, 4884-4909).
  • the interaction between small molecules or small molecule drugs and proteins in the physiological process is widely studied in biomedicine and clinical applications, which helps to further understand the physiological and metabolic processes of the body and guide the design and synthesis of drugs. Discovering and verifying the interaction of proteins with other molecules such as proteins, DNA, RNA, and small molecules is of great significance for revealing the inherent laws of life activities at the molecular level.
  • Immunoprecipitation methods include protein co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP) ) (Das PM et al., 2017. Biotechniques. 37(6): 961-9.), RNA immunoprecipitation (RNA Immunoprecipitation) (Gagliardi M et al. Methods Mol Biol. 2016; 1480:73-86.) , small molecule affinity chromatography (Sleno et al. 2008, Curr Opin Chem Biol, 12, 46-54) (Sato, et al., 2010, Chem Biol, 17, 616-623) and the like.
  • ChIP and CLIP can be used to identify the interaction between DNA and RNA and proteins, respectively.
  • cross-linking and immobilization of DNA or RNA and protein complexes such as formaldehyde can be used.
  • ChIP can enrich target protein and DNA complexes through antibodies.
  • CLIP can bind to specific RNAs and identify interacting proteins by mass spectrometry.
  • Co-IP enriches target proteins and their interacting proteins by antibodies, relying on non-covalent interactions between proteins.
  • Pull down technologies such as GST Pull Down, RNA pull down, small molecule affinity chromatography, etc., enrich target molecules by tags attached to proteins, RNAs or small molecules to obtain interacting proteins.
  • Chip technology has the advantages of high throughput, less sample consumption, and short reaction time. It can globally discover molecules that interact with target molecules in one experiment, and is an efficient tool for molecular/protein interaction research. However, this method also has certain limitations.
  • the signal detected by the chip is the result of the in vitro interaction of molecules. Considering the complexity and diversity of the in vivo environment, there will inevitably be false positives, which is also common in in vitro screening methods. existing problems.
  • Proximity tagging systems that have emerged in recent years can be used to identify interacting proteins of various molecules, such as BioID (proximity dependent biotin identification), APEX (engineered ascorbate peroxidase), PUP-IT (pupylation-based interaction tagging) (Liu et al., 2018. Nat.
  • Methods, 15, 715-722. can identify interacting proteins of known proteins, CasID (Schmidtmann et al., 2016, Nucleus, 7, 476-484) and CASPEX (Myers et al., 2018, Nat Methods, 15, 437-439) can identify known DNA-protein interactions, CRIUS (Ziheng Zhang et al., 2020, Nucleic Acids Res, 1) can identify known RNA-interacting proteins, etc.
  • BioID the enzyme with adjacent labeling function and the bait protein are fused and expressed in the cell, and a labeling molecule (such as biotin) is added to the cell culture medium, and the protein adjacent/interacting with the bait protein is covalently linked.
  • CasID, CASPEX and CRIUS combine dCas9 or dCas13a proteins with existing proximity labeling systems to enable the identification of known DNA or RNA interacting proteins.
  • PafA and dCas13a are fused and expressed, and localized to the target RNA under the action of sgRNA, and then PafA can label the biotin-labeled pup polypeptide on the RNA-binding protein; the disadvantage of this method is that it is highly dependent on sgRNA. targeting efficiency.
  • sgRNAs need to be designed for different target RNAs.
  • different sgRNA targeting efficiencies may easily lead to differences in the amount of PafA proteins that are targeted to bind, introducing systematic errors.
  • Proximity labeling systems can convert non-covalent molecule-protein interactions into covalent linkages between proteins and labeled molecules, enabling the capture of weak and transient interactions in real cellular environments.
  • these methods require the fusion and expression of the enzyme with the bait protein or dCas protein (dead Cas proteins).
  • the larger molecular weight of the enzyme may affect the original structure of the bait protein, or affect the interaction between the bait protein and other proteins due to steric hindrance; Moreover, the above proximity labeling system can only work in cells, and cannot be applied to primary cells and most passaged cells, so the above methods are not applicable to many proteins.
  • SPR Surface Plasmon Resonance
  • BLI Bio-Layer Interferometry
  • ITC isothermal titration calorimetry
  • DNA/RNA-protein complex has larger molecular mass and slower migration rate under the action of electric field.
  • the disadvantage is that it depends on the instrument, the operation is relatively complicated, and it is difficult to detect weak interactions.
  • the present invention proposes a system based on covalent connection.
  • the core of the system is the fusion and expression of four short peptides with streptavidin. It is known that the molecule is modified with biotin and then bound to streptavidin through the proximity effect of PafA enzyme.
  • the covalent attachment of short peptides to capture proteins can be used for detection and verification of interactions between proteins and various molecules such as proteins, DNA, RNA, and small molecules.
  • the purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a known molecule-protein interaction detection system based on covalent linkage and a method for identification or verification thereof.
  • the detection system can be applied to the discovery and verification of the interaction between known molecules and proteins.
  • the detection of weak interactions and transient interactions can be realized, which is expected to greatly improve the relationship between known molecules and proteins. Sensitivity, specificity, and success rate of protein interaction detection.
  • the method provided by the present invention utilizes biotin to label known molecules. After the known molecules interact with other proteins, the known molecules are efficiently captured by streptavidin coupled with Pup under mild conditions. Molecular interacting proteins. Then, under the catalysis of the adjacent labeling activity of the PafA-Pup system, the Pup is covalently linked to the interacting protein, so that the non-covalent binding between the known molecule and the interacting protein can be converted into streptavidin and the interacting protein. As a covalent link between proteins, extremely harsh conditions can be used for subsequent washing, so that the specificity can be significantly improved on the premise of ensuring sensitivity.
  • the method of the invention can realize the capture and detection of weak interaction and transient interaction on the basis of maintaining the original structure and activity of known molecules.
  • the present invention provides a known molecule-protein interaction detection system based on covalent linkage, and the detection system includes the following molecules:
  • Streptavidin-short peptide tetramer which is a fusion of streptavidin tetramer to express four short peptides, of which streptavidin can bind to biotin and biotin mediators, and short peptides can be catalyzed by PafA Covalently attached to adjacent proteins;
  • PafA enzymes which can covalently link specific short peptides to adjacent proteins.
  • the short peptide is a peptide containing 12-100 amino acids. Peptides less than 12 amino acids in length are generally not functional.
  • the short peptide includes a Pup molecule or a mutant molecule thereof, and the glutamine at the end of the Pup molecule is mutated to glutamic acid, and its sequence is shown in SEQ ID NO. 1; the reason is: in Mycobacterium tuberculosis In the ubiquitin-like proteasome system, Dop catalyzes the deamidation reaction of glutamine at the end of Pup to form glutamate, and PafA catalyzes the ligation reaction between Pup(E) and the substrate;
  • the mutant molecule of Pup is a Pup molecule with one or more mutations, and the sequence is shown in any one of SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.
  • the Pup molecule is derived from any one of Mycobacterium, Corynebacterium, Streptomyces, Cockerella, and Micrococcus, but is not limited thereto.
  • Pup molecules are derived from Mycobacterium tuberculosis, Mycobacterium smegmatis, Corynebacterium glutamicum, Bacillus leprae, Actinobacillus erythromycin, Corynebacterium diphtheriae, Streptomyces coelicolor, Corynebacterium rhizogenes, Garcinia cambogia Micrococcus etc.
  • the lysine on the surface of the streptavidin is mutated to arginine.
  • the streptavidin-short peptide tetramer is a streptavidin-Pup tetramer, and its amino acid sequence is shown in SEQ ID NO.5.
  • 7 lysines on the surface of the PafA enzyme are mutated to arginine, and the mutation sites are K162R, K202R, K320R, K361R, K423R, K435R and K446R, and the surface of the mutated PafA enzyme does not contain lysine, Non-specific covalent attachment of Pup molecules is avoided.
  • the mutated PafA enzyme sequence is shown in SEQ ID NO.7.
  • the PafA enzyme is derived from any one of Mycobacterium, Corynebacterium, Streptomyces, Cockerella, and Micrococcus, but is not limited thereto.
  • PafA enzymes are derived from Mycobacterium tuberculosis, Mycobacterium smegmatis, Corynebacterium glutamicum, Bacillus leprae, Actinobacillus erythromycin, Corynebacterium diphtheriae, Streptomyces coelicolor, Corynebacterium rhizogenes, Garcinia cambogia Micrococcus etc.
  • the known biotin-modified molecules include any one or more of proteins, DNA, RNA, and small molecules.
  • the protein includes at least one of proteins, peptides, modified peptides, antibodies, and lectins, which can be combined with streptavidin-short peptides;
  • the RNA includes at least one of messenger RNA, ribosomal RNA, long-chain non-coding RNA, and non-coding small RNA, which can be combined with streptavidin-short peptide;
  • the DNA includes at least one of double-stranded DNA and closed-circle DNA, which can be combined with streptavidin-short peptide;
  • the small molecules include at least one of biologically active oligonucleotides, amino acids, vitamins, secondary metabolites of animal and plant microorganisms, and chemically synthesized small molecules in the organism, which can be combined with streptavidin-short Peptide binding.
  • the small molecule is 50-1500 Da in size.
  • the biotin-modified site of the protein, DNA, and RNA is N-terminal, C-terminal or any other site, and the biotin-modified site of the small molecule is an optional non-critical active site.
  • the present invention also provides a method for identifying the interaction of a known molecule with a protein according to the aforementioned detection system, comprising the following steps:
  • step B Add streptavidin-short peptide tetramer to the mixture treated in step A, mix well and incubate at 25°C-35°C for 0-1h;
  • step C Add PafA enzyme to the mixture treated in step B, mix well and incubate at 25°C-35°C for 1min-6h;
  • step D Add biotin-labeled affinity medium to the mixture treated in step C to separate streptavidin-short peptide and its linked protein;
  • the sample to be tested includes at least one of proteins, living cells or tissues, membrane proteins, cell lysates, and tissue lysates.
  • the biotin-labeled affinity medium includes biotin magnetic beads and biotin agarose beads, but is not limited thereto.
  • the method is used to detect the interaction between known molecules and proteins in the sample to be tested.
  • the present invention also provides a method for verifying the interaction between known molecules and proteins according to the aforementioned detection system, comprising the following steps:
  • step S2 Add streptavidin-short peptide tetramer to the mixture treated in step S1, mix well and incubate at 25°C-35°C for 0-1h;
  • step S3 Add PafA enzyme to the mixture treated in step S2, mix well and incubate at 25°C-35°C for 1min-6h;
  • the known molecule to be verified is a known molecule modified with biotin, including any one or more of protein, DNA, RNA, and small molecules;
  • the protein includes at least one of proteins, peptides, modified peptides, antibodies, and lectins;
  • the RNA includes at least one of messenger RNA, ribosomal RNA, long non-coding RNA, and small non-coding RNA;
  • the DNA includes at least one of double-stranded DNA and closed-circle DNA;
  • the small molecule includes at least one of biologically active oligonucleotides, amino acids, vitamins, secondary metabolites of animal, plant and microorganisms and chemically synthesized small molecules in the organism.
  • the present invention has the following beneficial effects:
  • the known molecule-protein interaction detection system based on covalent linkage proposed by the present invention utilizes the strong affinity between biotin and streptavidin to realize the adjacent covalent linkage between known molecules and Pup molecules.
  • the known molecule is protein, DNA or RNA
  • the present invention avoids modified enzyme, bait protein or dCas protein to a certain extent.
  • the steric hindrance between dCas proteins can maintain the structure and activity of the modified enzyme and the bait protein, and detect protein interactions more accurately.
  • the known molecule-protein interaction detection system converts the non-covalent interaction between known molecules and proteins into Pup molecules through the covalent interaction between lysine and the captured protein, which can realize weak interaction and transient interaction capture with high sensitivity and low false positives.
  • the known molecule-protein interaction detection system provided by the present invention is applied to the verification of the known molecule-protein interaction, and immunoblotting analysis can be performed by co-coagulation of the interacting protein with the streptavidin-short peptide tetramer.
  • the results can be read from the apparent migration on the gel caused by the valence coupling, which is simple and does not depend on expensive instrumentation.
  • FIG. 1 is a schematic diagram of the application of the present invention in the discovery of interacting proteins; wherein SA is a streptavidin tetramer, Pup is a short peptide, PafA 7KR is a mutated PafA enzyme, Bait is the protein to be studied, and Prey is The captured interacting proteins, Biotin agarose are biotinylated agarose beads;
  • FIG. 2 is a schematic diagram of the application of the present invention in the verification of interacting proteins; wherein A and B are the interacting proteins to be verified, and protein A is a biotin-modified protein;
  • Figure 3 shows the self-ligation of GFP-Pup(E) under the enzymatic action of PafA
  • Figure 4a is a schematic diagram of the mutation site and amino acid sequence of SA m -Pup E
  • Figure 4b is the detection of biotin-binding activity of SA m -Pup E
  • Figure 4c is SA m -Pup E binding to biotin agarose beads in high salt buffer Stability testing in liquid;
  • Figure 5a is a schematic diagram of the mutation site and amino acid sequence of PafA 7KR enzyme
  • Figure 5b is the detection of the Pupylation of PafA 7KR enzyme to itself
  • Figure 5c is the detection of the ability of PafA 7KR enzyme to Pupylation of substrates
  • Figure 6a shows the interaction results between CheZ and CheAs (wild type) at different concentrations
  • Figure 6b shows the interaction results between CheZ and different mutant CheAs (WT, L126A, L123A)
  • Figure 6c shows the CheAs (wild type) detected by mass spectrometry type) covalently linking site to SA m -Pup E ;
  • Fig. 7a is a schematic diagram of detecting CobB-interacting proteins
  • Fig. 7b is a flow chart of detecting CobB-interacting proteins
  • Fig. 7c is a comparison between the CobB-interacting proteins obtained by this method and the existing results
  • Figure 8a shows the purification results of CobB and some interacting proteins
  • Figure 8b ⁇ f shows the interaction between the captured protein and CobB detected by BLI
  • Figure 8g ⁇ h shows the deacetylation function of CobB interacting with VacB and DksA;
  • Figure 9a is a schematic diagram of detecting the cell surface receptor of PD-1 protein
  • Figure 9b is a flow chart of detecting the cell surface receptor of PD-1 protein
  • Figure 9c is the verification of PD-1 protein and its cell surface receptor PD-L1 Interaction;
  • Figure 10a is a flow chart for detecting the interacting proteins of SARS-CoV-2 proteins
  • Figure 10b is a comparison between the interacting proteins of SARS-CoV-2 proteins obtained by this method and the existing results
  • Figure 10c is the verification of SARS-CoV-2 Interaction of protein ORF9b with human protein TOM70.
  • RNA is biotinylated RNA
  • Prey is the captured interacting protein
  • Biotin agarose is biotinylated agarose beads
  • RNA is biotinylated RNA
  • Prey is the captured interacting protein
  • Figure 13 shows the results of verifying the interaction of m6A with YTDHF1, YTDHF2, and YTDHF3;
  • Figure 14 is a schematic diagram of the application of the present invention in the discovery of DNA-protein interaction; wherein SA is a streptavidin tetramer, Pup is a short peptide, PafA 7KR is a mutated PafA enzyme, and DNA is biotinylated DNA, Prey is the captured interacting protein, and Biotin agarose is biotinylated agarose beads;
  • Figure 15 is a schematic diagram of the application of the present invention in the verification of the interaction between DNA and protein; wherein DNA is biotinylated DNA, and Prey is the captured interacting protein;
  • Figure 16 is the result of verifying the interaction between different DNA systems and EthR
  • Fig. 17 is to verify the interaction result of different DNA fragments and RutR;
  • Figure 18 is the result of verifying the interaction between different DNA systems and GCN4;
  • Figure 19 is a schematic diagram of the application of the present invention in the discovery of small molecule-protein interactions; wherein SA is a streptavidin tetramer, Pup is a short peptide, PafA 7KR is a mutated PafA enzyme, and SM is a small molecule to be studied.
  • SA is a streptavidin tetramer
  • Pup is a short peptide
  • PafA 7KR is a mutated PafA enzyme
  • SM small molecule to be studied.
  • Prey is the captured interacting protein
  • Biotin agarose is a biotinylated agarose bead;
  • Figure 20 is a schematic diagram of the application of the present invention in the verification of interacting small molecules and proteins; wherein A is a biotin-modified small molecule, and B is a protein to be verified;
  • Figure 21 shows the interaction between different small molecules and proteins, among which, Figure 21a shows the interaction results of different concentrations of small molecule C-di-GMP and ETHR, and Figure 21b shows the validation of small molecule C-di-GMP and CSP series of short peptides The result of the interaction, Figure 21c is the result of verification of the interaction between the small molecule Rapamycin and FKBP12;
  • SPIDER shown in each figure represents the abbreviation of the detection system of the present invention.
  • the schematic diagram for discovering protein interactions is shown in Figure 1.
  • the biotin-labeled bait protein is bound to the streptavidin-Pup tetramer (SA-Pup), and when the protein in the system interacts with the bait protein, the free PafA 7KR enzyme covalently links the C-terminus of Pup to the bait protein. on interacting proteins.
  • SA-Pup streptavidin-Pup tetramer
  • the SA-Pup covalently linked prey protein, which is the interacting protein of the bait protein, was enriched with biotin-labeled affinity media.
  • FIG. 1 A schematic diagram for verifying protein interactions is shown in Figure 2.
  • Biotinylated protein A was bound to SA-Pup, and when protein B interacted with protein A, the free PafA 7KR enzyme in the system covalently linked the C-terminus of Pup to protein B.
  • RNA interacting proteins The schematic diagram of discovering the interaction between RNA and protein is shown in Figure 11.
  • Biotin-labeled RNA is bound to streptavidin-Pup tetramer (SA-Pup), and when the protein in the system interacts with RNA, the free PafA 7KR enzyme covalently links the C-terminus of Pup to the interacting on the protein.
  • the capture proteins covalently linked to SA-Pup were enriched using biotin-labeled affinity mediators, that is, RNA interacting proteins.
  • FIG. 14 The schematic diagram for the discovery of DNA-protein interactions is shown in Figure 14.
  • Biotin-labeled DNA is bound to streptavidin-Pup tetramer (SA-Pup), and when the protein in the system interacts with DNA, the free PafA 7KR enzyme covalently links the C-terminus of Pup to the interacting on the protein.
  • Biotin-agarose beads were used to enrich the SA-Pup covalently linked capture proteins, which are DNA-interacting proteins.
  • FIG. 7 A schematic diagram of the discovery of small molecule-protein interactions is shown in Figure 19.
  • the biotin-labeled bait molecule is bound to streptavidin-Pup tetramer (SA-Pup).
  • SA-Pup streptavidin-Pup tetramer
  • the free PafA 7KR enzyme covalently binds the C-terminus of Pup. linked to interacting proteins.
  • the SA-Pup covalently linked capture protein was enriched with biotin-labeled affinity mediator, which is the interacting protein of the bait small molecule.
  • E.coli BL31 (DE3) strain was purchased from Quanshijin Biotechnology Co., Ltd.
  • HEK293T cells were purchased from the Chinese Academy of Sciences Cell Bank
  • pET28a vector, pTrc99a vector, pET32a vector are commonly used laboratory plasmids
  • biotin agar Sugar beads were purchased from Sigma-Aldrich Company
  • nickel columns were purchased from Zhongke Senhui Microsphere Technology Co., Ltd.
  • Annealing Buffer (5X) was purchased from Biyuntian Biotechnology Co., Ltd.
  • biotinylated C-di-GMP was purchased from Biolog Company
  • the biotin-modified lenalidomide small molecule was presented by Dong Jiajia, a teacher from the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, and the biotinylated Rapamycin was presented by Dang Yongjun, a teacher at the School of Basic Medicine, Fudan University.
  • Pup(E) refers to the mutation of glutamine (Q) at the C-terminus of wild-type Pup molecule (sequence shown in SEQ NO. 1) to glutamic acid (E). Pup(E) was fused and expressed at the N-terminus and C-terminus of GFP protein, respectively, GFP-Pup(E) and Pup(E)-GFP were constructed on pET28a and transformed into E.coli BL21(DE3) strain, wherein A 6xHis tag was attached to the end without Pup(E). Cultivate 1 L of bacterial solution, add IPTG when OD 600 ⁇ 0.6, induce overnight at 18°C, and purify with nickel column to obtain GFP-Pup(E) and Pup(E)-GFP proteins.
  • PafA is connected to pTrc99a vector and transformed into E.coli BL21(DE3) strain, wherein PafA C-terminal is connected with a 6 ⁇ His tag.
  • Cultivate 1 L of bacterial solution when OD 600 ⁇ 0.6, add IPTG, induce overnight at 18°C, and purify with nickel column to obtain PafA enzyme.
  • the protein concentration ratio is as follows: GFP-Pup(E) or Pup(E)-GFP (10 ⁇ M), PafA (0.5 ⁇ M), ATP (5mM), insufficient volume use reaction buffer (50mM Tris, PH 7.5, 100 mM NaCl, 20 mM MgCl 2 , 10% (v/v) glycerol) were added, and the reaction was performed at 30° C. for 6 h. SDS-PAGE and Coomassie brilliant blue staining.
  • the GFP-Pup(E) band migrated downward, indicating that GFP-Pup(E) self-ligated, PafA and Pup(E) had Pupylation activity, and the Pup(E)-GFP band did not Migration, indicating that PafA can only covalently link the Pup(E) molecule to the substrate through the C-terminus of the Pup(E) molecule.
  • the Pup sequence is shown in SEQ ID NO. 1
  • the surface of streptavidin protein and the lysine of Pup molecule were mutated to arginine, and the mutated streptavidin
  • the amino acid sequence (SEQ ID NO. 5) of the copolyvinyl-Pup tetramer (SA m -Pup E ) is shown in Figure 4a (wherein the Pup E sequence is shown in SEQ ID NO. 2).
  • SAm -Pup E was constructed into pET28a vector and transformed into E. coli BL31(DE3) strain.
  • SA m -Pup E protein and wild-type streptavidin (SA) purified in step 1 were mixed with biotin (Sanggong Bio, A600078) and incubated at room temperature for 1 hour, and detected by SDS-PAGE.
  • SA m -Pup E exhibited biotin-binding activity equivalent to that of wild-type streptavidin.
  • the SA-Pup purified in step 1 was added to low-salt buffer Buffer R (50mM Tris, pH 7.5, 100mM NaCl, 20mM MgCl 2 , 10% (v/v) glycerol) and 8M urea-containing high-salt buffer ( 50mM Tris-HCl, PH 8.0, 8M urea, 15mM DTT, 1mM EDTA, PH 8.0), mix well and aspirate the supernatant, then add biotin agarose beads and incubate at room temperature for 1 hour and then aspirate the supernatant. The obtained supernatant was detected by SDS-PAGE. As shown in Figure 4c, SA m -Pup E still stably bound to biotin-agarose beads in high-salt buffer.
  • Buffer R 50mM Tris, pH 7.5, 100mM NaCl, 20mM MgCl 2 , 10% (v/v) glycerol
  • This embodiment also provides a modified streptavidin-Pup tetrameric protein, which is prepared by the method of step 1, the only difference is that the mutant molecular sequence of Pup is used, such as SEQ ID NO.3 or SEQ ID NO.3 As shown in ID No. 4, the corresponding streptavidin-Pup tetrameric proteins SA m -Pup E-1 and SA m -Pup E-2 were thus prepared.
  • PafA 7KR A site-directed mutagenesis kit (Agilent) constructed seven point-mutated PafA (named as PafA 7KR ), ligated into pTrc99a vector and transformed into E.coli BL21 (DE3) strain, in which PafA 7KR C-terminus was linked with a segment of 6 ⁇ His Label.
  • PafA 7KR A site-directed mutagenesis kit
  • the PafA 7KR enzyme was incubated with SA m -Pup E or Pup E at 30°C for 4 h, and the degree of Pupylation was detected by WB. As shown in Figure 5b, compared with wild-type PafA, the Pupylation connection of PafA 7KR to itself was significantly reduced , only a small amount of self-connection occurs.
  • PafA 7KR was incubated with Pup and substrate PanB at 30°C for 6h, and detected by SDS-PAGE. As shown in Figure 5c, PafA 7KR exhibited substrate Pupylation efficiency equivalent to that of wild-type PafA.
  • the CheZ sequence with the Avi tag at the end was constructed into the pET32a vector, and the BirA enzyme with biotin labeling function was constructed into the pET28a vector, and the two plasmids were co-transformed into E. coli BL21 (DE3) strain. Cultivate 1 L of bacterial solution, OD 600 ⁇ 0.6, add IPTG, induce overnight at 18°C, and purify with nickel column to obtain the biotin-modified protein CheZ.
  • CheAs wild-type and mutant (L126A, L123A) sequences were connected with 6 ⁇ His and Flag tags to obtain the CheAs-Flag-His sequence.
  • the 6 ⁇ His tag was used for protein purification, and the Flag tag was used for immunoblotting detection.
  • the CheAs-Flag-His sequence was constructed on the pET28a vector and transformed into E. coli BL21(DE3) strain. Cultivate 1 L of bacterial solution, add IPTG when OD 600 ⁇ 0.6, induce 3 hours at 37°C, and obtain CheAs protein by nickel column purification.
  • Biotinylated protein CheZ was mixed well with wild-type protein CheAs (0.2 ⁇ M) and SA m -Pup E.
  • the concentration gradient of biotinylated protein CheZ was 0, 0.1 ⁇ M, 0.2 ⁇ M, 0.4 ⁇ M.
  • Biotinylated protein CheZ (0.4 ⁇ M) was mixed well with protein CheAs (0.2 ⁇ M) and SA m -Pup E.
  • the CheAs included wild-type (WT) and mutant (L126A, L123A).
  • Biotinylated CobB protein binds to SA m -Pup E , and when the protein in cell lysate interacts with CobB, PafA 7KR exerts proximity labeling activity to covalently link the C-terminus of SA m -Pup E to the interacting protein of CobB superior.
  • the purified biotinylated CobB protein was constructed and reacted with SA m -Pup E , PafA 7KR enzyme and E.coli SLIAC (Lys/Arg heavy label) cell lysate (experimental group), and SLIAC heavy label cell lysate in the control group Change to E.coli ordinary cell lysate.
  • the experimental group and the control group were mixed evenly, and biotin agarose beads were used to enrich SA m -Pup E and its covalently linked capture protein, and mass spectrometry identified the capture protein and removed non-specific binding.
  • CobB sequence with N-terminal Avi tag was constructed into pET32a vector, and BirA with biotin labeling function was constructed into pET28a vector, and the two vectors were transformed into E.coli BL21(DE3) strain at the same time.
  • E.coli ordinary cells and SLIAC (Lys/Arg re-labeled) cells were cultured, and the cell lysate was obtained by high-pressure crushing method.
  • the following reaction system was prepared: 1 ⁇ M biotinylated CobB protein, 5 ⁇ M SA m -Pup E , 0.5 ⁇ M PafA 7KR enzyme, 5 mM ATP, add 5 mg of E. coli common cell lysate or SILAC cell lysate, lysis buffer (50 mM Tris 8.0, 0.5M NaCl, 20mM MgCl2 , 10% (v/v) Glycerol, 10mM imidazole) to make up to 5ml. The system was incubated at 30°C for 6 hours, and biotin agarose beads were added to incubate at 4°C overnight.
  • lysis buffer 50 mM Tris 8.0, 0.5M NaCl, 20mM MgCl2 , 10% (v/v) Glycerol, 10mM imidazole
  • Wash buffer 1 (8M urea, 50mM Tris 8.0, 200mM NaCl, 0.2% SDS), Wash buffer 2 (8M urea, 50mM Tris 8.0, 200mM NaCl, 2% SDS), Wash buffer 3 (8M urea, 50mM Tris 8.0, 200 mM NaCl), Wash buffer 4 (50 mM Tris 8.0, 0.5 mM EDTA, 1 mM DTT), and Wash buffer 5 (50 mM NH 4 HCO 3 ) were incubated at room temperature with rotation for 5 min, and centrifuged at 1500 rpm for 4 min to remove the supernatant.
  • the discovered interacting proteins were picked for purification, and the interaction with CobB was verified.
  • the purified protein results are shown in Figure 8a.
  • the KD value of the interaction between the protein and CobB detected by BLI is between 25 and 772 nM, as shown in Figure 8b to f. shown. It is shown that this method can detect protein interactions with a wide range of affinity.
  • CobB has deacetylase activity.
  • CobB was co-incubated with the detected VacB and DksA proteins, and the acetylation levels of the proteins were detected by immunoblotting analysis of acetylated antibodies.
  • Figure 8g ⁇ h the acetylation level of a group of proteins added with CobB was significantly reduced, indicating that CobB exerts deacetylation on VacB and DksA, which functionally proves the interaction of CobB with VacB and DksA.
  • Biotinylated PD-1 protein binds to SA m -Pup E , and when PD-1 interacts with cell surface receptors, PafA 7KR exerts proximity labeling activity to covalently link the C-terminus of SA m -Pup E to the receptor superior.
  • the purified biotinylated PD-1 protein was constructed and reacted with live HEK293T cells in a petri dish; after the reaction was completed, the cells were lysed to obtain a lysate, and then biotin agarose beads were used to enrich SA m -Pup E and its covalent linkage
  • the capture protein was identified by mass spectrometry.
  • the PD-L1 plasmid was transiently transfected into HEK293T cells using Lipofectamine 2000 (ThermoFisher 118668), and the viable cells were harvested after 48 h of culture.
  • the following reaction system was prepared: 1 ⁇ M biotinylated PD-1 protein, 5 ⁇ M SA m -Pup E , 0.5 ⁇ M PafA 7KR enzyme, 5 mM ATP, and the HEK293T live cells overexpressing PD-L1 were added to react in a dish, and incubated at 30 °C for 6 hours , add biotin agarose beads and incubate overnight at 4°C.
  • Wash buffer 1 (8M urea, 50mM Tris 8.0, 200mM NaCl, 0.2% SDS), Wash buffer 2 (8M urea, 50mM Tris 8.0, 200mM NaCl, 2% SDS), Wash buffer 3 (8M urea, 50mM Tris 8.0, 200 mM NaCl), Wash buffer 4 (50 mM Tris 8.0, 0.5 mM EDTA, 1 mM DTT), and Wash buffer 5 (50 mM NH 4 HCO 3 ) were incubated at room temperature with rotation for 5 min, and centrifuged at 1500 rpm for 4 min to remove the supernatant.
  • the present invention is used to verify the interaction between PD-1 and PD-L1. If there is an interaction, PafA 7KR enzyme will covalently link the C-terminus of SA m -Pup E to PD-L1. As shown in Figure 9c, when PD-L1-overexpressing cell lysates were co-incubated with PD-1, immunoblotting showed an upshifting band above PD-L1, demonstrating the interaction of PD-1 with PD-L1.
  • Example 7 Detection of interacting proteins of SARS-CoV-2 proteins
  • biotin-modified SARS-CoV-2 protein Construct the SARS-CoV-2 protein sequence linked to the N-terminal Avi tag to the pET32a vector, and construct the BirA with biotin labeling function into the pET28a vector, and co-transform the two constructed plasmids into E. coli BL21 (DE3) in the strain. Cultivate 1 L of bacterial solution, when OD 600 ⁇ 0.6, add IPTG, induce overnight at 18°C, and purify with nickel column to obtain biotin-modified SARS-CoV-2 protein.
  • the following reaction system was prepared: 1 ⁇ M biotinylated bait protein, 5 ⁇ M SA m -Pup E , 0.5 ⁇ M PafA, 5 mM ATP, 5 mg of HEK293T common cell lysate or SILAC cell lysate was added, and the M-PER lysate was supplemented to 5 ml.
  • the system was incubated at 30°C for 6 hours, and biotin agarose beads were added to incubate at 4°C overnight.
  • Wash buffer 1 (8M urea, 50mM Tris 8.0, 200mM NaCl, 0.2% SDS), Wash buffer 2 (8M urea, 50mM Tris 8.0, 200mM NaCl, 2% SDS), Wash buffer 3 (8M urea, 50mM Tris 8.0, 200 mM NaCl), Wash buffer 4 (50 mM Tris 8.0, 0.5 mM EDTA, 1 mM DTT), and Wash buffer 5 (50 mM NH4HCO3) were incubated at room temperature with rotation for 5 min, and centrifuged at 1500 rpm for 4 min to remove the supernatant.
  • the purified biotinylated ORF9b was co-incubated with SA m -Pup E , cell lysate overexpressing TOM70, PafA and ATP, and the detection of the covalently linked band between TOM70 and SA m -Pup E monomer indicated that the proteins interacted with each other. effect.
  • ORF9b interacted with TOM70, while Nsp9, another protein of SARS-CoV-2 in the control group, did not interact with TOM70.
  • Example 8 Identification of interacting proteins of biotinylated m6A RNA
  • the 5'-end biotin-modified m6A RNA (ie biotinylated m6A RNA) was synthesized by Nanjing GenScript Company, and the RNA sequence was CGUCUCGGCUCGGCUGCU (SEQ ID NO.8).
  • Wash buffer 1 (8M urea, 50mM Tris 8.0, 200mM NaCl, 0.2% SDS), Wash buffer 2 (8M urea, 50mM Tris 8.0, 200mM NaCl, 2% SDS), Wash buffer 3 (8M urea, 50mM Tris 8.0, 200 mM NaCl), Wash buffer 4 (50 mM Tris 8.0, 0.5 mM EDTA, 1 mM DTT), and Wash buffer 5 (50 mM NH 4 HCO 3 ) were incubated at room temperature with rotation for 5 min, and centrifuged at 1500 rpm for 4 min to remove the supernatant.
  • Example 9 Verify the interaction between biotinylated m6A RNA and YTDHF1, YTDHF2, YTDHF3 proteins effect
  • the 5'-end biotin-modified m6A RNA (ie biotinylated m6A RNA) was synthesized by Nanjing GenScript Company, and the RNA sequence was CGUCUCGGCUCGGCUGCU (SEQ ID NO.8). Obtain cell lysates overexpressing YTDHF1, YTDHF2, and YTDHF3 proteins.
  • GFP tags were attached to the sequences of YTDHF1, YTDHF2, and YTDHF3, and the GFP tags were used for immunoblot detection.
  • the YTDHF1-GFP, YTDHF2-GFP, YTDHF3-GFP sequences were constructed on the pCDNA3.1 vector, and transiently transfected into HEK293T cells using Lipofectamine 2000 (ThermoFisher 118668). After culturing for 48 hours, the cells overexpressing YTDHF1, YTDHF2, and YTDHF3 were extracted. Lysate.
  • the cell lysates overexpressing YTDHF1, YTDHF2, and YTDHF3 were thoroughly mixed with biotinylated m6A RNA (0.5 ⁇ M) and SA m -Pup E , respectively, and PafA 7KR (10 mM) and ATP (5 mM) were added to the system, and mixed thoroughly. Homogenize and incubate at 30°C for 4-6h. Immunoblot analysis using GFP antibody.
  • proteins YTDHF1, YTDHF2, YTDHF3 interact with biotinylated m6A RNA
  • a complex band (>120kDa) between the protein and SA m -Pup E can be detected; if proteins YTDHF1, YTDHF2, YTDHF3 interact with biotinylated m6A Without RNA interaction, only YTDHF1, YTDHF2, YTDHF3 bands (about 100 kDa) were detected.
  • the complex band exists only in the presence of biotinylated m6A RNA, indicating that the present invention can specifically verify the interaction between biotinylated RNA and protein.
  • This example also verifies the streptavidin-Pup tetrameric protein prepared by using the Pup(E) described in Example 1 (the preparation method is the same as that in Example 2, except that Pup E is replaced by Pup(E) ) was used to verify the interaction of biotinylated m6A RNA with YTDHF1, YTDHF2, YTDHF3 proteins, and the results were similar to those in Figure 13.
  • biotinylated DNAs were used, and the interacting proteins were identified after mixing them.
  • the target sequences of the four biotinylated DNAs were: CGGCAGATGCATAACAAAGGTG (SEQ ID NO.9), CACCTTTGTTATGCATCTGCCG (SEQ ID NO.10) ), CCTTTGTTATGCAAAT (SEQ ID NO. 11), ATATGCAAATT (SEQ ID NO. 12).
  • 5X Annealing Buffer
  • reaction system 1 ⁇ M mixed biotinylated DNA, 5 ⁇ M SA m -Pup E , 0.5 ⁇ M PafA 7KR enzyme, 5 mM ATP, 5 mg of HEK293T common cell lysate or SILAC cell lysate, lysis buffer (50 mM Tris 8.0, 0.5 M NaCl, 20 mM MgCl 2 , 10% (v/v) Glycerol, 10 mM imidazole) to make up to 5 ml.
  • the system was incubated at 30°C for 6 hours, and biotin agarose beads were added to incubate at 4°C overnight.
  • Wash buffer 1 (8M urea, 50mM Tris 8.0, 200mM NaCl, 0.2% SDS), Wash buffer 2 (8M urea, 50mM Tris 8.0, 200mM NaCl, 2% SDS), Wash buffer 3 (8M urea, 50mM Tris 8.0) were added in sequence , 200 mM NaCl), Wash buffer 4 (50 mM Tris 8.0, 0.5 mM EDTA, 1 mM DTT), and Wash buffer 5 (50 mM NH 4 HCO 3 ) were incubated at room temperature with rotation for 5 min, and centrifuged at 1500 rpm for 4 min to remove the supernatant.
  • Nanjing GenScript Biotechnology Co., Ltd. synthesized the 5'-end biotin-modified DNA target sequence and its complementary sequence.
  • the DNA target sequence is:
  • DNA target sequence DNA oligo A
  • DNA oligo B DNA oligo B
  • the DNA target sequence DNA oligo A
  • DNA oligo B DNA oligo B
  • the above systems were mixed and placed in a PCR machine to perform annealing reaction: 95 °C for 2 min, drop 0.1 °C every 8 seconds, and then drop to 25 °C to obtain double-stranded target DNA ( i.e. biotinylated DNA).
  • EthR-Flag-His sequences were obtained by connecting 6 ⁇ His and Flag tags to the sequences encoding the EthR protein, wherein the 6 ⁇ His tags were used for protein purification and the Flag tags were used for immunoblotting detection.
  • the EthR-Flag-His sequence was constructed on the pET28a vector and transformed into E. coli BL21(DE3) strain. Cultivate 1 L of bacterial solution, when OD 600 ⁇ 0.6, add IPTG, induce overnight at 18 °C, and use nickel column purification to obtain EthR protein.
  • biotinylated DNA molecules are used to react with EthR protein, wherein biotinylated DNA molecules are used as the experimental group, and poly dIdC molecules can reduce the non-specific binding of DNA and proteins.
  • mutated biotinylated DNA (sequence: CATGGATCCACGCTATCAACGTAATGTCGAGGCCGTCAACAAGATAAGCCCCCTATCGACACGTAGTAAGCTGCCAGATGACAAA, SEQ ID NO.14) is used to verify DNA sequence specificity, Several systems were added: biotinylated DNA (1 ⁇ M), biotinylated DNA (1 ⁇ M) and poly dI dC mixture, biotinylated DNA (1 ⁇ M) and non-biotin modified and identical sequence DNA (10 ⁇ M) mixture, Mutated biotinylated DNA (1 ⁇ M).
  • EthR protein 0.2 ⁇ M
  • SA m -Pup E The DNA fragments bound by different types of EthR were mixed well with EthR protein (0.2 ⁇ M) and SA m -Pup E. Add PafA 7KR (10 mM) and ATP (5 mM) to the system, mix well, and incubate at 30°C for 4-6 h. Immunoblot analysis using Flag antibody. If the protein EthR interacts with DNA, a complex band (about 50 kDa) between EthR and SA m -Pup E monomer is detected; if the protein EthR does not interact with DNA, an EthR band (about 32 kDa) is detected. As shown in Figure 16, the complex band was the thickest only in the presence of biotinylated DNA, indicating that the present invention can specifically verify the interaction between biotinylated DNA and protein.
  • Nanjing GenScript Biotechnology Co., Ltd. synthesized the 5'-end biotin-modified DNA target sequence and its complementary sequence.
  • the two DNA target sequences were TTGACCACATGGACCAAACAGTCTG (SEQ ID NO.15, corresponding to the DNA sequence of biotin-D1 or D1) and TTGACCACATAGACCGACTGGTCTA (SEQ ID NO. 16, corresponding to the DNA sequence of biotin-D2 or D2 for short below).
  • the DNA target sequence (DNA oligo A) and its corresponding complementary sequence (DNA oligo B) were respectively prepared into 50 ⁇ M with ultrapure water, and the following reaction system was set up: Nuclease-Free Water 40 ⁇ l, Annealing Buffer (5X) 20 ⁇ l, DNA oligo A (50 ⁇ M) 20 ⁇ l, DNA oligo B (50 ⁇ M) 20 ⁇ l, the above systems were mixed and placed in a PCR machine to perform annealing reaction: 95 °C for 2 min, drop 0.1 °C every 8 seconds, and then drop to 25 °C to obtain double-stranded target DNA ( i.e. biotinylated DNA).
  • the RutR-Flag-His sequence was obtained by connecting 6 ⁇ His and Flag tags to the sequence encoding RutR protein, wherein the 6 ⁇ His tag was used for protein purification, and the Flag tag was used for immunoblotting detection.
  • the RutR-Flag-His sequence was constructed on the pET28a vector and transformed into E. coli BL21(DE3) strain. Cultivate 1 L of bacterial solution, add IPTG when OD 600 ⁇ 0.6, induce overnight at 18°C, and obtain RutR protein by nickel column purification.
  • biotinylated DNA biotin-D1, biotin-D2
  • no biotin modification and sequence Consistent DNA D1, D2
  • irrelevant sequence D3 the sequence of D3 is: CAACCCATGAGTCATAC, SEQ ID NO.
  • biotin-D3 biotin-labeled D3 (biotin-D3); in different reaction systems, add: biotin-D1 ( 1 ⁇ M), biotin-D1 (1 ⁇ M) and D1 (10 ⁇ M), biotin-D2 (1 ⁇ M), biotin-D2 (1 ⁇ M) and D2 (10 ⁇ M), biotin-D3 (1 ⁇ M), biotin-D3 (1 ⁇ M) and D3 ( 10 ⁇ M) (as shown in Figure 17).
  • biotin-D1 1 ⁇ M
  • biotin-D1 (1 ⁇ M) and D1 10 ⁇ M
  • biotin-D2 (1 ⁇ M
  • biotin-D2 (1 ⁇ M) and D2
  • biotin-D3 1 ⁇ M
  • biotin-D3 biotin-D3
  • D3 10 ⁇ M
  • the DNA target sequence was CAACCCATGAGTCATAC (SEQ ID NO.17).
  • the DNA target sequence (DNA oligo A) and its corresponding complementary sequence (DNA oligo B) were respectively prepared into 50 ⁇ M with ultrapure water, and the following reaction system was set up: Nuclease-Free Water 40 ⁇ l, Annealing Buffer (5X) 20 ⁇ l, DNA oligo A (50 ⁇ M) 20 ⁇ l, DNA oligo B (50 ⁇ M) 20 ⁇ l, the above systems were mixed and placed in a PCR machine to perform annealing reaction: 95 °C for 2 min, drop 0.1 °C every 8 seconds, and then drop to 25 °C to obtain double-stranded target DNA ( i.e. biotinylated DNA).
  • the GCN4-Flag-His sequence was obtained by connecting 6 ⁇ His and Flag tags to the sequence encoding the GCN4 protein, wherein the 6 ⁇ His tag was used for protein purification, and the Flag tag was used for immunoblotting detection.
  • the GCN4-Flag-His sequence was constructed on the pET28a vector and transformed into E. coli BL21(DE3) strain. Cultivate 1 L of bacterial solution, add IPTG when OD 600 ⁇ 0.6, induce overnight at 18°C, and obtain GCN4 protein by nickel column purification.
  • the biotinylated DNA molecule (1 ⁇ M) was thoroughly mixed with GCN4 protein (0.2 ⁇ M) and SA m -Pup E , and a high concentration of non-biotin-modified DNA (10 ⁇ M) with the same sequence was added to the other system.
  • Add PafA 7KR (10 mM) and ATP (5 mM) to the system, mix well, and incubate at 30°C for 4-6 hours. Immunoblot analysis using Flag antibody.
  • Example 14 Identification of interacting proteins of lenalidomide small molecules
  • lenalidomide small molecule was presented by Dong Jiajia, a teacher from the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.
  • lenalidomide is a conventional small molecule with a size of 259.261Da.
  • HEK293T cells were cultured using Thermo Fisher 78501 Mammalian Protein Extraction lysis to obtain cell lysate.
  • reaction system 1 ⁇ M mixed biotinylated lenalidomide, 5 ⁇ M SA m -Pup E , 0.5 ⁇ M PafA 7KR enzyme, 5 mM ATP, 5 mg of HEK293T common cell lysate or SILAC cell lysate, lysis buffer (50 mM Tris 8.0, 0.5 M NaCl, 20 mM MgCl 2 , 10% (v/v) Glycerol, 10 mM imidazole) to make up to 5 ml.
  • the system was incubated at 30°C for 6 hours, and biotin agarose beads were added to incubate at 4°C overnight.
  • Wash buffer 1 (8M urea, 50mM Tris 8.0, 200mM NaCl, 0.2% SDS), Wash buffer 2 (8M urea, 50mM Tris 8.0, 200mM NaCl, 2% SDS), Wash buffer 3 (8M urea, 50mM Tris 8.0) were added in sequence , 200 mM NaCl), Wash buffer 4 (50 mM Tris 8.0, 0.5 mM EDTA, 1 mM DTT), and Wash buffer 5 (50 mM NH 4 HCO 3 ) were incubated at room temperature with rotation for 5 min, and centrifuged at 1500 rpm for 4 min to remove the supernatant.
  • Biotin-modified c-di-GMP was purchased from Biolog Company, the product number is B098-005, and the molecular size is 1172 Da.
  • ETHR-Flag-His sequence was obtained by connecting 6 ⁇ His and Flag tags to the sequences encoding ETHR protein, wherein 6 ⁇ His tags were used for protein purification, and Flag tags were used for immunoblot detection.
  • the ETHR-Flag-His sequence was constructed on the pET28a vector and transformed into E. coli BL21(DE3) strain. Cultivate 1 L of bacterial solution, when OD600 ⁇ 0.6, add IPTG, induce overnight at 18°C, and use nickel column to purify to obtain ETHR protein.
  • biotinylated small molecule c-di-GMP ie Biotin-c-di-GMP
  • the concentration gradient of biotinylated small molecule c-di-GMP was 0, 0.5 ⁇ M, 2 ⁇ M.
  • the bait small molecule c-di-GMP has no interaction with the protein ETHR, only the ETHR protein band (about 32KDa) is detected; if the small molecule c-di-GMP interacts with the protein, ETHR and SA m are detected - Complex band of Pup E monomer (about 50 KDa). As shown in Fig. 21a, as the concentration of Biotin-C-di-GMP increases, the detected complex bands are thicker, indicating that the protein interaction verified by the present invention is in a concentration-dependent manner.
  • the fourth group of reaction systems added an excess of non-biotinylated c-di-GMP (ie, c-di-GMP in Figure 21a) to compete for the binding of biotinylated c-di-GMP, and the complex band remained basically unchanged, indicating that the The system specifically binds its interacting proteins through biotinylated c-di-GMP.
  • Example 16 Validation of the interaction between c-di-GMP small molecules and CSP series of short peptides
  • Biotin-modified c-di-GMP was purchased from Biolog Company under the catalog number B098-005.
  • the N-terminus of the sequences encoding CSP1, CSP2, and CSP3 proteins were all tagged with Flag, and constructed on a PET32a vector, which was fused with thioredoxin for expression.
  • the recombinant vector was transformed into E. coli BL21(DE3) strain. Cultivate 1 L of bacterial solution, when OD600 ⁇ 0.6, add IPTG, induce 4 h at 37°C, and purify with nickel column to obtain CSP1, CSP2, and CSP3 proteins.
  • the CSP series of short peptide sequences are:
  • CSP2 GGSGDRRFNSADYKGPRRRKAD (SEQ ID NO. 19)
  • Biotinylated small molecule c-di-GMP (2 ⁇ M), CSP series protein (5 ⁇ M) and SA m -Pup E were thoroughly mixed, incubated at 30°C for 20min, and then PafA 7KR (1 ⁇ M) and ATP ( 10 mM), mixed well, and incubated at 30 °C for 6 h. Immunoblot analysis using Flag antibody. Compared with the c-di-GMP without biotinylated in the system, the CSP series protein bands in the experimental group shifted significantly. It shows that the system starts to work after connecting with the whole system through biotinylated c-di-GMP.
  • Example 17 Verify the interaction between Rapamycin small molecule and FKBP12 protein
  • Biotin-modified Rapamycin was donated by Mr. Dang Yongjun from the School of Basic Medicine of Fudan University. It is a conventional known small molecule with a molecular size of 914.19Da.
  • the FKBP12-V5-His sequence was obtained by connecting 6 ⁇ His and V5 tags to the sequences encoding the FKBP12 protein, where the 6 ⁇ His tag was used for protein purification and the V5 tag was used for immunoblotting detection.
  • the FKBP12-V5-His sequence was constructed on the pET28a vector and transformed into E. coli BL21(DE3) strain. Cultivate 1 L of bacterial solution, when OD600 ⁇ 0.6, add IPTG, induce overnight at 18°C, and purify FKBP12 protein by nickel column.

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Abstract

公开了一种基于共价连接的已知分子与蛋白质相互作用检测系统及其鉴定或验证方法，所述检测系统包括：a）链霉亲和素-短肽四聚体；b）PafA酶；c）生物素修饰的已知分子。本方法采用生物素标记已知分子，已知分子与蛋白质发生相互作用后，通过链霉亲和素-短肽四聚体在温和的条件下高效地捕获已知分子的相互作用蛋白质，再在PafA酶的催化下，将短肽与已知分子的相互作用蛋白质共价连接，从而将相互作用的已知分子与蛋白质之间的非共价结合转换为链霉亲和素与蛋白质之间的共价连接，从而进行分析，分离和鉴定。

Description

一种基于共价连接的已知分子与蛋白质相互作用检测系统及其鉴定或验证方法 技术领域
本发明属于分子生物学技术领域，涉及一种已知分子与蛋白质相互作用检测系统，尤其涉及一种基于共价连接的已知分子与蛋白质相互作用检测系统及其鉴定或验证方法。
背景技术
蛋白质是生命活动的执行者。超过80％的蛋白质通过与其他分子相互作用发挥功能，包括胚胎发育、细胞通讯、受体-配体结合、信号传导等广泛的生命过程。无序、失控的蛋白质-分子相互作用可能引发癌症、神经退行性疾病等(kesin et al.,2016.Chem.Rev.,116,4884-4909)。生理过程中小分子或小分子药物与蛋白质的相互作用在生物医学和临床应用中研究广泛，有助于对机体生理代谢过程的进一步理解以及指导药物设计与合成。发现和验证蛋白质与其他分子如蛋白、DNA、RNA、小分子的相互作用，对于在分子层面揭示生命活动的内在规律具有重要意义。蛋白质-分子的相互作用研究一直存在两个难点，一是由于细胞内环境的复杂性，相互作用网络错综复杂，每一种分子存在多种相互作用；二是蛋白质与分子间的相互作用具有强弱和时长的差异，弱相互作用和瞬时相互作用通常难以捕获。
经典的蛋白质-分子相互作用鉴定技术包括免疫沉淀技术、Pull Down、芯片技术等，免疫沉淀法包括蛋白质免疫共沉淀(Co-Immunoprecipitation，Co-IP)、染色质免疫共沉淀(Chromatin immunoprecipitation assay,ChIP)(Das PM et al.,2017.Biotechniques.37(6):961-9.)、RNA免疫共沉淀(RNA Immunoprecipitation)(Gagliardi M et al.Methods Mol Biol.2016；1480:73-86.)、小分子亲和层析(Sleno et al.2008,Curr Opin Chem Biol,12,46-54)(Sato,et al.,2010,Chem Biol,17,616-623)等。ChIP和CLIP可分别用于鉴定DNA和RNA与蛋白质的相互作用，在活细胞状态下利用甲醛等交联固定DNA或RNA与蛋白质的复合物，ChIP可通过抗体富集目标蛋白与DNA复合物，CLIP则可以结合特定RNA并通过质谱鉴定互作蛋白。Co-IP通过抗体富集目标蛋白及其相互作用蛋白质，依赖于蛋白质间的非共价相互作用。Pull down技术如GST Pull Down、RNA pull down、小分子亲和层析等则通过连接在蛋白、RNA或小分子上的标签富集目标分子从而获得 相互作用蛋白。以上两类技术分别用于体内及体外的蛋白质-分子相互作用发现及验证，方法易于重复，操作简单且成本较低，但抗体或亲和介质的非特异性结合容易导致高背景信号，对于弱相互作用和瞬时相互作用难以检测，得到的结果需要使用其他方法来进一步验证(louche et al.,2017.Methods Mol.Biol.,1615,247-255.)。蛋白质芯片(Deng et al.,2014.Cell Rep.,9,2317-2329.)或基因芯片(Hu et al.,2009,Cell,139,610-622)分别将多个蛋白或DNA片段点制在芯片上，以蛋白质芯片为例，将带标签的目标分子(蛋白、DNA、RNA、小分子)与蛋白质芯片共孵育后，洗涤去掉非特异性结合，后续可鉴定分子与芯片上蛋白的相互作用。芯片技术具有通量高，样品用量少，反应时间短等优点，能够在一次实验中全局性地发现与目的分子相互作用的分子，是一种高效的分子/蛋白质相互作用研究工具。但同时该方法也具有一定的局限性，芯片检测到的信号是分子的体外相互作用结果，考虑到生物体内环境的复杂性和多样性，不可避免会存在假阳性，这也是体外筛选方法中普遍存在的问题。
近年来出现的邻近标记系统可用于鉴定多种分子的相互作用蛋白质，如BioID(proximity dependent biotin identification)、APEX(engineered ascorbate peroxidase)、PUP-IT(pupylation-based interaction tagging)(Liu et al.,2018.Nat.Methods,15,715-722.)等可以鉴定已知蛋白质的相互作用蛋白，CasID(Schmidtmann et al.,2016,Nucleus,7,476-484)和CASPEX(Myers et al.,2018,Nat Methods,15,437-439)可以鉴定已知DNA与蛋白质的相互作用，CRIUS(Ziheng Zhang et al.,2020,Nucleic Acids Res,1)可以鉴定已知RNA的相互作用蛋白质等。以BioID为例，将具有邻近标记功能的酶与诱饵蛋白在细胞内融合表达，同时在细胞培养液中加入标记分子(如biotin)，与诱饵蛋白邻近/相互作用的蛋白质即被共价连接上标记分子，再通过质谱鉴定出带有标记分子的捕获蛋白即为可能的相互作用蛋白。CasID、CASPEX及CRIUS将dCas9或dCas13a蛋白与已有邻近标记系统结合实现已知DNA或RNA的相互作用蛋白质的鉴定。以CRIUS为例，将PafA与dCas13a融合表达，在sgRNA的作用下定位到靶RNA,随即PafA可将带有生物素标签的pup多肽标记在RNA结合蛋白上；该方法的缺点是高度依赖sgRNA的靶向效率。在一次性鉴定多个RNA互作的蛋白应用场景中，需要针对不同的目标RNA设计不同的sgRNA，此时不同的sgRNA靶向效率容易带来靶向结合的PafA蛋白量的差异，引入系统误差。邻近标记系统能够将非共价的分子与蛋白质间相互作用转化为蛋白与标记分子的共价连接，能够在真实细胞环境中实现弱相互作用和瞬时相互作用的捕获。但这些方法需要将酶与诱饵蛋白或dCas蛋白(dead Cas proteins)融合表达，酶的分子量较大可能 会影响诱饵蛋白原有的结构，或由于空间位阻影响诱饵蛋白与其他蛋白的相互作用；且以上邻近标记系统只能在细胞中发挥作用，无法适用于原代细胞及大部分传代细胞，因而以上方法对很多蛋白质并不适用。
已有的蛋白质与DNA/RNA/小分子相互作用验证技术包括表面等离子共振(Surface Plasmon Resonance，SPR)以及生物膜干涉(Bio-Layer Interferometry，BLI)、等温滴定量热法(ITC)等。SPR是蛋白质与小分子、蛋白质、抗体等相互作用评估的金标准，可以实现实时、定量以及高灵敏度的检测，且无需标记蛋白，样品易于制备。但该方法依赖于精密仪器，操作较为复杂(Olaru et al.,2015.Crit.Rev.Anal.Chem.,45,97-105.)。BLI可以用于蛋白及多种分子之间的相互作用动力学参数测定，具有实时、定量以及高灵敏的特点，但该方法需要提前标记配体蛋白或分子，且依赖于精密仪器，目前的BLI仪器的温度控制范围非常有限，不适用于确定热力学参数(Desai et al.,2019.J.Vis.Exp.(149),e59687)。免疫沉淀、Pull down也能够应用于蛋白质相互作用的验证。基于电泳的技术如Far-Western和EMSA(Cai et al.,2012,Amino Acids,43,1141-1146)可分别用于验证蛋白质或DNA/RNA与蛋白质的相互作用，以EMSA为例，紫外交联形成DNA/RNA与蛋白质的复合物，在电场作用下DNA/RNA-蛋白复合物相较于DNA/RNA单分子的分子质量大、迁移速率慢。其缺点是依赖仪器，操作相对复杂并且对于弱相互作用检测难度大。
综上，当前存在的多种技术需结合使用实现蛋白质与分子相互作用的鉴定和验证。本发明提出了一个基于共价连接的系统，该系统核心为链霉亲和素融合表达四个短肽，已知分子生物素修饰后结合到链霉亲和素上，通过PafA酶的邻近效应实现短肽与捕获蛋白的共价连接，可以用于蛋白质与多种分子如蛋白质、DNA、RNA、小分子相互作用检测和验证。
发明内容
本发明的目的是为了克服现有技术的不足，提供一种基于共价连接的已知分子与蛋白质相互作用检测系统及其鉴定或验证方法。该检测系统能够应用于已知分子与蛋白质相互作用的发现及验证，在保持已知分子原有结构和活性的基础上，实现弱相互作用和瞬时相互作用的检测，有望大大提高已知分子与蛋白质相互作用检测的灵敏度，特异性以及成功率。
本发明提供的方法利用生物素标记已知分子，已知分子与其它蛋白发生相互作用后，再通过偶联有Pup的链霉亲和素在温和的条件下高效地捕获生物素标记的已知分子的 相互作用蛋白。进而在PafA-Pup系统的邻近标记活力的催化下，将Pup与互作蛋白共价连接，从而可将已知分子与互作蛋白之间的非共价结合转换为链霉亲和素与互作蛋白间的共价连接，后续可采用极其严苛的条件进行清洗，从而可以在保证灵敏度的前提下显著地提高特异性。本发明的方法可在保持已知分子原有结构和活性的基础上，实现弱相互作用和瞬时相互作用的捕获与检测。
本发明的目的是通过以下技术方案实现的：
本发明提供了一种基于共价连接的已知分子与蛋白质相互作用检测系统，所述检测系统包括以下分子：
a)链霉亲和素-短肽四聚体，为链霉亲和素四聚体融合表达四条短肽，其中链霉亲和素能够结合生物素及生物素介质，短肽能够被PafA催化共价连接到邻近蛋白质上；
b)PafA酶，可将特定的短肽共价连接到邻近的蛋白质上。
c)生物素化的已知分子，能够结合到链霉亲和素-短肽四聚体上。
优选地，所述链霉亲和素-短肽四聚体中，短肽为含有12-100个氨基酸的肽。长度小于12个氨基酸的肽一般没有功能。
优选地，所述短肽包括Pup分子或其突变分子，且所述Pup分子末端的谷氨酰胺突变为谷氨酸，其序列如SEQ ID NO.1所示；原因是：在结核分枝杆菌的类泛素蛋白酶体系统中，Dop酶催化Pup末端谷氨酰胺脱酰氨基反应形成谷氨酸，PafA再催化Pup(E)与底物之间的连接反应；
所述Pup的突变分子为存在一个或多个突变的Pup分子，序列如SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4中的任一序列所示。
所述Pup分子来源于分枝杆菌属、棒状杆菌属、链霉菌属、考克氏菌属、微球菌属中的任一种，但不限于此。例如Pup分子来源自结核分枝杆菌、耻垢分枝杆菌、谷氨酸棒状杆菌、麻风杆菌、红霉素放线菌、白喉棒状杆菌、天蓝色链霉菌、嗜根考克氏菌、藤黄微球菌等。
优选地，所述链霉亲和素表面的赖氨酸突变为精氨酸。
优选地，所述链霉亲和素-短肽四聚体为链霉亲和素-Pup四聚体，其氨基酸序列如SEQ ID NO.5所示。
优选地，所述PafA酶表面的7个赖氨酸突变为精氨酸，突变位点为K162R，K202R，K320R，K361R，K423R，K435R和K446R，突变后的PafA酶表面不含赖氨酸，避免 了Pup分子的非特异性共价连接。突变后的PafA酶序列如SEQ ID NO.7所示。
所述PafA酶来源于分枝杆菌属、棒状杆菌属、链霉菌属、考克氏菌属、微球菌属中的任一种，但不限于此。例如PafA酶来源自结核分枝杆菌、耻垢分枝杆菌、谷氨酸棒状杆菌、麻风杆菌、红霉素放线菌、白喉棒状杆菌、天蓝色链霉菌、嗜根考克氏菌、藤黄微球菌等。
优选地，所述生物素修饰的已知分子包括蛋白、DNA、RNA、小分子中的任一种或多种。
优选地，所述蛋白包括蛋白质、肽、修饰肽、抗体、凝集素中的至少一种，可与链霉亲和素-短肽结合；
所述RNA包括信使RNA、核糖体RNA、长链非编码RNA、非编码小RNA中的至少一种，可与链霉亲和素-短肽结合；
所述DNA包括双链DNA、闭环DNA中的至少一种，可与链霉亲和素-短肽结合；
所述小分子包括生物体中具有生物活性的寡核苷酸、氨基酸、维生素、动植物微生物的次级代谢产物以及化学合成的小分子中的至少一种，可与链霉亲和素-短肽结合。
更优选地，所述小分子的大小为50-1500Da。
优选地，所述蛋白、DNA、RNA的生物素修饰位点为N端，C端或其他任意位点，所述小分子的生物素饰位点为可选的非关键活性位点。
本发明还提供了一种根据前述的检测系统用于鉴定已知分子与蛋白质相互作用的方法，包括以下步骤：
A、将生物素化的已知分子及待测样品充分混匀，并于25℃-35℃孵育0-1h；
B、在步骤A处理后的混合物中加入链霉亲和素-短肽四聚体，充分混匀并于25℃-35℃孵育0-1h；
C、在步骤B处理后的混合物中加入PafA酶，充分混匀并于25℃-35℃孵育1min-6h；
D、在步骤C处理后的混合物中加入生物素标记的亲和介质，分离出链霉亲和素-短肽及其连接的蛋白质；
E、质谱鉴定。
优选地，所述待测样品包括蛋白质，活细胞或组织，膜蛋白，细胞裂解液，组织裂解液中的至少一种。
优选地，所述生物素标记的亲和介质包括生物素磁珠、生物素琼脂糖珠，但不限于此。
通过所述方法用于检测待测样品中已知分子与蛋白质的相互作用。
本发明还提供了一种根据前述的检测系统用于验证已知分子与蛋白质相互作用的方法，包括以下步骤：
S1、将待验证已知分子与待验证蛋白充分混匀，并于25℃-35℃孵育0-1h；
S2、在步骤S1处理后的混合物中加入链霉亲和素-短肽四聚体，充分混匀并于25℃-35℃孵育0-1h；
S3、在步骤S2处理后的混合物中加入PafA酶，充分混匀并于25℃-35℃孵育1min-6h；
S4、免疫印迹分析检测待验证已知分子与待验证蛋白的相互作用。
优选地，所述待验证已知分子为生物素修饰的已知分子，包括蛋白、DNA、RNA、小分子中的任一种或多种；
所述蛋白包括蛋白质、肽、修饰肽、抗体、凝集素中的至少一种；
所述RNA包括信使RNA、核糖体RNA、长链非编码RNA、非编码小RNA中的至少一种；
所述DNA包括双链DNA、闭环DNA中的至少一种；
所述小分子包括生物体中具有生物活性的寡核苷酸、氨基酸、维生素、动植物微生物的次级代谢产物以及化学合成的小分子中的至少一种。
与现有技术相比，本发明具有如下的有益效果：
1.本发明提出的基于共价连接的已知分子与蛋白质相互作用检测系统，利用生物素与链霉亲和素间的强亲和力实现已知分子与Pup分子的邻近共价连接。针对已知分子为蛋白、DNA、RNA时，相比于现有的邻近标记技术，即利用融合表达修饰酶诱饵蛋白或dCas蛋白的方法，本发明在一定程度上避免了修饰酶及诱饵蛋白或dCas蛋白之间的空间位阻问题，能够保持修饰酶及诱饵蛋白的结构和活性，更准确地检测蛋白质相互作用。
2.本发明提供的已知分子与蛋白质相互作用检测系统将已知分子与蛋白质间的非共价相互作用转化为Pup分子通过赖氨酸与捕获蛋白质的共价相互作用，可以实现弱相互作用和瞬时相互作用的捕获，灵敏度高，假阳性低。
3.本发明提供的已知分子与蛋白质相互作用检测系统应用于已知分子与蛋白质相互作用的验证，免疫印迹分析即可通过互作蛋白与链霉亲和素-短肽四聚体的共价偶联所导致的在凝胶上的明显迁移来读取结果，操作简单且不依赖于昂贵仪器。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为本发明在相互作用蛋白发现中的应用原理图；其中SA为链霉亲和素四聚体，Pup为短肽，PafA 7KR为突变的PafA酶，Bait为需要研究的蛋白，Prey为捕获到的相互作用蛋白，Biotin agarose为生物素化的琼脂糖珠；
图2为本发明在相互作用蛋白验证中的应用原理图；其中A和B为待验证的相互作用蛋白，A蛋白为生物素修饰的蛋白；
图3为GFP-Pup(E)在PafA的酶活作用下发生自连；
图4a为SA m-Pup E的突变位点示意图及氨基酸序列，图4b是SA m-Pup E的生物素结合活性检测，图4c是SA m-Pup E结合生物素琼脂糖珠在高盐缓冲液中的稳定性检测；
图5a为PafA 7KR酶突变位点示意图及氨基酸序列，图5b是PafA 7KR酶对自身的Pup化连接检测，图5c是PafA 7KR酶对底物Pup化的能力检测；
图6a为验证不同浓度CheZ与CheAs(野生型)的相互作用结果，图6b为验证CheZ与不同突变型CheAs(WT,L126A,L123A)的相互作用结果，图6c为质谱检测到的CheAs(野生型)与SA m-Pup E共价连接位点；
图7a为检测CobB相互作用蛋白质的原理图，图7b为检测CobB相互作用蛋白质的流程图；图7c为本方法得到的CobB相互作用蛋白与已有结果对比；
图8a为CobB及部分相互作用蛋白纯化结果；图8b～f为BLI检测捕获到蛋白质与CobB的相互作用；图8g～h为CobB与VacB及DksA相互作用发挥去乙酰化功能验证；
图9a为检测PD-1蛋白的细胞表面受体的原理图，图9b为检测PD-1蛋白的细胞表面受体的流程图，图9c为验证PD-1蛋白与其细胞表面受体PD-L1的相互作用；
图10a为检测SARS-CoV-2蛋白质的相互作用蛋白质的流程图，图10b为本方法得到的SARS-CoV-2蛋白质的相互作用蛋白与已有结果对比；图10c为验证SARS-CoV-2蛋白ORF9b与人蛋白TOM70的相互作用。
图11为本发明在RNA与蛋白质相互作用发现中的应用原理图；其中SA为链霉亲和素四聚体，Pup为短肽，PafA 7KR为突变的PafA酶，RNA为生物素化RNA，Prey为捕获到的相互作用蛋白，Biotin agarose为生物素化的琼脂糖珠；
图12为本发明在RNA与蛋白质相互作用验证中的应用原理图；其中RNA为生物素化RNA，Prey为捕获到的相互作用蛋白；
图13为验证m6A与YTDHF1、YTDHF2、YTDHF3的相互作用结果；
图14为本发明在DNA与蛋白质相互作用发现中的应用原理图；其中SA为链霉亲和素四聚体，Pup为短肽，PafA 7KR为突变的PafA酶，DNA为生物素化DNA，Prey为捕获到的相互作用蛋白，Biotin agarose为生物素化的琼脂糖珠；
图15为本发明在DNA与蛋白质相互作用验证中的应用原理图；其中DNA为生物素化DNA，Prey为捕获到的相互作用蛋白；
图16为验证不同DNA体系与EthR的相互作用结果；
图17为验证不同DNA片段与RutR的相互作用结果；
图18为验证不同DNA体系与GCN4的相互作用结果；
图19为本发明在小分子与蛋白质相互作用发现中的应用原理图；其中SA为链霉亲和素四聚体，Pup为短肽，PafA 7KR为突变的PafA酶，SM为需要研究的小分子，Prey为捕获到的相互作用蛋白，Biotin agarose为生物素化的琼脂糖珠；
图20为本发明在相互作用小分子与蛋白验证中的应用原理图；其中A为生物素修饰的小分子，B为待验证的蛋白；
图21为不同小分子与蛋白质的相互作用，其中，图21a为验证不同浓度小分子C-di-GMP与ETHR的相互作用结果，图21b为验证小分子C-di-GMP与CSP系列短肽相互作用的结果图，图21c为验证小分子Rapamycin与FKBP12的相互作用结果图；
其中，各图中显示的“SPIDER”表示本发明检测系统的简称。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
本发明的原理说明如下：
1.发现蛋白质相互作用的原理图如图1所示。生物素标记的诱饵蛋白结合到链霉亲和素-Pup四聚体(SA-Pup)上，体系中的蛋白与诱饵蛋白相互作用时，游离的PafA 7KR酶将Pup的C末端共价连接到互作蛋白上。利用生物素标记的亲和介质富集SA-Pup共价连接的捕获蛋白，即为诱饵蛋白的相互作用蛋白。
2.验证蛋白质相互作用的原理图如图2所示。生物素化的蛋白A结合到SA-Pup上，当蛋白B与蛋白A相互作用时，游离在体系中的PafA 7KR酶将Pup的C末端共价连接到蛋白B上。
3.发现RNA与蛋白质相互作用的原理图如图11所示。生物素标记的RNA结合到链霉亲和素-Pup四聚体(SA-Pup)上，体系中的蛋白与RNA相互作用时，游离的PafA 7KR酶将Pup的C末端共价连接到互作蛋白上。利用生物素标记的亲和介质富集SA-Pup共价连接的捕获蛋白，即为RNA的相互作用蛋白。
4.验证RNA与蛋白质相互作用的原理图如图12所示。生物素化的RNA结合到SA-Pup上，当蛋白与RNA相互作用时，游离在体系中的PafA 7KR酶将Pup的C末端共价连接到蛋白上。
5.发现DNA与蛋白质相互作用的原理图如图14所示。生物素标记的DNA结合到链霉亲和素-Pup四聚体(SA-Pup)上，体系中的蛋白与DNA相互作用时，游离的PafA 7KR酶将Pup的C末端共价连接到互作蛋白上。利用生物素琼脂糖珠富集SA-Pup共价连接的捕获蛋白，即为DNA的相互作用蛋白。
6.验证DNA与蛋白质相互作用的原理图如图15所示。生物素化的DNA结合到SA-Pup上，当蛋白与DNA相互作用时，游离在体系中的PafA 7KR酶将Pup的C末端共价连接到蛋白上。
7.发现小分子与蛋白质相互作用的原理图如图19所示。生物素标记的诱饵小分子结合到链霉亲和素-Pup四聚体(SA-Pup)上，体系中的蛋白与诱饵小分子相互作用时，游离的PafA 7KR酶将Pup的C末端共价连接到互作蛋白上。利用生物素标记的亲和介质富集SA-Pup共价连接的捕获蛋白，即为诱饵小分子的相互作用蛋白。
8.验证小分子与蛋白质相互作用的原理图如图20所示。生物素化的诱饵小分子结合到SA-Pup上，当蛋白与诱饵小分子相互作用时，游离在体系中的PafA 7KR酶将Pup的C末端共价连接到蛋白上。
以下实施例中，采用的E.coli BL31(DE3)菌株购自全式金生物技术有限公司，HEK293T细胞购自中科院细胞库，pET28a载体、pTrc99a载体、pET32a载体为实验室常用质粒，生物素琼脂糖珠购自Sigma-Aldrich公司，镍柱购自中科森辉微球技术有限公司，Annealing Buffer(5X)购自碧云天生物科技有限公司，生物素化的C-di-GMP购于biolog公司，生物素修饰的lenalidomide小分子由中科院上海有机化学研究所董佳家老师赠送，生物素化的Rapamycin由复旦大学基础医学院党永军老师赠送。
实施例一：PafA及Pup(E)的反应活性验证
1.获取GFP-Pup(E)及Pup(E)-GFP蛋白
Pup(E)是指野生型Pup分子(序列如SEQ NO.1所示)C末端的谷氨酰胺(Q)突变为谷氨酸(E)。将Pup(E)分别融合表达在GFP蛋白的N端和C端，GFP-Pup(E)和Pup(E)-GFP分别构建到pET28a上并转化到E.coli BL21(DE3)菌株中，其中无Pup(E)的末端连接一段6×His标签。培养1L菌液，OD 600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化得到GFP-Pup(E)及Pup(E)-GFP蛋白。
2.获取PafA酶
野生型PafA序列如SEQ NO.6所示，PafA连接到pTrc99a载体上并转化到E.coli BL21(DE3)菌株中，其中PafA C末端连接一段6×His标签。培养1L菌液，OD 600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化得到PafA酶。
3.验证PafA及Pup(E)的反应活性
配制10μL酶活反应体系，蛋白浓度比例如下：GFP-Pup(E)或Pup(E)-GFP(10μM),PafA(0.5μM),ATP(5mM),体积不足用反应buffer(50mM Tris,PH 7.5,100mM NaCl,20mM MgCl 2,10％(v/v)glycerol)补齐，30℃反应6h。SDS-PAGE及考马斯亮蓝染色检测。如图3所示，GFP-Pup(E)条带向下迁移，说明GFP-Pup(E)发生自连，PafA及Pup(E)具有Pup化反应活性，Pup(E)-GFP条带无迁移，说明PafA只能通过Pup(E)分子C末端发挥作用将Pup(E)分子共价连接到底物上。
实施例二：链霉亲和素-Pup的改造及活性验证
1.获取改造的链霉亲和素-Pup四聚体蛋白
为避免链霉亲和素-Pup自连(Pup序列如SEQ ID NO.1所示)，将链霉亲和素蛋白表面及Pup分子的赖氨酸突变为精氨酸，突变的链霉亲和素-Pup四聚体(SA m-Pup E)氨基酸序列(SEQ ID NO.5)如图4a所示(其中Pup E序列如SEQ ID NO.2所示)。将SA m-Pup E构建到pET28a载体上并转化到E.coli BL31(DE3)菌株中。培养1L菌液，OD 600≈0.6时，加入终浓度0.5mM的IPTG，37℃诱导4小时，使用包涵体变复性方法(Michael T.Jacobsen et al.,2017.Cell.Chem.Bio.,2017 Aug 17；24(8):1040-1047)纯化得到SA m-Pup E蛋白。
2.检测SA m-Pup E的生物素结合活性，如图4b所示。
将步骤1纯化出的SA m-Pup E蛋白及野生型链霉亲和素(SA)分别与生物素(生工生物，A600078)混匀室温孵育1小时，SDS-PAGE检测。由图4b可见，SA m-Pup E表现出与野生型链霉亲和素同等的生物素结合活性。
3.检测SA m-Pup E与生物素琼脂糖珠结合的稳定性，如图4c所示。
将步骤1纯化出的SA-Pup分别加入低盐缓冲液Buffer R(50mM Tris,PH 7.5,100mM NaCl,20mM MgCl 2,10％(v/v)glycerol)和含8M尿素的高盐缓冲液(50mM Tris-HCl,PH 8.0,8M urea,15mM DTT,1mM EDTA,PH 8.0)中，混匀后吸取上清，之后加入生物素琼脂糖珠室温旋转孵育1小时后吸取上清。得到的上清SDS-PAGE检测。如图4c所示，SA m-Pup E与生物素琼脂糖珠在高盐缓冲液中仍能稳定结合。
本实施例还提供了一种改造的链霉亲和素-Pup四聚体蛋白，其采用步骤1的方法制备，不同之处仅在于：采用Pup的突变分子序列如SEQ ID NO.3或SEQ ID NO.4所示，由此制得相应的链霉亲和素-Pup四聚体蛋白SA m-Pup E-1和SA m-Pup E-2。
实施例三：PafA酶的改造及活性验证
1.获取改造的PafA酶，序列(SEQ ID NO.7)如图5a所示。
为避免PafA(序列如SEQ ID NO.6所示)自连，将其表面的7个赖氨酸位点突变为精氨酸，突变位点为K162R，K202R，K320R，K361R，K423R，K435R和K446R。用
Figure PCTCN2021073899-appb-000001
定点诱变试剂盒(安捷伦公司)构建7个点突变的PafA(命名为PafA 7KR)连接到pTrc99a载体上并转化到E.coli BL21(DE3)菌株中，其中PafA 7KR C末端连接一段6×His标签。培养1L菌液，OD 600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化得到PafA 7KR酶。
2.检测PafA 7KR酶对自身的Pup化活性，如图5b所示。
将PafA 7KR酶与SA m-Pup E或Pup E在30℃共孵育4h，WB检测其Pup化程度，如图5b所示，与野生型PafA相比，PafA 7KR对自身的Pup化连接明显降低，仅发生少量自连。
3.检测PafA 7KR对底物Pup化的能力，如图5c所示。
将PafA 7KR与Pup及底物PanB在30℃共孵育6h，SDS-PAGE检测。如图5c所示，PafA 7KR表现出与野生型PafA同等的底物Pup化效率。
实施例四：验证CheAs蛋白与CheZ蛋白的相互作用
1.获取生物素修饰的蛋白CheZ。
构建末端连接Avi标签的CheZ序列到pET32a载体上，同时将具有生物素标记功能的BirA酶构建到pET28a载体上，将两个质粒共转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD 600≈0.6，加入IPTG，18℃诱导过夜，采用镍柱纯化得到生物素修饰的蛋白CheZ。
2.获取待验证蛋白CheAs
CheAs野生型及突变型(L126A,L123A)序列上分别连接6×His及Flag标签得到CheAs-Flag-His序列，其中6×His标签用于蛋白纯化，Flag标签用于免疫印迹检测。将CheAs-Flag-His序列构建在pET28a载体上并转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD 600≈0.6时，加入IPTG，37℃诱导3小时，采用镍柱纯化获取CheAs蛋白。
3.验证不同浓度CheZ蛋白与野生型CheAs蛋白的相互作用，如图6a所示。
将生物素化的蛋白CheZ与野生型蛋白CheAs(0.2μM)及SA m-Pup E充分混匀。其中生物素化的蛋白CheZ设置浓度梯度为0,0.1μM,0.2μM,0.4μM。在体系中加入PafA 7KR(10mM)及ATP(5mM)，充分混匀，30℃孵育6h。使用Flag抗体免疫印迹分析。若蛋白CheAs与蛋白CheZ无相互作用，检测到CheAs条带(截短型，约19kDa)；若两蛋白有相互作用，检测到CheAs与SA m-Pup E单体的复合物条带。如图6a所示，随CheZ浓度升高，检测到的复合物条带越粗，表明本发明验证蛋白质相互作用呈浓度依赖方式。
4.验证CheZ蛋白与不同突变型CheAs蛋白的相互作用，如图6b所示。
将生物素化的蛋白CheZ(0.4μM)与蛋白CheAs(0.2μM)及SA m-Pup E充分混匀。其中CheAs包含野生型(WT)及突变型(L126A,L123A)。在体系中加入PafA 7KR(10mM)及ATP(5mM)，充分混匀，30℃孵育6h。使用Flag抗体免疫印迹分析。如图6b所示，突变后CheAs与CheZ亲和力降低，检测到的复合物条带更细，表明本发明可用于验证不同亲和力的蛋白质相互作用。
5.质谱鉴定CheAs蛋白形成的复合物，如图6c所示。
LC-MS/MS检测CheAs蛋白形成的复合物，得出SA m-Pup E的C末端连接到CheAs的多个位点，其中包括K146位点，如图6c所示。
本实施例还验证了采用实施例一所述的Pup(E)制备的链霉亲和素-Pup四聚体蛋白(制备方法与实施例二相同，仅是将Pup E替换为Pup(E))用于验证CheAs蛋白与CheZ蛋白的相互作用，其结果与图6结果相似。
本实施例还验证了采用实施例二所述的SA m-Pup E-1和SA m-Pup E-2用于验证CheAs蛋白与CheZ蛋白的相互作用，其结果与图6结果相似。
实施例五：检测CobB的相互作用蛋白质
1.使用本方法检测CobB相互作用蛋白质的原理如图7a所示。
生物素化的CobB蛋白与SA m-Pup E结合，细胞裂解液中的蛋白与CobB相互作用时，PafA 7KR发挥邻近标记活性将SA m-Pup E的C末端共价连接到CobB的相互作用蛋白上。
2.使用本方法检测CobB相互作用蛋白质的流程如图7b所示。
首先构建纯化生物素化的CobB蛋白，与SA m-Pup E、PafA 7KR酶及E.coli SLIAC(Lys/Arg重标)细胞裂解液(实验组)反应，对照组中SLIAC重标细胞裂解液换为E.coli普通细胞裂解液。反应完成后将实验组和对照组体系混匀，使用生物素琼脂糖珠富集SA m-Pup E及其共价连接的捕获蛋白，质谱鉴定捕获蛋白并去除非特异性结合。
具体步骤如下：
2.1获取生物素修饰的CobB蛋白。构建N端连接Avi标签的CobB序列到pET32a载体上，同时将具有生物素标记功能的BirA构建到pET28a载体上，将两个载体同时转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD 600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化得到生物素修饰的CobB蛋白。
2.2细胞裂解液样品制备
培养E.coli普通细胞及SLIAC(Lys/Arg重标)细胞，采用高压破碎法裂解获取细胞裂解液。
2.3蛋白质相互作用的捕获及检测
配制如下反应体系：1μM生物素化CobB蛋白，5μM SA m-Pup E，0.5μM PafA 7KR酶，5mM ATP，加入E.coli普通细胞裂解液或SILAC细胞裂解液5mg，裂解buffer(50mM Tris 8.0,0.5M NaCl,20mM MgCl 2,10％(v/v)Glycerol,10mM imidazole)补齐至5ml。体系于30℃孵育6小时，加入生物素琼脂糖珠4℃孵育过夜。依次加入Wash buffer 1(8M urea,50mM Tris 8.0,200mM NaCl,0.2％SDS)、Wash buffer2(8M urea,50mM Tris 8.0,200mM NaCl,2％SDS)、Wash buffer 3(8M urea,50mM Tris 8.0,200mM NaCl)、Wash buffer 4(50mM Tris 8.0,0.5mM EDTA,1mM DTT)、Wash buffer 5(50mM NH 4HCO 3)于室温旋转孵育5min，1500rpm离心4min去上清。转移生物素琼脂糖珠到1.5mL离心管，加入1mL Wash buffer 5，1500rpm 4min水平离心机离心并用Wash buffer 5重复洗一次，得到反应产物。胰酶裂解并进行质谱鉴定。
3.结果分析
将本方法得到的CobB相互作用蛋白与已有研究比较，结果如图7c所示，本方法共检测到CobB的相互作用蛋白261个，其中122个与已有研究一致，34个与已有的两个研究一致，139个相互作用蛋白首次被检测到。
4.相互作用蛋白验证
挑取发现的相互作用蛋白进行纯化，并验证与CobB的相互作用，纯化蛋白结果如图8a所示，BLI检测蛋白质与CobB相互作用的K D值在25～772nM之间，如图8b～f所示。表明了本方法可检测到较大亲和力范围的蛋白质相互作用。
CobB具有去乙酰化酶的活性，将CobB与检测到的VacB及DksA蛋白共孵育，乙酰化抗体免疫印迹分析检测蛋白质的乙酰化水平。如图8g～h所示，加入CobB的一组蛋白质乙酰化水平明显降低，表明CobB对VacB及DksA发挥去乙酰化作用，从功能上证明了CobB与VacB及DksA的相互作用。
实施例六：检测PD-1蛋白质的细胞表面受体
1.使用本方法检测PD-1蛋白细胞表面受体的原理如图9a所示。
生物素化的PD-1蛋白与SA m-Pup E结合，当PD-1与细胞表面受体相互作用时，PafA 7KR发挥邻近标记活性将SA m-Pup E的C末端共价连接到受体上。
2.使用本方法检测PD-1蛋白的细胞表面受体的流程如图9b所示。
首先构建纯化生物素化的PD-1蛋白，与HEK293T活细胞在培养皿中反应；反应完成后裂解细胞获取裂解液，之后使用生物素琼脂糖珠富集SA m-Pup E及其共价连接的捕获蛋白，质谱鉴定捕获蛋白。
具体步骤如下：
2.1构建N端连接Avi标签的PD-1序列到pET32a载体上，同时将具有生物素标记功能的BirA构建到pET28a载体上，将构建的两个质粒共转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD 600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化得到生物素修饰的PD-1蛋白。
2.2 HEK293T细胞制备
使用Lipofectamine 2000(ThermoFisher 118668)将PD-L1质粒瞬时转染进入HEK293T细胞中,培养48h后，收取活细胞。
2.3细胞表面受体的捕获
配制如下反应体系：1μM生物素化PD-1蛋白，5μM SA m-Pup E，0.5μM PafA 7KR 酶，5mM ATP，加入过表达PD-L1的HEK293T活细胞在平皿中反应，30℃孵育6小时，加入生物素琼脂糖珠4℃孵育过夜。依次加入Wash buffer 1(8M urea,50mM Tris 8.0,200mM NaCl,0.2％SDS)、Wash buffer2(8M urea,50mM Tris 8.0,200mM NaCl,2％SDS)、Wash buffer 3(8M urea,50mM Tris 8.0,200mM NaCl)、Wash buffer 4(50mM Tris 8.0,0.5mM EDTA,1mM DTT)、Wash buffer 5(50mM NH 4HCO 3)于室温旋转孵育5min，1500rpm离心4min去上清。转移生物素琼脂糖珠到1.5mL离心管，加入1mL Wash buffer 5，1500rpm 4min水平离心机离心并用Wash buffer 5重复洗一次，得到反应产物。胰酶裂解并进行质谱鉴定。
3.PD-1与PD-L1相互作用验证
使用本发明验证PD-1与PD-L1的相互作用，若存在相互作用，PafA 7KR酶将SA m-Pup E C末端共价连接到PD-L1上。如图9c所示，当PD-L1过表达的细胞裂解液与PD-1共同孵育时，免疫印迹显示PD-L1上方有上移条带，证明了PD-1与PD-L1的相互作用。
实施例七：检测SARS-CoV-2蛋白质的相互作用蛋白质
1.使用本方法检测SARS-CoV-2部分蛋白质的相互作用蛋白质流程如图10a所示。
获取生物素修饰的SARS-CoV-2蛋白。构建N端连接Avi标签的SARS-CoV-2蛋白序列到pET32a载体上，同时将具有生物素标记功能的BirA构建到pET28a载体上，将构建的两个质粒共转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD 600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化得到生物素修饰的SARS-CoV-2蛋白。
2.待测样品准备
培养HEK293T普通细胞及SLIAC(Lys/Arg重标)细胞，采用Thermo Fisher 78501
Figure PCTCN2021073899-appb-000002
Mammalian Protein Extraction裂解获取细胞裂解液。
3.蛋白质相互作用的捕获及检测
配制如下反应体系：1μΜ生物素化诱饵蛋白，5μΜSA m-Pup E，0.5μΜ PafA，5mΜ ATP，加入HEK293T普通细胞裂解液或SILAC细胞裂解液5mg，M-PER裂解液补齐至5ml。体系于30℃孵育6小时，加入生物素琼脂糖珠4℃孵育过夜。依次加入Wash buffer 1(8M urea,50mM Tris 8.0,200mM NaCl,0.2％SDS)、Wash buffer2(8M urea,50mM Tris 8.0,200mM NaCl,2％SDS)、Wash buffer 3(8M urea,50mM Tris 8.0,200mM NaCl)、Wash buffer 4(50mM Tris 8.0,0.5mM EDTA,1mM DTT)、Wash buffer 5(50mM NH4HCO3)于室温旋转孵育5min，1500rpm离心4min去上清。转移生物素琼脂糖珠到 1.5mL离心管，加入1mL Wash buffer 5，1500rpm 4min水平离心机离心并用Wash buffer 5重复洗一次，得到反应产物。胰酶裂解并进行质谱鉴定。
4.结果读取
将本方法得到的SARS-CoV-2蛋白相互作用蛋白与已有方法比较，结果如图10b所示，本方法共检测到SARS-CoV-2蛋白的相互作用蛋白113个，其中检测到新的相互作用蛋白96个，17个与已有研究一致。
5.相互作用蛋白验证
使用纯化出的生物素化ORF9b与SA m-Pup E、过表达TOM70的细胞裂解液及PafA、ATP共孵育，检测到TOM70与SA m-Pup E单体共价连接的条带即表明蛋白质相互作用。如图10c所示，ORF9b与TOM70存在相互作用，而对照组SARS-CoV-2另一蛋白Nsp9与TOM70无相互作用。
实施例八：鉴定生物素化m6A RNA的相互作用蛋白
5.获取生物素修饰的RNA。
由南京金斯瑞公司合成5’端生物素修饰m6A RNA(即生物素化m6A RNA)，RNA序列为CGUCUCGGCUCGGCUGCU(SEQ ID NO.8)。
2.待测样品准备
培养HEK293T普通细胞及SLIAC(Lys/Arg重标)细胞，采用Thermo Fisher 78501
Figure PCTCN2021073899-appb-000003
Mammalian Protein Extraction裂解获取细胞裂解液。
3.m6A RNA相互作用蛋白的捕获及检测
配制如下反应体系：1μM生物素化m6A RNA，5μM SA m-Pup E，0.5μM PafA 7KR酶，5mM ATP，加入HEK293T普通细胞裂解液或SILAC细胞裂解液5mg，裂解buffer(50mM Tris 8.0,0.5M NaCl,20mM MgCl 2,10％(v/v)Glycerol,10mM imidazole)补齐至5ml。体系于30℃孵育6小时，加入生物素琼脂糖珠4℃孵育过夜。依次加入Wash buffer 1(8M urea,50mM Tris 8.0,200mM NaCl,0.2％SDS)、Wash buffer2(8M urea,50mM Tris 8.0,200mM NaCl,2％SDS)、Wash buffer 3(8M urea,50mM Tris 8.0,200mM NaCl)、Wash buffer 4(50mM Tris 8.0,0.5mM EDTA,1mM DTT)、Wash buffer 5(50mM NH 4HCO 3)于室温旋转孵育5min，1500rpm离心4min去上清。转移生物素琼脂糖珠到1.5mL离心管，加入1mL Wash buffer 5，1500rpm 4min水平离心机离心并用Wash buffer 5重复洗一次，得到反应产物。胰酶裂解并进行质谱鉴定。根据质谱结果得到3个可信的m6A结合蛋白YTDHF1、YTDHF2、YTDHF3。
实施例九：验证生物素化m6A RNA与YTDHF1、YTDHF2、YTDHF3蛋白的相互 作用
1.获取生物素修饰的m6A RNA。
由南京金斯瑞公司合成5’端生物素修饰m6A RNA(即生物素化m6A RNA)，RNA序列为CGUCUCGGCUCGGCUGCU(SEQ ID NO.8)。获取过表达YTDHF1、YTDHF2、YTDHF3蛋白的细胞裂解液。
2.制备待验证样品
在YTDHF1、YTDHF2、YTDHF3序列上连接GFP标签,GFP标签用于免疫印迹检测。将YTDHF1-GFP、YTDHF2-GFP、YTDHF3-GFP序列构建在pCDNA3.1载体上，使用Lipofectamine 2000(ThermoFisher 118668)瞬时转染进入HEK293T细胞中,培养48h后，提取过表达YTDHF1、YTDHF2、YTDHF3的细胞裂解液。
3.验证生物素化m6A RNA与YTDHF1、YTDHF2、YTDHF3蛋白的相互作用，如图13所示。
分别将过表达YTDHF1、YTDHF2、YTDHF3的细胞裂解液与生物素化m6A RNA(0.5μM)及SA m-Pup E充分混匀,在体系中加入PafA 7KR(10mM)及ATP(5mM)，充分混匀，30℃孵育4～6h。使用GFP抗体免疫印迹分析。若蛋白YTDHF1、YTDHF2、YTDHF3与生物素化m6A RNA有相互作用，则能检测到蛋白与SA m-Pup E的复合物条带(>120kDa)；若蛋白YTDHF1、YTDHF2、YTDHF3与生物素化m6A RNA无相互作用，则只能检测到YTDHF1、YTDHF2、YTDHF3条带(约100kDa)。如图13所示，仅在生物素化m6A RNA存在的条件下存在复合物条带，表明本发明能够特异性验证生物素化RNA与蛋白质的相互作用。
本实施例还验证了采用实施例一所述的Pup(E)制备的链霉亲和素-Pup四聚体蛋白(制备方法与实施例二相同，仅是将Pup E替换为Pup(E))用于验证生物素化m6A RNA与YTDHF1、YTDHF2、YTDHF3蛋白的相互作用，其结果与图13结果相似。
本实施例还验证了采用实施例二所述的SA m-Pup E-1和SA m-Pup E-2用于验证生物素化m6A RNA与YTDHF1、YTDHF2、YTDHF3蛋白的相互作用，其结果与图13结果相似。
实施例十：鉴定生物素化DNA的相互作用蛋白
本实施例中使用了四段生物素化DNA，将其混合后鉴定相互作用蛋白，四段生物素化DNA的目标序列分别为：CGGCAGATGCATAACAAAGGTG(SEQ ID NO.9)、 CACCTTTGTTATGCATCTGCCG(SEQ ID NO.10)、CCTTTGTTATGCAAAT(SEQ ID NO.11)、ATATGCAAATT(SEQ ID NO.12)。
1.获取生物素修饰的DNA
南京金斯瑞生物科技有限公司合成上述4段5’端生物素修饰的DNA目标序列及其对应的4段互补序列，将DNA目标序列(DNA oligo A)及其对应的互补序列(DNA oligo B)分别用超纯水配制成50μM，设置如下反应体系：Nuclease-Free Water 40μl、Annealing Buffer(5X)20μl、DNA oligo A(50μM)20μl、DNA oligo B(50μM)20μl，以上体系混匀后置于PCR仪执行退火反应：95℃2min、每8秒下降0.1℃，降至25℃，即可获取双链目标DNA(即生物素化DNA)。
2.获取待检测样品
培养小鼠细胞，采用Thermo Fisher 78501
Figure PCTCN2021073899-appb-000004
Mammalian Protein Extraction裂解获取细胞裂解液。
3.生物素化DNA相互作用蛋白的捕获及检测
配制如下反应体系：1μM混合的生物素化DNA，5μM SA m-Pup E，0.5μM PafA 7KR酶，5mM ATP，加入HEK293T普通细胞裂解液或SILAC细胞裂解液5mg，裂解buffer(50mM Tris 8.0,0.5M NaCl,20mM MgCl 2,10％(v/v)Glycerol,10mM imidazole)补齐至5ml。体系于30℃孵育6小时，加入生物素琼脂糖珠4℃孵育过夜。依次加入Wash buffer 1(8M urea,50mM Tris 8.0,200mM NaCl,0.2％SDS)、Wash buffer 2(8M urea,50mM Tris 8.0,200mM NaCl,2％SDS)、Wash buffer 3(8M urea,50mM Tris 8.0,200mM NaCl)、Wash buffer 4(50mM Tris 8.0,0.5mM EDTA,1mM DTT)、Wash buffer 5(50mM NH 4HCO 3)于室温旋转孵育5min，1500rpm离心4min去上清。转移生物素琼脂糖珠到1.5mL离心管，加入1mL Wash buffer 5，1500rpm 4min水平离心机离心并用Wash buffer 5重复洗一次，得到反应产物。胰酶裂解并进行质谱鉴定。根据质谱结果得到多个可信的生物素DNA结合蛋白，如Sox2、HNRNPAB、Sub1、Arid3a等。
实施例十一：验证生物素化DNA与EthR蛋白的相互作用
6.获取生物素修饰的DNA
南京金斯瑞生物科技有限公司合成5’端生物素修饰的DNA目标序列及其互补序列，DNA目标序列为：
CATGGATCCACGCTATCAACGTAATGTCGAGGCCGTCAACGAGATGTCGACACTATCGACACGTAGTAAGCTGCCAGATGACAAA(SEQ ID NO.13)。将DNA目标序列(DNA  oligo A)及其对应的互补序列(DNA oligo B)分别用超纯水配制成50μM，设置如下反应体系：Nuclease-Free Water 40μl、Annealing Buffer(5X)20μl、DNA oligo A(50μM)20μl、DNA oligo B(50μM)20μl，以上体系混匀后置于PCR仪执行退火反应：95℃2min、每8秒下降0.1℃，降至25℃，即可获取双链目标DNA(即生物素化DNA)。
7.获取DNA结合蛋白EthR
在编码EthR蛋白的序列上分别连接6×His及Flag标签得到EthR-Flag-His序列，其中6×His标签用于蛋白纯化，Flag标签用于免疫印迹检测。将EthR-Flag-His序列构建在pET28a载体上并转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD 600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化获取EthR蛋白。
8.验证生物素化DNA与EthR的相互作用，如图16所示。
为验证本发明特异性捕获生物素化DNA与EthR蛋白相互作用，使用不同类型的DNA分子与EthR蛋白反应，其中生物素化DNA分子作为实验组、poly dIdC分子可降低DNA与蛋白质的非特异结合、高浓度无生物素修饰且序列相同的DNA分子用于竞争结合低浓度生物素化DNA、突变的生物素化DNA(序列为：CATGGATCCACGCTATCAACGTAATGTCGAGGCCGTCAACAAGATAAGCCCCCTATCGACACGTAGTAAGCTGCCAGATGACAAA，SEQ ID NO.14)用于验证DNA序列特异性，几个体系中分别添加：生物素化DNA(1μM)、生物素化DNA(1μM)与poly dI dC混合物、生物素化DNA(1μM)与无生物素修饰且序列相同的DNA(10μM)混合物、突变的生物素化DNA(1μM)。将不同类型EthR结合的DNA片段与EthR蛋白(0.2μM)及SA m-Pup E充分混匀。在体系中加入PafA 7KR(10mM)及ATP(5mM)，充分混匀，30℃孵育4～6h。使用Flag抗体免疫印迹分析。若蛋白EthR与DNA有相互作用，检测到EthR与SA m-Pup E单体的复合物条带(约50kDa)；若蛋白EthR与DNA无相互作用，检测到EthR条带(约32kDa)。如图16所示，仅在生物素化DNA存在的条件下复合物条带最粗，表明本发明能够特异性验证生物素化DNA与蛋白质的相互作用。
实施例十二：验证DNA与RutR相互作用的特异性
1.获取生物素修饰的DNA。
南京金斯瑞生物科技有限公司合成5’端生物素修饰DNA目标序列及其互补序列，两段DNA目标序列分别为TTGACCACATGGACCAAACAGTCTG(SEQ ID NO.15，对应以下简称biotin-D1或D1的DNA序列)和TTGACCACATAGACCGACTGGTCTA(SEQ ID NO.16，对应以下简称biotin-D2或D2的DNA序列)。将DNA目标序列(DNA oligo  A)及其对应的互补序列(DNA oligo B)分别用超纯水配制成50μM，设置如下反应体系：Nuclease-Free Water 40μl、Annealing Buffer(5X)20μl、DNA oligo A(50μM)20μl、DNA oligo B(50μM)20μl，以上体系混匀后置于PCR仪执行退火反应：95℃2min、每8秒下降0.1℃，降至25℃，即可获取双链目标DNA(即生物素化DNA)。
2.获取DNA结合蛋白RutR
在编码RutR蛋白的序列上分别连接6×His及Flag标签得到RutR-Flag-His序列，其中6×His标签用于蛋白纯化，Flag标签用于免疫印迹检测。将RutR-Flag-His序列构建在pET28a载体上并转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD 600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化获取RutR蛋白。
3.验证生物素化DNA与RutR的相互作用，如图17所示。
为验证本发明可特异性捕获生物素化DNA与RutR蛋白的相互作用，使用不同类型的DNA分子与RutR蛋白反应：生物素化DNA(biotin-D1、biotin-D2)、无生物素修饰且序列一致的DNA(D1、D2)、无关序列D3(D3的序列为：CAACCCATGAGTCATAC，SEQ ID NO.17)及生物素标记的D3(biotin-D3)；不同的反应体系中分别添加：biotin-D1(1μM)、biotin-D1(1μM)与D1(10μM)、biotin-D2(1μM)、biotin-D2(1μM)与D2(10μM)、biotin-D3(1μM)、biotin-D3(1μM)与D3(10μM)(如图17所示)。将几个DNA片段与RutR蛋白(0.2μM)及SA m-Pup E充分混匀。在体系中加入PafA 7KR(10mM)及ATP(5mM)，充分混匀，30℃孵育4～6h。使用Flag抗体免疫印迹分析。若蛋白RutR与DNA有相互作用，检测到RutR与SA m-Pup E单体的复合物条带(约52kDa)；若蛋白RutR与DNA无相互作用，检测到RutR条带(约30kDa)。如图17所示，仅在生物素化DNA(biotin-D1、biotin-D2)存在的条件下出现RutR与SA m-Pup E单体的复合物条带，表明本发明能够特异性验证生物素化DNA与蛋白质的相互作用。
实施例十三：验证生物素化DNA与GCN4的相互作用
1.获取生物素修饰的DNA。
南京金斯瑞生物科技有限公司合成5’端生物素修饰DNA目标序列及其互补序列，DNA目标序列为CAACCCATGAGTCATAC(SEQ ID NO.17)。将DNA目标序列(DNA oligo A)及其对应的互补序列(DNA oligo B)分别用超纯水配制成50μM，设置如下反应体系：Nuclease-Free Water 40μl、Annealing Buffer(5X)20μl、DNA oligo A(50μM)20μl、DNA oligo B(50μM)20μl，以上体系混匀后置于PCR仪执行退火反应：95℃2min、每8秒下降0.1℃，降至25℃，即可获取双链目标DNA(即生物素化DNA)。
2.获取DNA结合蛋白GCN4
在编码GCN4蛋白的序列上分别连接6×His及Flag标签得到GCN4-Flag-His序列，其中6×His标签用于蛋白纯化，Flag标签用于免疫印迹检测。将GCN4-Flag-His序列构建在pET28a载体上并转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD 600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化获取GCN4蛋白。
3.验证生物素化DNA与GCN4的相互作用，如图18所示。
将生物素化的DNA分子(1μM)与GCN4蛋白(0.2μM)及SA m-Pup E充分混匀，另一体系中额外加入高浓度无生物素修饰且序列一致的DNA(10μM)，用于验证SPIDER技术特异性捕获生物素化的DNA与GCN4蛋白相互作用。体系中加入PafA 7KR(10mM)及ATP(5mM)，充分混匀，30℃孵育4～6h。使用Flag抗体免疫印迹分析。若蛋白GCN4与DNA有相互作用，检测到GCN4与SA m-Pup E单体的复合物条带(约40kDa)；若蛋白GCN4与DNA无相互作用，检测到GCN4条带(约20kDa)。如图18所示，仅在生物素化的DNA存在的条件下出现GCN4与SA m-Pup E单体的复合物条带，表明本发明能够特异性验证生物素化DNA与蛋白质的相互作用。
实施例十四：鉴定lenalidomide小分子的相互作用蛋白
1.获取生物素修饰的lenalidomide(来那度胺)小分子
生物素修饰的lenalidomide小分子由中科院上海有机化学研究所董佳家老师赠送。lenalidomide为常规小分子，大小为259.261Da。
2.获取待检测样品
培养HEK293T细胞，采用Thermo Fisher 78501
Figure PCTCN2021073899-appb-000005
Mammalian Protein Extraction裂解获取细胞裂解液。
3.生物素化lenalidomide相互作用蛋白的捕获及检测
配制如下反应体系：1μM混合的生物素化lenalidomide，5μM SA m-Pup E，0.5μM PafA 7KR酶，5mM ATP，加入HEK293T普通细胞裂解液或SILAC细胞裂解液5mg，裂解buffer(50mM Tris 8.0,0.5M NaCl,20mM MgCl 2,10％(v/v)Glycerol,10mM imidazole)补齐至5ml。体系于30℃孵育6小时，加入生物素琼脂糖珠4℃孵育过夜。依次加入Wash buffer 1(8M urea,50mM Tris 8.0,200mM NaCl,0.2％SDS)、Wash buffer 2(8M urea,50mM Tris 8.0,200mM NaCl,2％SDS)、Wash buffer 3(8M urea,50mM Tris 8.0,200mM NaCl)、Wash buffer 4(50mM Tris 8.0,0.5mM EDTA,1mM DTT)、Wash buffer 5(50mM NH 4HCO 3)于室温旋转孵育5min，1500rpm离心4min去上清。转移生物素琼脂 糖珠到1.5mL离心管，加入1mL Wash buffer 5，1500rpm 4min水平离心机离心并用Wash buffer 5重复洗一次，得到反应产物。胰酶裂解并进行质谱鉴定。根据质谱结果得到多个可信的生物素lenalidomide结合蛋白，如PRDX2、ADD3、TRIM25、PSME1、PHB、WDR18、HK2等。
实施例十五验证c-di-GMP小分子与ETHR蛋白的相互作用
9.获取生物素修饰的c-di-GMP。
生物素修饰的c-di-GMP购买于Biolog公司，货号为B098-005，分子大小为1172Da。
10.获取待验证蛋白
将编码ETHR蛋白的序列上分别连接6×His及Flag标签得到ETHR-Flag-His序列，其中6×His标签用于蛋白纯化，Flag标签用于免疫印迹检测。将ETHR-Flag-His序列构建在pET28a载体上并转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化获取ETHR蛋白。
11.验证不同浓度c-di-GMP小分子与ETHR蛋白的相互作用，如图21a所示。
将生物素化的小分子c-di-GMP(即Biotin-c-di-GMP)与ETHR蛋白及SA m-Pup E充分混匀。其中生物素化的小分子c-di-GMP设置浓度梯度为0,0.5μM,2μM。在各浓度体系中加入PafA 7KR(1μM)及ATP(10mM)，充分混匀，30℃孵育4～6h。使用Flag抗体免疫印迹分析。若诱饵小分子c-di-GMP与蛋白ETHR无相互作用，则仅检测到ETHR蛋白条带(约32KDa)；若小分子c-di-GMP与蛋白有相互作用，则检测到ETHR与SA m-Pup E单体的复合物条带(约50KDa)。如图21a所示，随Biotin-C-di-GMP浓度升高，检测到的复合物条带越粗，表明本发明验证蛋白质相互作用呈浓度依赖方式。第四组反应体系添加过量的未生物素化c-di-GMP(即图21a中的c-di-GMP)竞争结合生物素化c-di-GMP，复合物条带基本不变，表明该系统是通过生物素化c-di-GMP特异性结合其互作蛋白。
实施例十六：验证c-di-GMP小分子与CSP系列短肽的相互作用
1.获取生物素修饰的c-di-GMP。
生物素修饰的c-di-GMP购买于Biolog公司，货号为B098-005。
2.获取待验证蛋白
将编码CSP1,CSP2，CSP3蛋白的序列N端均带Flag标签，并构建于PET32a载体上，与硫氧还蛋白融合表达。将重组载体转化到E.coli BL21(DE3)菌株中。培养1L菌液， OD600≈0.6时，加入IPTG，37℃诱导4h采用镍柱纯化获取CSP1,CSP2,CSP3蛋白。
CSP系列短肽序列分别为：
CSP1:GGSGDRRRFNSADYKGPRRRKAD(SEQ ID NO.18)
CSP2:GGSGDRRFNSADYKGPRRRKAD(SEQ ID NO.19)
CSP3:GGSGDRRRFNSADYKAPRRRKAD(SEQ ID NO.20)
3.验证c-di-GMP小分子与CSP系列蛋白的相互作用，如图21b所示。
将生物素化的小分子c-di-GMP(2μM)与CSP系列蛋白(5μM)及SA m-Pup E充分混匀，30℃孵育20min，之后在体系中加入PafA 7KR(1μM)及ATP(10mM)，充分混匀，30℃孵育6h。使用Flag抗体免疫印迹分析。与体系中不添加生物素化的c-di-GMP相比，实验组的CSP系列蛋白条带发生明显迁移。表明该系统是通过生物素化c-di-GMP与整个体系连接后启动工作的。
实施例十七：验证Rapamycin小分子与FKBP12蛋白的相互作用
3.获取生物素修饰的Rapamycin。
生物素修饰的Rapamycin由复旦大学基础医学院党永军老师赠送，为常规的一种已知小分子，分子大小为914.19Da。
2.获取待验证蛋白
将编码FKBP12蛋白的序列上分别连接6×His及V5标签得到FKBP12-V5-His序列，其中6×His标签用于蛋白纯化，V5标签用于免疫印迹检测。将FKBP12-V5-His序列构建在pET28a载体上并转化到E.coli BL21(DE3)菌株中。培养1L菌液，OD600≈0.6时，加入IPTG，18℃诱导过夜，采用镍柱纯化获取FKBP12蛋白。
将生物素化的小分子Rapamycin(2μM)与FKBP12蛋白(5μM)及SA m-Pup E充分混匀，30℃孵育20min，之后在体系中加入PafA 7KR(1μM)及ATP(10mM)，充分混匀，30℃孵育6h。使用Flag抗体免疫印迹分析。与体系中不添加生物素化的Rapamycin相比，实验组的FKBP12蛋白条带发生明显迁移。表明该系统是通过生物素化Rapamycin与整个体系连接后启动工作的,如图21c所示。
本发明具体应用途径很多，以上所述仅是本发明的优选实施方式。应当指出，以上实施例仅用于说明本发明，而并不用于限制本发明的保护范围。对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进，这些改进也应视为本发明的保护范围。

Claims (10)

  1. 一种基于共价连接的已知分子与蛋白质相互作用检测系统，其特征在于，所述检测系统包括以下分子：
    a)链霉亲和素-短肽四聚体；
    b)PafA酶；
    c)生物素修饰的已知分子。
  2. 根据权利要求1所述的基于共价连接的已知分子与蛋白质相互作用检测系统，其特征在于，所述链霉亲和素-短肽四聚体中，短肽为含有12-100个氨基酸的肽链。
  3. 根据权利要求2所述的基于共价连接的已知分子与蛋白质相互作用检测系统，其特征在于，所述短肽包括Pup分子或其突变分子，且所述Pup分子末端的谷氨酰胺突变为谷氨酸，其序列如SEQ ID NO.1所示；
    所述Pup的突变分子为存在一个或多个突变的Pup分子，序列如SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4中的任一序列所示。
  4. 根据权利要求1所述的基于共价连接的已知分子与蛋白质相互作用检测系统，其特征在于，所述PafA酶表面的7个赖氨酸突变为精氨酸，突变位点为K162R，K202R，K320R，K361R，K423R，K435R和K446R。
  5. 根据权利要求1所述的基于共价连接的已知分子与蛋白质相互作用检测系统，其特征在于，所述生物素修饰的已知分子包括蛋白、DNA、RNA、小分子中的任一种或多种。
  6. 根据权利要求5所述的基于共价连接的已知分子与蛋白质相互作用检测系统，其特征在于，所述蛋白包括蛋白质、肽、修饰肽、抗体、凝集素中的至少一种；
    所述RNA包括信使RNA、核糖体RNA、长链非编码RNA、非编码小RNA中的至少一种；
    所述DNA包括双链DNA、闭环DNA中的至少一种；
    所述小分子包括生物体中具有生物活性的寡核苷酸、氨基酸、维生素、动植物微生物的次级代谢产物以及化学合成的小分子中的至少一种。
  7. 一种根据权利要求1所述的检测系统用于鉴定已知分子与蛋白质相互作用的方法，其特征在于，包括以下步骤：
    A、将生物素化的已知分子及待测样品充分混匀，并于25℃-35℃孵育0-1h；
    B、在步骤A处理后的混合物中加入链霉亲和素-短肽四聚体，充分混匀并于25℃-35℃孵育0-1h；
    C、在步骤B处理后的混合物中加入PafA酶，充分混匀并于25℃-35℃孵育1min-6h；
    D、在步骤C处理后的混合物中加入生物素标记的亲和介质，分离出链霉亲和素-短肽及其连接的蛋白质；
    E、质谱鉴定。
  8. 根据权利要求7所述的用于鉴定已知分子与蛋白质相互作用的方法，其特征在于，所述待测样品包括蛋白质，活细胞或组织，膜蛋白，细胞裂解液，组织裂解液中的至少一种。
  9. 一种根据权利要求1所述的检测系统用于验证已知分子与蛋白质相互作用的方法，其特征在于，包括以下步骤：
    S1、将待验证已知分子与待验证蛋白充分混匀，并于25℃-35℃孵育0-1h；
    S2、在步骤S1处理后的混合物中加入链霉亲和素-短肽四聚体，充分混匀并于25℃-35℃孵育0-1h；
    S3、在步骤S2处理后的混合物中加入PafA酶，充分混匀并于25℃-35℃孵育1min-6h；
    S4、免疫印迹分析检测待验证已知分子与待验证蛋白的相互作用。
  10. 根据权利要求9所述的检测系统用于验证已知分子与蛋白质相互作用的方法，其特征在于，所述待验证已知分子为生物素修饰的已知分子，包括蛋白、DNA、RNA、小分子中的任一种或多种；
    所述蛋白包括蛋白质、多肽、修饰肽、抗体、凝集素中的至少一种；
    所述RNA包括信使RNA、核糖体RNA、长链非编码RNA、非编码小RNA中的至少一种；
    所述DNA包括双链DNA、闭环DNA中的至少一种；
    所述小分子包括生物体中具有生物活性的寡核苷酸、氨基酸、维生素、动植物微生物的次级代谢产物以及化学合成的小分子中的至少一种。
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