An anti-histone modification antibody, covalently coupled t5 complex and application thereof

Abstract

The application discloses an anti-histone modification antibody, a covalently coupled Tn5 complex and application thereof. The anti-histone modification antibody specifically binds to any one or a combination of at least two of the following histone modifications: (1) histone H3 lysine acetylation modification H3K27ac at the 27th position; (2) histone H3 lysine trimethylation H3K27me3 at the 27th position; (3) histone H3 lysine monomethylation H3K4me1 at the 4th position; and (4) histone H3 lysine trimethylation H3K4me3 at the 4th position. The application forms an in-vitro stable recombinant antibody-Tn5 covalent isopeptide bond through a Catcher-Tag system, completely eliminates cross contamination in multi-target detection, perfectly retains the activity of the anti-histone modification antibody and the transposase, and realizes high-fidelity, low-background joint detection of a multi-epigenetic regulation network.

Description

Technical Field

[0001] This invention belongs to the field of gene transcription regulation and epigenetic analysis technology, and relates to an anti-histone modification antibody, a covalently coupled Tn5 complex, and their applications. Specifically, this invention covers the screening, preparation, and sequence identification of promoter and enhancer-specific histone modification-specific monoclonal mouse antibodies H3K27ac, H3K27me3, H3K4me1, and H3K4me3. Simultaneously, this invention also relates to a novel multi-channel CUT&Tag detection technology, which achieves joint detection of multiple DNA-protein interactions and epigenetic modifications within the same system through specific covalent coupling of antibodies and transposases. Background Technology

[0002] In eukaryotes, the spatiotemporal specific expression of genes is largely regulated by cis-regulatory elements in non-coding regions (such as promoters and enhancers). Promoters are control sequences typically located near the transcription start site of a gene; they can be recognized and bound by RNA polymerases and transcription factors, and their core function is to initiate and drive the transcription of downstream target sequences. Enhancers, on the other hand, are cis-acting elements that do not usually initiate transcription directly, but can significantly amplify and enhance the transcriptional activity of associated promoters by binding to specific transcriptional regulatory proteins. Furthermore, their function is generally independent of the absolute position and orientation of the sequence relative to the target gene.

[0003] Accurately identifying the activity status of these two regulatory elements in specific cell types is crucial for understanding gene regulatory networks. The activity status of promoters and enhancers is finely regulated by histone modifications. For example, H3K4me3 is enriched in active promoters, H3K4me1 is a marker of enhancers, H3K27ac represents highly open chromatin (active promoter / enhancer), while H3K27me3 is a repressive modification. In actual biological processes, these modifications often function synergistically or antagonistically. Therefore, detecting only a single modification cannot comprehensively and accurately characterize the true activity status of regulatory elements; joint analysis of multiple recombinant protein modifications is necessary.

[0004] Currently, next-generation in situ epigenetic detection technologies, represented by CUT&Tag, perform excellently in single-target detection, but face significant bottlenecks in "multi-target co-detection": 1. Existing detection antibodies lack sequence basis: Most commercially available histone-modified antibodies are polyclonal or hybridoma antibodies with unknown sequences, making it impossible to genetically engineer them to incorporate novel bio-coupled tags. 2. Traditional covalent cross-linking destroys biological activity: The use of chemical cross-linking agents (such as glutaraldehyde) to covalently link Tn5 to antibodies has high randomness, easily blocking enzyme active sites or destroying antibody affinity. 3. Unstable probe coupling leads to cross-contamination: Existing multiplex CUT&Tag relies on the non-covalent binding of Protein A / G to antibodies. When multiple antibodies are mixed, probe exchange easily occurs, leading to severe background noise and signal cross-contamination.

[0005] Therefore, there is an urgent need in this field to develop a new technology that can achieve stable covalent coupling of transposases and antibodies without affecting their activity, so as to truly achieve high-fidelity, low-background joint detection of multiple epigenetic regulatory networks. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides an anti-histone modification antibody, a covalently coupled Tn5 complex, and its applications. It aims to solve the problems of existing detection antibodies lacking clear sequences and high specificity, providing a material basis for constructing a standardized detection system. It also addresses the difficulty of existing detection methods in simultaneously considering multi-target co-detection and probe (Tn5-antibody) coupling stability. By using a more stable covalent coupling method to connect proteins from different sources, it solves the problem that single-group protein modification detection cannot comprehensively and accurately reflect promoter and enhancer activity, providing a highly efficient, high signal-to-noise ratio multi-target co-detection platform.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides an anti-histone modification antibody, which specifically binds to any one or at least two of the following histone modifications: (1) acetylation of lysine at position 27 of histone H3 (H3K27ac); (2) trimethylation of lysine at position 27 of histone H3 (H3K27me3); (3) monomethylation of lysine at position 4 of histone H3 (H3K4me1); (4) trimethylation of lysine at position 4 of histone H3 (H3K4me3); The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-histone modified antibody are shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively; the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10, respectively; or, The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-histone modified antibody are shown in SEQ ID NO. 11, SEQ ID NO. 12, and SEQ ID NO. 13, respectively; the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 16, respectively; or, The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-histone modified antibody are shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19, respectively; the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22, respectively; or, The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-histone modified antibody are shown in SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25, respectively; the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28, respectively.

[0008] This invention completely eliminates cross-contamination in multi-target detection by forming stable covalent isopeptide bonds through the Catcher-Tag system, perfectly preserving the activity of anti-histone modification antibodies and transposases, and achieving high-fidelity, low-background joint detection of multiple epigenetic regulatory networks.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the anti-histone modified antibody is the sequence shown in SEQ ID NO.29, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.30; or, The amino acid sequence of the heavy chain variable region of the anti-histone modified antibody is the sequence shown in SEQ ID NO. 31, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO. 32; or, The amino acid sequence of the heavy chain variable region of the anti-histone modified antibody is the sequence shown in SEQ ID NO. 33, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO. 34; or, The amino acid sequence of the heavy chain variable region of the anti-histone modified antibody is the sequence shown in SEQ ID NO.35, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.36.

[0010] In a second aspect, the present invention provides a covalently coupled complex comprising the antihistone-modifying antibody and the Tn5 transposase described in the first aspect, wherein the antihistone-modifying antibody and the Tn5 transposase are covalently coupled via a Catcher-Tag system.

[0011] Preferably, the Catcher-Tag system includes SpyCatcher and SpyTag; SpyTag is fused to the C-terminus of the heavy chain of the anti-histone-modified antibody, SpyTag is fused to the C-terminus of the heavy chain of the anti-histone-modified antibody via a flexible linker peptide, and SpyCatcher is fused to the N-terminus of the Tn5 transposase.

[0012] Preferably, the Tn5 transposase is a highly active mutant Tn5 transposase, and the amino acid sequence of the highly active mutant Tn5 transposase includes the sequence shown in SEQ ID NO.37.

[0013] Preferably, the covalently coupled complex is further connected to a sequencing adapter, the sequencing adapter containing a barcode sequence for distinguishing different antibody targets; the barcode sequence includes sequences as shown in SEQ ID NO.38-SEQ ID NO.41.

[0014] SEQ ID NO.5: GYAFTSYN.

[0015] SEQ ID NO.6: IDPYNGDT.

[0016] SEQ ID NO.7: CSISGGGKDYW.

[0017] SEQ ID NO.8: QSLLYSNGKTY.

[0018] SEQ ID NO.9: QVS.

[0019] SEQ ID NO.10: CLQYYGTPHT.

[0020] SEQ ID NO.11: GFTFSNYW.

[0021] SEQ ID NO.12: IRLKSNNYAT.

[0022] SEQ ID NO. 13: CSGYADDGVDYW.

[0023] SEQ ID NO.14: QNVDID.

[0024] SEQ ID NO.15: SAS.

[0025] SEQ ID NO.16: CQNNQYYPYTF

[0026] SEQ ID NO.17: GYTFTDYV.

[0027] SEQ ID NO.18: VYPGSVNT.

[0028] SEQ ID NO.19:AKWSSDGGPFDY。

[0029] SEQ ID NO.20:QSLFNNRDQKTY。

[0030] SEQ ID NO.21: WTS.

[0031] SEQ ID NO.22: QNSSDYPFT.

[0032] SEQ ID NO.23: GYTFTNYG.

[0033] SEQ ID NO.24:INTYTGEP.

[0034] SEQ ID NO.25: ARNYEGDYAMDY.

[0035] SEQ ID NO.26: QSLLYSNGKTY.

[0036] SEQ ID NO.27: LVS.

[0037] SEQ ID NO.28: VQHFGTPQT.

[0038] SEQ ID NO.29: EIQLQQSGPDLVKPGASVKVSCKASGYAFTSYNMYWVKQSHGKSLEWIGYIDPYNGDTSYNQKFKGKATLVDKSSSTAYMHLNSLTSEDSAVYYCSISGGGKDYWGQGTTLTVSS。

[0039] SEQ ID NO.30: DVVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQAPKHLIYQVSKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGVYYCLQYYGTPHTFGGGTKLEIK。

[0040] SEQ ID NO.31: EVKFEESGGGLVQPGGSMKLSCVASGFTFSNYWINWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSGVYLQMNNLRAEDTAIYYCSGYADDGVDYWGQGTSVTVSS。

[0041] SEQ ID NO.32: DIVMTQSQKFMPTSVGDRVSVTCKASQNVDIDVAWYQQKPGQSPKTLIYSASYRYSGVPDRFTGSGSGTDFTLTISNVRVEDLADYFCQNNQYYPYTFGGGTRLEIE。

[0042] SEQ ID NO.33: QVHLQQSGPELVKPGASVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGEVYPGSVNTYYNEKFKGKATLTADKSSNTAYIQLSSLTSEDSAVYFCAKWSSDGGPFDYWGQGTTLTVSS。

[0043] SEQ ID NO.34: DIVMTQSPSSLTVTAGEKVTVNCKSSQSLFNNRDQKTYLTWFQQKPGQPPKLLIYWTSTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNSSDYPFTFGSGTKLEIK。

[0044] SEQ ID NO.35: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARNYEGDYAMDYWGQGTSVTVSS。

[0045] SEQ ID NO.36: DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQHFGTPQTFGGGTKLEIK。

[0046] SEQ ID NO.37: MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQD KSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQ ISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILS FVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLDGFLAAKDLMAQGIKI.

[0047] SEQ ID NO. 38: TCGTCGGCAGCGTCATCACAAGATGTGTATAAGAGACAG.

[0048] SEQ ID NO. 39: GTCTCGTGGGCTCGGATCACAAGATGTGTATAAGAGACAG.

[0049] SEQ ID NO. 40: TCGTCGGCAGCGTCCGATGTAGATGTGTATAAGAGACAG.

[0050] SEQ ID NO. 41: GTCTCGTGGGCTCGGCGATGTAGATGTGTATAAGAGACAG.

[0051] Thirdly, the present invention provides a multichannel chromatin analysis method, the method comprising the following steps: (1) Provide at least two covalently coupled complexes as described in the second aspect, wherein the anti-histone modification antibodies in the different covalently coupled complexes recognize different histone modifications and are each connected to a sequencing adapter containing a different barcode sequence. (2) Incubate at least two covalently coupled complexes described in step (1) with permeabilized cell samples simultaneously; (3) Activate Tn5 transposase to cut genomic DNA and insert it into sequencing adapters; (4) Extract DNA fragments and perform high-throughput sequencing; (5) Based on the barcode sequence split sequencing data, obtain the whole genome distribution map of different histone modifications.

[0052] Preferably, the histone modification includes at least two of H3K27ac, H3K27me3, H3K4me1, or H3K4me3.

[0053] Preferably, the mixed incubation in step (2) includes overnight incubation at 0°C-4°C (e.g., 0°C, 2°C or 4°C).

[0054] Preferably, the activation of Tn5 transposase in step (3) is achieved by using a method containing Mg 2+ Incubation was carried out in a buffer solution at 35°C–38°C (e.g., 35°C, 37°C, or 38°C).

[0055] Preferably, the method further includes preloading the covalently coupled complex with the sequencing adapter prior to step (2).

[0056] Fourthly, the present invention provides a method for identifying promoter and / or enhancer activity, wherein the method comprises obtaining a genome-wide distribution map of different histone modifications using the method described in the third aspect, and identifying promoter and / or enhancer activity based on the following criteria: (1) Active enhancer: A chromatin region that is enriched with both H3K4me1 and H3K27ac signals; (2) Bivalent promoter: a transcription start site region that is enriched with both H3K4me3 and H3K27me3 signals; (3) Active promoter: a transcription start site region that is enriched with both H3K4me3 and H3K27ac signals.

[0057] Fifthly, the present invention provides the use of the anti-histone modification antibody described in the first aspect or the covalently coupled complex described in the second aspect in the preparation of a substance for detecting histone modification.

[0058] Compared with the prior art, the present invention has the following beneficial effects: (1) Completely eliminate cross-contamination: The extremely stable covalent isopeptide bonds formed by the Catcher-tag system. This invention uses the Catcher-tag system to lock the anti-histone modified antibody and Tn5 together, completely eliminating false positive signals caused by antibody exchange in complex mixed systems, and realizing high-fidelity multi-target co-detection. (2) Site-directed coupling perfectly preserves dual-effect activity: This invention avoids the destruction of activity caused by traditional random chemical cross-linking, and protects the high specific affinity of anti-histone modified antibodies and the efficient cleavage and linker insertion activity of Tn5; (3) Realistically restores the epigenetic network: This invention can simultaneously associate multiple histone modifications in a single experiment and under the same microscopic environment, which greatly improves the accuracy of identifying complex regulatory states such as "active enhancers" and "bivalent promoters" and reduces the consumption of precious samples. Attached Figure Description

[0059] Figure 1 This image shows the expression, purification, and identification of the recombinant monoclonal antibody-Tag protein.

[0060] Figure 2 This image shows the expression, purification, and identification of the Tn5-Catcher protein.

[0061] Figure 3 The graph shows the efficiency of covalent conjugation of antibody-Tn5 and Catcher-Tag and the verification of their in vitro activity.

[0062] Figure 4 This is a schematic diagram of the workflow for multi-channel CUT & Tag joint detection.

[0063] Figure 5 This is a genome-wide colocalization map of four histone modifications in the promoter and enhancer regions. Figure A shows the genome-wide colocalization map of the four histone modifications individually in the promoter and enhancer regions, while Figure B shows the genome-wide colocalization map of H3K27ac and H3K27me3, and H3K4me1 and H3K4me3 in pairs in the promoter and enhancer regions. Detailed Implementation

[0064] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0065] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0066] Example 1 Preparation and sequence identification of H3K27ac, H3K27me3, H3K4me1 and H3K4me3 specific monoclonal mouse antibodies.

[0067] 1. Synthesize histone-modified polypeptides with specific modifications (Seq ID NO.1-Seq ID NO.4), conjugate them to KLH carrier proteins, and obtain the antigen.

[0068] Seq ID NO.1: KQLATKAAR K SAPATGGVKC, where K The symbol {Lys-Ac} indicates that an acetyl group is attached to the lysine residue at this site, forming acetylated lysine.

[0069] Seq ID NO.2: KQLATKAAR K SAPATGGVKC, where K The value {Lys(Me3)} indicates that three methyl groups have been added to the amino group of the lysine side chain at this site.

[0070] Seq ID NO.3: ART K QTARKSTGC, where K The value {Lys(Me1)} indicates that a methyl group has been added to the amino group of the lysine side chain at this site.

[0071] Seq ID NO.4: ART K QTARKSTGC, where K The value {Lys(Me3)} indicates that three methyl groups have been added to the amino group of the lysine side chain at this site.

[0072] 2. Take 6-8 week old female BALB / c mice and administer the first subcutaneous multi-point immunization with Freund's complete adjuvant (FCA) (50 µg antigen per mouse); then administer booster immunizations with Freund's incomplete adjuvant (FIA) every 2 weeks for a total of 3-4 times.

[0073] 3. Three days after the final immunization, mouse spleen cells and SP2 / 0 myeloma cells were fused at a ratio of 5:1 under PEG 1500-mediated fusion, seeded in 96-well plates, and selectively cultured in HAT medium.

[0074] 4. After 14 days of culture, the supernatant was collected for indirect ELISA screening. Taking anti-H3K27ac antibody screening as an example, the microplate was coated with: ① H3K27ac peptide (target); ② H3K27me3 peptide (interference 1); ③ unmodified H3K27 peptide (interference 2). Only wells with OD values ​​corresponding to the target were selected.450 Values ​​greater than 1.0, and for interference aperture OD 450 Strongly positive, highly specific clones with a value less than 0.1 were subjected to three limiting dilutions for subcloning. The screening of anti-H3K27me3, anti-H3K4me1, and anti-H3K4me3 antibodies was performed using the same method.

[0075] 5. Collect the cell pellet of the above monoclonal cell lines, extract total RNA, and reverse transcribe it into cDNA.

[0076] 6. Using multiple degenerate primers, the heavy chain variable region (VH) and light chain variable region (VL) gene fragments of the antibody were amplified respectively.

[0077] 7. After cloning the amplified product into the T vector, Sanger sequencing was performed. Sequence analysis using the IMGT tool yielded the following variable regions: heavy chain variable regions CDR1, CDR2, and CDR3 (SEQ ID NO. 5-SEQ ID NO. 7) and light chain variable regions CDR1, CDR2, and CDR3 (SEQ ID NO. 8-SEQ ID NO. 10) for anti-H3K27ac antibody; heavy chain variable regions CDR1, CDR2, and CDR3 (SEQ ID NO. 11-SEQ ID NO. 13) and light chain variable regions CDR1, CDR2, and CDR3 (SEQ ID NO. 14-SEQ ID NO. 16) for anti-H3K27me3 antibody; heavy chain variable regions CDR1, CDR2, and CDR3 (SEQ ID NO. 17-SEQ ID NO. 19) and light chain variable regions CDR1, CDR2, and CDR3 (SEQ ID NO. 20-SEQ ID NO. 22) for anti-H3K4me1 antibody; and heavy chain variable regions CDR1, CDR2, and CDR3 (SEQ ID NO. 10-SEQ ID NO. 10) for anti-H3K27me3 antibody. ID NO.23-SEQ ID NO.25), light chain variable regions CDR1, CDR2 and CDR3 (SEQ ID NO.26-SEQ ID NO.28).

[0078] Example 2 Preparation of site-specific covalent conjugates of antihistone-modified antibodies-Tn5 based on the Catcher-Tag system and isopeptide bonds.

[0079] 1. Construction of recombinant fusion expression vectors and protein expression (1) Antibody-Tag Construction: The VH and VL sequences (SEQ ID NO.29-SEQ ID NO.36) of the four anti-histone modified antibodies obtained in Example 1 were spliced ​​with the mouse IgG1 constant region sequence (SEQ ID NO.42). The SpyTag domain sequence (SEQ ID NO.43) was fused to the C-terminus of the heavy chain using a flexible linker peptide (GGGGS). Expi293 cells were transfected for serum-free suspension expression. The supernatant was collected and purified by Protein A affinity chromatography to obtain high-purity antibody-Tag protein. Lane 1 contains the antibody-Tag protein obtained after sample denaturation, and lane 2 contains the antibody-Tag protein obtained without sample denaturation. Figure 1 ).

[0080] (2) Construction of Tn5-Catcher: The SpyCatcher polypeptide sequence (SEQ ID NO.44) was fused to the N-terminus of a highly active mutant Tn5 transposase. Expression was induced in a prokaryotic system, and the protein was purified by CBD agarose gel affinity chromatography to obtain high-purity Tn5-Catcher protein. Figure 2 ).

[0081] Mouse IgG1 constant region sequence (SEQ ID NO.42): AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0082] SpyTag domain sequence (SEQ ID NO.43): GGGGSGGGGSLPSTGSGSMRGVPHIVMVDAYKRYK.

[0083] SpyCatcher polypeptide sequence (SEQ ID NO.44): MVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSGGGGSGGGGSGGGGSGGGGS.

[0084] 2. Non-destructive covalent coupling reaction (formation of isopeptide bonds) (1) The purified antibody-Tag and Tn5-Catcher were mixed in a reaction buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) at a molar ratio of 1:4.

[0085] (2) Incubate at room temperature for 2 hours. Under these mild conditions, the Asp residues of SpyCatcher and the Lys residues of SpyTag spontaneously undergo a nucleophilic substitution reaction to form an extremely stable covalent isopeptide bond.

[0086] 3. Coupling efficiency and activity verification (1) Conjugation efficiency: The reaction products (antibody K27ac, barcode sequences SEQ ID NO.38 and SEQ ID NO.39) were subjected to reducing SDS-PAGE analysis, and the detection results are as follows: Figure 3 As shown, Tn5 is completely conjugated to the antibody.

[0087] (2) Retention of activity: The results of in vitro DNA fragmentation experiments are as follows Figure 3 As shown, the covalently bound Tn5 still retains the ability to efficiently cleave and insert sequencing adapters.

[0088] Example 3 Multichannel CUT & Tag detection and chromatin analysis of complex stromal cells.

[0089] This embodiment provides a standard operating procedure for multiplex CUT & Tag based on the anti-histone modified antibody-Tn5 site-directed covalent conjugate described in Example 2. This procedure completely eliminates antibody exchange interference caused by the dynamic shedding of the covalent conjugate probe. A schematic diagram of the multi-channel CUT & Tag joint detection workflow is shown below. Figure 4 As shown.

[0090] 1. Sequencing adapter preloading (1) Design chimeric primer pairs containing different embedded barcodes. Barcode-A (Seq ID NO.38) corresponds to H3K27ac, Barcode-B (Seq ID NO.39) corresponds to H3K27me3, Barcode-C (Seq ID NO.40) corresponds to H3K4me1, and Barcode-D (Seq ID NO.41) corresponds to H3K4me3.

[0091] (2) After annealing the four barcode adapters (Seq ID NO.38- Seq ID NO.41) into double strands, they were incubated with their corresponding antihistone-modified antibody-Tn5 site-specific covalent conjugates at room temperature for 1 hour to prepare “covalent conjugate probe-adaptor complexes” with different identity tags.

[0092] 2. Cell sample preparation and permeabilization treatment (1) Collect 100,000 cells (HEK293T). Wash twice with washing buffer (20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 150 mM sodium chloride, 0.5 mM spermidine, 1× protease inhibitor mixture).

[0093] (2) The cells were resuspended and attached to magnetic beads coated with concanavalin A (ConA).

[0094] 3. Multi-target co-incubation and rigorous washing (1) Resuspend the cells bound to the magnetic beads in a buffer containing 0.05% digitoxin (purchased from Sigma, catalog number D141) (wash buffer + 0.05% digitoxin).

[0095] (2) Add the two covalently coupled probe complexes loaded with different barcodes (1 µg of each) to the same reaction tube. Incubate overnight at 4°C after mixing.

[0096] (3) After incubation, wash thoroughly three times with a washing buffer containing 0.05% digitalis saponin (20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 150 mM sodium chloride, 0.5 mM spermidine, 1× protease inhibitor mixture).

[0097] 4. In situ activation of transposition and DNA purification (1) Resuspend the magnetic beads in transposase buffer containing 10 mM MgCl2 (purchased from Novizan, catalog number S602-01) and incubate at 37°C for 1 hour to activate Tn5 transposase to cut and insert the adapter into genomic DNA near the target site.

[0098] (2) Add EDTA (final concentration 16 mM), SDS (final concentration 0.1%) and proteinase K (50 µg), incubate at 50°C for 1 hour to terminate the reaction and digest the protein.

[0099] (3) Use magnetic beads to extract and purify broken DNA fragments.

[0100] 5. Library amplification and sequencing The purified DNA was amplified by PCR for 14 cycles using universal P5 and P7 primers. The PCR products were then purified using AMPure XP magnetic beads and ready for high-throughput sequencing (Illumina platform).

[0101] Example 4 This example provides a standard operating procedure for normal cut & tag of anti-histone modified antibody based on the description in Example 2.

[0102] 1. Cell sample preparation and permeabilization (1) Collect 100,000 cells (HEK293T). Wash twice with washing buffer (20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 150 mM sodium chloride, 0.5 mM spermidine, 1× protease inhibitor mixture).

[0103] (2) The cells were resuspended and attached to magnetic beads coated with concanavalin A (ConA).

[0104] 2. Co-incubation and rigorous washing (1) Resuspend the cells bound to the magnetic beads in a buffer containing digitoxin (wash buffer + 0.05% digitoxin).

[0105] (2) Add the anti-histone modified antibodies (1 µg each) to the reaction solution. Mix well and incubate overnight at 4°C.

[0106] (3) After incubation, wash thoroughly three times with a washing buffer containing 0.05% digitalis saponin (20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 150 mM sodium chloride, 0.5 mM spermidine, 1× protease inhibitor mixture).

[0107] 3. Co-incubation with PG-Tn5 (1) Add pG-Tn5 transposase (final concentration approximately 0.04 µM) and dissolve it in 100 µL of washing buffer containing 0.05% digitalis saponin (20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 150 mM sodium chloride, 0.5 mM spermidine, 1× protease inhibitor mixture).

[0108] (2) Incubate at room temperature by rotating for 1 hour.

[0109] (3) After incubation, wash thoroughly three times with a high-salt washing buffer containing 0.05% digitalis saponin (20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 300 mM sodium chloride, 0.5 mM spermidine, 1× protease inhibitor mixture).

[0110] 4. Activation of transposition and DNA purification (1) Resuspend the magnetic beads in transposase buffer containing 10 mM MgCl2 (purchased from Novizan, catalog number S602-01) and incubate at 37°C for 1 hour to activate Tn5 transposase to cut and insert the adapter into genomic DNA near the target site.

[0111] (2) Add EDTA (final concentration 16 mM), SDS (final concentration 0.1%) and proteinase K (50 µg), incubate at 50°C for 1 hour to terminate the reaction and digest the protein.

[0112] (3) Use magnetic beads to extract and purify broken DNA fragments.

[0113] 5. Library amplification and sequencing The purified DNA was amplified by PCR for 14 cycles using universal P5 and P7 primers. The PCR products were then purified using AMPure XP magnetic beads and ready for high-throughput sequencing (Illumina platform).

[0114] Example 5 Precise correlation identification between bioinformatics analysis and promoter / enhancer activity.

[0115] This embodiment illustrates the multi-channel data splitting algorithm, the analysis of the biological significance of co-localization of different histone modifications, and the comparison with normal CUT&TAG data of single antibodies.

[0116] 1. Individual CUT & TAG data for recombinant antibodies After obtaining the raw data (Fastq file), the split reads were independently aligned to the species reference genome. The genome-wide colocalization map of the four histone modifications in promoter and enhancer regions is shown below. Figure 5 As shown in Figure A.

[0117] 2. Multi-channel data splitting and comparison (1) After obtaining the raw data (Fastq file), based on the embedded barcode A / B / C / D in the read sequence, the single tube sequencing data is accurately split into four independent Fastq datasets (corresponding to channels H3K27ac, H3K27me3, H3K4me1, and H3K4me3 respectively).

[0118] (2) The reads after data splitting were independently aligned to the species reference genome. The genome-wide colocalization map of the four histone modifications in the promoter and enhancer regions is shown below. Figure 5 As shown in Figure B.

[0119] 3. Peak call and cross-contamination assessment The overlap between the K27ac and K27me3 peaks was analyzed. Compared to the traditional "free pA-Tn5 mixing method," the sequencing data generated in this embodiment showed almost no false positives or abnormal co-localization of K27ac (representing activation) and K27me3 (representing complete inhibition) in the same cell population, demonstrating that the covalent coupling system of this invention has extremely high specificity and signal-to-noise ratio in multiplex detection.

[0120] 4. Multidimensional Identification of the Activity of Transcriptional Cis-Regulatory Elements (CREs) (1) Identification of active enhancers: Extract chromatin regions in the intergenic or intronic regions that simultaneously have H3K4me1 signal enrichment and strong H3K27ac signal enrichment.

[0121] (2) Identification of bivalent promoters: Locate the ±2 kb region upstream and downstream of the transcription start site (TSS) and extract target genes that simultaneously enrich the activating H3K4me3 and repressive H3K27me3 signals. These genes are usually located at key regulatory nodes of differentiation and development.

[0122] (3) Identification of active promoters: Extract regions near TSS that simultaneously have strong H3K4me3 and strong H3K27ac signals, indicating that the gene is in a state of strong transcriptional activation.

[0123] In summary, this invention, through the stable covalent isopeptide bonds formed by the Catcher-Tag system, completely eliminates cross-contamination in multi-target detection, perfectly preserves the activity of anti-histone modification antibodies and transposases, and achieves high-fidelity, low-background joint detection of multiple epigenetic regulatory networks.

[0124] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-histone modification antibody, characterized in that, The anti-histone modification antibody specifically binds to any one or at least two of the following histone modifications: (1) acetylation of lysine at position 27 of histone H3 (H3K27ac); (2) trimethylation of lysine at position 27 of histone H3 (H3K27me3); (3) monomethylation of lysine at position 4 of histone H3 (H3K4me1); (4) trimethylation of lysine at position 4 of histone H3 (H3K4me3). The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-histone modified antibody are shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively. The amino acid sequences of the light chain variable regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively; or, The amino acid sequences of the heavy chain variable regions CDR1, CDR2 and CDR3 of the anti-histone modified antibody are shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively; The amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively; or, The amino acid sequences of the heavy chain variable regions CDR1, CDR2 and CDR3 of the anti-histone modified antibody are shown in SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.19, respectively; The amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22, respectively; or, The amino acid sequences of the heavy chain variable regions CDR1, CDR2 and CDR3 of the anti-histone modified antibody are shown in SEQ ID NO. 23, SEQ ID NO. 24 and SEQ ID NO. 25, respectively; The amino acid sequences of the light chain variable regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, respectively.

2. The anti-histone modified antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-histone modified antibody is the sequence shown in SEQ ID NO. 29, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO. 30; or, The amino acid sequence of the heavy chain variable region of the anti-histone modified antibody is the sequence shown in SEQ ID NO. 31, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO. 32; or, The amino acid sequence of the heavy chain variable region of the anti-histone modified antibody is the sequence shown in SEQ ID NO. 33, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO. 34; or, The amino acid sequence of the heavy chain variable region of the anti-histone modified antibody is the sequence shown in SEQ ID NO.35, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.

36.

3. A covalently coupled complex, characterized in that, The covalently coupled complex comprises the antihistone-modified antibody and Tn5 transposase as described in claim 1 or 2, wherein the antihistone-modified antibody and the Tn5 transposase are covalently coupled via a Catcher-Tag system.

4. The covalently coupled complex according to claim 3, characterized in that, The Catcher-Tag system includes SpyCatcher and SpyTag; SpyTag is expressed via flexible linker peptide fusion at the C-terminus of the heavy chain of the anti-histone modified antibody, and SpyCatcher is expressed via fusion at the N-terminus of the Tn5 transposase.

5. The covalently coupled complex according to claim 3 or 4, characterized in that, The Tn5 transposase is a highly active mutant Tn5 transposase, and the amino acid sequence of the highly active mutant Tn5 transposase includes the sequence shown in SEQ ID NO.

37.

6. The covalently coupled complex according to any one of claims 3-5, characterized in that, The covalently coupled complex is further connected to a sequencing adapter containing a barcode sequence for distinguishing different antibody targets; the barcode sequence includes sequences as shown in SEQ ID NO.38-SEQ ID NO.

41.

7. A multichannel chromatin analysis method, characterized in that, The method includes the following steps: (1) Provide at least two covalently coupled complexes according to any one of claims 3-6, wherein the anti-histone modification antibodies in the different covalently coupled complexes recognize different histone modifications and are each connected to a sequencing adapter containing a different barcode sequence. (2) Incubate at least two covalently coupled complexes described in step (1) with permeabilized cell samples simultaneously; (3) Activate Tn5 transposase to cut genomic DNA and insert it into sequencing adapters; (4) Extract DNA fragments and perform high-throughput sequencing; (5) Based on the barcode sequence split sequencing data, obtain the whole genome distribution map of different histone modifications.

8. The method according to claim 7, characterized in that, The histone modifications include at least two of H3K27ac, H3K27me3, H3K4me1, or H3K4me3.

9. A method for identifying promoter and / or enhancer activity, characterized in that, The method for identifying promoter and / or enhancer activity includes obtaining a genome-wide distribution map of different histone modifications using the method of claim 7 or 8, and identifying promoter and / or enhancer activity based on the following criteria: (1) Active enhancer: A chromatin region that is enriched with both H3K4me1 and H3K27ac signals; (2) Bivalent promoter: a transcription start site region that is enriched with both H3K4me3 and H3K27me3 signals; (3) Active promoter: a transcription start site region that is enriched with both H3K4me3 and H3K27ac signals.

10. The use of the anti-histone modification antibody of claim 1 or 2 or the covalently coupled complex of any one of claims 3-6 in the preparation of products for detecting histone modifications.

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