Method for detecting protein-DNA interaction based on deaminase

Abstract

The invention belongs to the technical field of biology. Specifically, the invention relates to a method for detecting protein-DNA interaction based on deaminase.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a method for detecting protein-DNA interaction based on deaminase. Background of the Invention

[0003] Epigenetic research is the focus of gene regulation research. Epigenetics is usually defined as a stable heritable mechanism caused by changes in chromosomes without DNA sequence changes. Epigenetic mechanisms include histone modification and incorporation of histone variants; 5'-adenosine triphosphate (ATP)-dependent chromatin remodeling; regulation of transcription factors; methylation and demethylation mediated by DNA methylases; non-protein coding RNA (ncRNA); RNA modification, etc.

[0004] Protein-DNA interactions mainly include the interactions between histones, transcription factors, DNA methylases, and chromatin remodeling complexes in the above-mentioned epigenetic mechanisms and chromatin DNA. Protein-DNA interactions are the key to gene transcription regulation and the prerequisite for initiating gene transcription. These mechanisms all meet the conditions for changing gene function without changing the DNA sequence, representing dynamic changes at all structural levels of important cellular activities including transcription and translation, and their subtle changes may lead to changes in local chromatin configuration, openness, or nuclear structure in cells.

[0005] A series of protein-DNA interaction detection methods based on second-generation sequencing have been developed and widely recognized, among which the most representative are chromatin immunoprecipitation sequencing (ChIP seq), CUT&RUN and CUT&Tag. ChIPseq enriches the target protein in fragmented chromatin through immune binding reaction, and in this process, the DNA fragments bound to the target protein are co-precipitated and enriched. Through second-generation sequencing, the localization distribution of DNA binding proteins and histone modifications in the whole genome can be analyzed. CUT&RUN and CUT&Tag are both relatively new protein-DNA interaction detection technologies. They fuse the antibody binding functional domain of protein A / G to the highly active microspherical nuclease or transposase, so that they can bind to the antibody targeting the target protein, so that the microspherical nuclease or transposase is anchored in situ at the binding or action site of the target protein and completes the reaction. At this time, the chromatin DNA will be fragmented at a specific site by the microspherical nuclease or transposase. The DNA in the fragmented chromatin is collected and a library is established to obtain the protein-DNA interaction information. In addition, there are many other methods such as DamID and DiMeLo-seq based on third-generation sequencing. These methods have become important tools for studying gene regulation and protein-DNA interactions.

[0006] So far, most genomic-level detections of protein-DNA interactions are performed on a large number of cells as samples, and the data obtained based on these methods are the average of the cell samples. The characteristic information and differences of individual cells are masked by the average value, and the heterogeneity between cells is difficult to reflect. For example, in samples with high heterogeneity such as cancer samples and neural tissue samples, which are difficult to identify or separate individual cell populations, this averaged information masking has a huge impact. Or when studying cell populations such as stem cells or yeast populations in different spatiotemporal states, the transcriptome and epigenetic levels of the same type of cell populations in different states of life activities are in dynamic change, and this type of method is difficult to distinguish. Or for some life activities such as early embryonic development and egg meiosis, it is often impossible to collect a large number of cell samples, and these schemes are also incompatible. The advantages and disadvantages of the above-mentioned methods are currently summarized in the following table.

[0007] 1. sc-ChIP 2.sc-CUT&Tag 3.sc-DamID 4. DiMeLo Single cell level √ √ √ × No enrichment loss × × √ √ PCR-amplification √ √ √ × Quantification × × × √ Single cell sensitivity × × × ×

[0008] Therefore, there is still a need in the art for improved methods for detecting protein-DNA interactions, especially at the whole-genome level, methods that can detect protein-DNA interactions based on a small number of cells or even a single cell or a single nucleus. Brief description of the invention

[0010] The present invention relates to a method of qDEPI-seq (Quantitative DNA Deaminase Based Protein-DNA Interaction Mapping Assay Using Sequencing), by fusing a protein A / G antibody binding domain on a highly active double-stranded DNA dCTP deaminase DddA, the protein A / G domain can bind to an antibody targeting a target protein, so that the double-stranded DNA dCTP deaminase is anchored in situ on the binding or action site of the target protein, and the DNA is subjected to a deamination reaction near the site to complete the conversion of dCTP to dUTP. The extracted DNA is then used to prepare a library for sequencing. During the PCR amplification process of the library preparation, the in situ dUTP formed by the deamination reaction of dCTP on the genome will be paired with dATP, thereby ultimately achieving the conversion of the sequence from CG to TA. In the subsequent library data analysis, bioinformatics methods are used to find and statistically calculate the number, position, conversion efficiency and other information of the above-mentioned conversion sites to obtain information such as the binding sites of the target protein and DNA interaction. This method can use the complete genomic DNA of a small amount of cells to a single cell nucleus as the starting input, and perform whole genome sequencing or specific gene region sequencing. Compared with other existing detection methods, the starting DNA input amount is greatly reduced, and the loss of DNA in the library construction process is reduced at the same time, and the library construction efficiency is improved, and then a very small amount of cells or even a single cell nucleus can be used to detect the protein-DNA interaction of cells at the whole genome level. The data detected using the method of the present invention can cover most of the regions on the genome, and the protein-DNA interaction of single cells can be detected very effectively. And in the library construction and sequencing means based on the whole genome, because the genome information includes the sequence information of conversion and unconverted, the method can realize the simple quantification of protein-DNA interactions such as epigenetic modification and transcription factor binding, and in the library of a small amount of cell starting amount, the quasi-quantitative statistics in the cell population can be performed for the site of interest of the target protein.

[0011] This application includes but is not limited to the following implementation plans:

[0012] Embodiment 1. A method for detecting the interaction between a target protein and genomic DNA in a cell, the method comprising:

[0013] a) treating at least one of the cells with a permeabilization buffer, or isolating a cell nucleus from at least one of the cells;

[0014] b) contacting the permeabilized cells or isolated cell nuclei obtained in step a) with a first antibody that specifically binds to the target protein, thereby allowing the first antibody to fully bind to the target protein in the cell and form a complex;

[0015] c) contacting a fusion protein comprising a DNA deaminase and protein A or protein G with the product of step b), allowing the fusion protein to fully bind to the complex while the activity of the DNA deaminase is inhibited;

[0016] d) subjecting the product of step c) to a reaction under conditions that restore the activity of the DNA deaminase, thereby allowing the DNA deaminase to deaminize one or more cytidines (C) in or near the region of the genomic DNA that interacts with the target protein;

[0017] e) isolating genomic DNA from the product of step d); and

[0018] f) sequencing the isolated genomic DNA or a portion thereof, and determining the interaction between the target protein and the genomic DNA based on the sequencing result.

[0019] Embodiment 2. The method of embodiment 1, wherein in step a), the proteins and DNA in the cells are cross-linked, for example by UV cross-linking, fixation by formaldehyde or paraformaldehyde.

[0020] Embodiment 3. The method of embodiment 1 or 2, wherein the permeabilization buffer comprises a detergent such as Digitonin, for example, the permeabilization buffer comprises about 0.02% Digitonin.

[0021] Embodiment 4. The method of embodiment 3, wherein the permeabilization buffer comprises about 20 mM HEPES pH 7.5, about 150 mM NaCl, about 0.5 mM Spermidine, about 0.1% BSA, about 1X PIC, about 2 mM EDTA, and about 0.02% Digitonin.

[0022] Embodiment 5. The method of any one of embodiments 1-4, wherein step a) of "treating at least one of the cells with a permeabilization buffer comprising a detergent" is carried out at about 20°C to about 37°C, preferably at room temperature (e.g., about 25°C).

[0023] Embodiment 6. The method of any one of embodiments 1-5, wherein in step a), at least one of the cells is treated with a permeabilization buffer comprising a detergent for about 5 minutes to about 10 minutes.

[0024] Embodiment 7. The method of any one of embodiments 1-6, wherein the at least one cell is about 1 cell to about 5000 cells or more,

[0025] Preferably, the at least one cell is no more than 5000 cells, no more than 2500 cells, no more than 1000 cells, no more than 500 cells, no more than 100 cells, no more than 50 cells, no more than 10 cells, more preferably, the at least one cell is 1 cell.

[0026] Embodiment 8. The method of any one of Embodiments 1-7, wherein the DNA deaminase can be single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA) or a functional fragment thereof, for example, the DNA deaminase is DddA or a functional fragment thereof, preferably DddA tox.

[0027] Embodiment 9. The method of embodiment 8, wherein the DddA tox comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:1.

[0028] Embodiment 10. The method of any one of embodiments 1-9, wherein protein A comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity to SEQ ID NO:2; or protein A comprises the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity to SEQ ID NO:3.

[0029] Embodiment 11. The method of any one of embodiments 1-10, wherein the DNA deaminase is fused to Protein A or Protein G directly, or via a peptide linker.

[0030] Embodiment 12. The method of embodiment 11, wherein the peptide linker is a flexible peptide linker or a rigid peptide linker, preferably a rigid peptide linker, for example, the rigid peptide linker comprises the amino acid sequence shown in SEQ ID NO:4, and the flexible peptide linker comprises the amino acid sequence shown in SEQ ID NO:5.

[0031] Embodiment 13. The method according to any one of embodiments 1 to 12, wherein the fusion protein of DNA deaminase and protein A comprises the amino acid sequence shown in SEQ ID NO: 6 or 7.

[0032] Embodiment 14. The method according to any one of embodiments 1 to 13, wherein a second antibody may be further added in step b), wherein the second antibody specifically binds to the first antibody, or specifically binds to a different epitope of the target protein.

[0033] Embodiment 15. The method of any one of embodiments 1-14, wherein step b) is carried out at low temperature. For example, step b) is carried out at about 4°C to about 10°C, preferably about 4°C.

[0034] Embodiment 16. The method of any one of embodiments 1-15, wherein step b) is performed in a first binding buffer, for example, the first binding buffer comprises a detergent selected from Ttriton x 100, Digitonin or sodium deoxycholate, preferably, step b) the first binding buffer comprises approximately 0.02% Digitonin.

[0035] Embodiment 17. The method of embodiment 16, wherein the first binding buffer

[0036] i) further comprises a protease inhibitor;

[0037] ii) does not contain serum albumin such as BSA; and / or

[0038] iii) contains about 90 mM to about 200 mM, preferably about 150 mM, of a metal salt such as NaCl.

[0039] Embodiment 18. The method of any one of Embodiments 1-17, wherein step b) is performed for about 1 hour to about 12 hours or longer, for example, for about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours or longer.

[0040] Embodiment 19. The method of any one of embodiments 1-18, wherein step c) is carried out at low temperature, for example, step c) is carried out at about 4°C to about 10°C, preferably about 4°C.

[0041] Embodiment 20. The method of any one of embodiments 1-19, wherein step c) is performed in a second binding buffer, for example, the second binding buffer comprises about 90 mM to about 200 mM, preferably about 150 mM, of a salt such as NaCl.

[0042] Embodiment 21. The method of embodiment 20, wherein the second binding buffer

[0043] i) does not contain serum albumin such as BSA;

[0044] ii) comprising a detergent selected from Ttriton x 100, Digitonin or sodium deoxycholate, for example comprising about 0.02% Digitonin; and / or

[0045] iii) contains a protease inhibitor.

[0046] Embodiment 22. The method according to any one of embodiments 1 to 21, wherein the concentration of the fusion protein in step c) is about 0.02 to about 0.24 μM or higher, preferably about 0.02 to about 0.08 μM.

[0047] Embodiment 23. The method of any one of Embodiments 1-21, wherein step c) is performed for about 0.5 hours to about 2 hours or longer, such as for about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours or longer.

[0048] Embodiment 24. The method of any one of Embodiments 1-23, wherein step d) comprises exchanging the buffer of the product of step c) with a deamination reaction buffer, for example, the deamination reaction buffer comprises no more than about 50 mM, about 10 mM, no more than about 5 mM, preferably no more than about 2 mM of a metal salt such as NaCl or MgCl2, preferably, the deamination reaction buffer comprises about 50 mM NaCl and / or 2 mM MgCl2.

[0049] Embodiment 25. The method of any one of embodiments 1-24, wherein the reaction in step d) is carried out at about 20°C to about 40°C, preferably, the reaction in step d) is carried out at about 37°C.

[0050] Embodiment 26. The method of any one of Embodiments 1-25, wherein the reaction of step d) is carried out for about 10 minutes to about 2 hours or longer, preferably, the reaction of step d) is carried out for at least 30 minutes.

[0051] Embodiment 27. The method of any one of embodiments 1-26, wherein step f) comprises amplifying a specific portion of the genomic DNA and sequencing it to determine the interaction between the specific portion of the cell genome and the target protein.

[0052] Embodiment 28. The method of embodiment 27, wherein the amplification is PCR amplification, such as PCR amplification performed using a U-tolerant DNA polymerase, for example, the U-tolerant DNA polymerase is selected from: HiFi V3 (component in the Novozymes single-stranded library construction kit, NE103), Phusion U (Thermo Scientific F555L), Phanta Uc (vazyme, P507-01), Q5U (NEB, M0515L) and Q6U (Biyuntian, D7239M).

[0053] Embodiment 29. The method of any one of embodiments 1-26, wherein step f) comprises establishing a whole genome DNA library from the isolated genomic DNA, and performing sequencing based on the whole genome library, thereby determining the interaction between the target protein and the genomic DNA at the whole genome level.

[0054] Embodiment 30. The method of embodiment 29, wherein a U-tolerant DNA polymerase is used for DNA amplification in the establishment of the library, for example, the U-tolerant DNA polymerase is selected from the group consisting of: HiFi V3, Phusion U, PhantaUc, Q5U and Q6U.

[0055] Embodiment 31. The method of any one of embodiments 1-30, wherein the interaction of the target protein with the cellular genomic DNA or a portion thereof is determined by analyzing the presence of CG to TA transitions (i.e., C to T and G to A) in the cellular genomic DNA or a portion thereof relative to a control genomic DNA or a portion thereof.

[0056] Embodiment 32. The method of any one of embodiments 1-31, wherein the target protein is a DNA binding protein, for example, the target protein is a transcription factor, preferably CTCF; or preferably, the target protein is a histone.

[0057] Embodiment 33. The method of any one of Embodiments 1-32, wherein the cell is an animal cell, a plant cell or a microbial cell.

[0058] Embodiment 34. The method of embodiment 33, wherein the cell is a mammalian cell, including but not limited to cells of humans, mice, rats, cats, dogs, pigs, and cows; or, the cell is a monocotyledonous or dicotyledonous plant cell, such as cells of rice, corn, wheat, sorghum, soybean, potato, tomato, etc.; or, the cell is a fungus such as a yeast cell.

[0059] Embodiment 35. The method of embodiment 33, wherein the cell is a cell line cell; or, the cell is a primary cell from a different organ or tissue, for example, the cell is from blood, cerebrospinal fluid, biopsy tissue. The cell can be a hepatocyte, a cardiomyocyte, a neuronal cell, a fibroblast, an epithelial cell; or, the cell is a tumor cell, a stem cell such as an embryonic stem cell or an induced pluripotent stem cell.

[0060] Embodiment 36. The method of embodiment 33, wherein the cell is a cell treated with a specific substance or condition or a cell at a specific developmental stage, for example, the specific substance is a drug.

[0061] Embodiment 37. A kit for detecting the interaction between a target protein and genomic DNA in a cell, comprising at least a fusion protein comprising DNA deaminase and protein A or protein G, a first antibody that specifically binds to the target protein, and optionally a second antibody that specifically binds to the first antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 .Schematic diagram of the qDEPI-seq workflow.

[0064] Figure 2 The library constructed in Example 1 was tested by the Agilent 2100 biochip analysis system, showing the size distribution of the library fragments.

[0065] Figure 3 The next-generation sequencing results of the libraries obtained from 500 cells and 5000 cells samples. The CG to TA ratios upstream and downstream of the peak center of the public ChIP-seq data show the location of the target protein on the genome.

[0066] Figure 4 . Figure 3 The experiment was optimized under different surfactant digitonin concentrations (0.012%, 0.02%, 0.04%).

[0067] Figure 5 .The quality inspection results of the short fragment library obtained from a single MII oocyte sample using the Agilent 2100 biochip analysis system show the size distribution of the library fragments.

[0068] Figure 6 .The library fragment size distribution is shown in the quality inspection results of the Agilent 2100 biochip analysis system for the long fragment library obtained from a single MII oocyte sample.

[0069] Figure 7.Next-generation sequencing results of the library obtained from a single MII oocyte sample. The CG to TA ratios upstream and downstream of the peak center in the public ChIP-seq data show the location of the target protein on the genome. .

[0070] Figure 8 The editing efficiency of TC sites upstream and downstream of some CTCF motifs was close to 100%, indicating the feasibility of quasi-quantification.

[0071] Fig. 9 .Optimization of Protein A-DddA fusion protein.

[0072] Fig.10 .Optimization of buffer system for in situ binding of antibodies to target proteins.

[0073] Fig.11 .Optimization of fusion protein and antibody binding.

[0074] Fig.12 .Optimization of deamination reaction buffer and reaction time.

[0075] Fig.13 .The impact of amplification bias on detection methods. DETAILED DESCRIPTION OF THE INVENTION

[0077] I. Definition

[0078] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0079] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0080] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered at this time, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0081] Sequence "identity" has a recognized meaning in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule.

[0082] "Genome" as used herein encompasses not only the chromosomal DNA present in the cell nucleus, but also the organellar DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0083] 2. Deaminase-based method for detecting protein-DNA interactions

[0084] In one aspect, the present invention provides a method for detecting the interaction between a target protein and genomic DNA in a cell, the method comprising:

[0085] a) treating at least one of the cells with a permeabilization buffer, or isolating a cell nucleus from at least one of the cells;

[0086] b) contacting the permeabilized cells or isolated cell nuclei obtained in step a) with a first antibody that specifically binds to the target protein, thereby allowing the first antibody to fully bind to the target protein in the cell and form a complex;

[0087] c) contacting a fusion protein comprising a DNA deaminase and protein A or protein G with the product of step b), allowing the fusion protein to fully bind to the complex while the activity of the DNA deaminase is inhibited;

[0088] d) subjecting the product of step c) to a reaction under conditions that restore the activity of the DNA deaminase, thereby allowing the DNA deaminase to deaminize one or more cytidines (C) in or near the region of the genomic DNA that interacts with the target protein;

[0089] e) isolating genomic DNA from the product of step d); and

[0090] f) sequencing the isolated genomic DNA or a portion thereof, and determining the interaction between the target protein and the genomic DNA based on the sequencing result.

[0091] In some embodiments, the at least one cell is immobilized on a solid support, such as magnetic beads.

[0092] In order to retain the in situ interaction information of the target protein and genomic DNA to the maximum extent, the permeabilization buffer should be mild, which can destroy or partially destroy the cell wall / cell membrane, allowing antibodies and fusion proteins to enter the cells, but not significantly destroy the protein-DNA interaction in the cells. Cell nucleus separation should also be mild and not significantly destroy the protein-DNA interaction.

[0093] However, the proteins and DNA in the cells may also be cross-linked in step a). The cross-linking methods include, but are not limited to, UV cross-linking, formaldehyde and paraformaldehyde fixation, etc. In some embodiments, the cells are treated with formaldehyde at a final concentration of about 0.1% to cross-link the proteins and DNA in the cells.

[0094] In some embodiments, the permeabilization buffer comprises a detergent. In some embodiments, the detergent is Digitonin. In some embodiments, the permeabilization buffer comprises approximately 0.02% Digitonin.

[0095] In some embodiments, the permeabilization buffer comprises about 20 mM HEPES pH 7.5, about 150 mM NaCl, about 0.5 mM Spermidine, about 0.1% BSA, about IX PIC, about 2 mM EDTA, and about 0.02% Digitonin.

[0096] In some embodiments, step a) of "treating at least one of the cells with a permeabilization buffer comprising a detergent" is performed at about 20° C. to about 37° C. In some preferred embodiments, step a) is performed at room temperature (eg, about 25° C.).

[0097] In some embodiments, at least one of the cells is treated with a permeabilization buffer comprising a detergent for about 5 minutes to about 10 minutes in step a).

[0098] In some embodiments, the at least one cell is about 1 cell to about 5000 cells or more. In some preferred embodiments, the at least one cell is no more than 5000 cells, no more than 2500 cells, no more than 1000 cells, no more than 500 cells, no more than 100 cells, no more than 50 cells, no more than 10 cells. In some preferred embodiments, the at least one cell is 1 cell.

[0099] "Deaminase" refers to an enzyme that catalyzes a deamination reaction. As used herein, "DNA deaminase" refers to a deaminase that can accept DNA (single-stranded or double-stranded) as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. The DNA deaminase can deaminate C in regions of genomic DNA that are not bound by the target protein (e.g., regions on both sides of the site of interaction between the genomic DNA and the target protein) to U, so in the subsequent amplification reaction for sequencing, the CG of the original genomic DNA is converted to TA. By detecting the conversion of CG to TA, the deamination site can be detected.

[0100] The DNA deaminase described in the present invention can be single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA) or a functional fragment thereof. The DNA deaminase described in the present invention can be from different species, such as from Burkholderia cenocepacia and Simiaoa sunii.

[0101] As used herein, "functional fragment" refers to a fragment of a DNA deaminase that substantially retains its deamination activity, such as DddA tox or SsdA tox known in the art.

[0102] In some embodiments, the DNA deaminase is DddA or a functional fragment thereof, preferably DddA tox. In some embodiments, the DddA tox comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity with SEQ ID NO: 1.

[0103] Protein A or Protein G is well known in the art, and it can specifically bind to the constant region of immunoglobulin (antibody) and is commonly used to purify antibodies. Various variants of Protein A or Protein G are also known in the art, which are also encompassed within the scope of Protein A or Protein G of the present invention.

[0104] Exemplary protein A useful herein comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity to SEQ ID NO: 2. In some embodiments, protein A comprises the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity to SEQ ID NO: 3.

[0105] The DNA deaminase can be fused directly to protein A or protein G, or fused via a peptide linker. In some embodiments, the peptide linker is a rigid peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 4 or 5, preferably comprises the amino acid sequence shown in SEQ ID NO: 4.

[0106] In some embodiments, the fusion protein also contains a tag, such as a tag for separation and / or purification of the fusion protein. Methods for recombinant protein production are known in the art. And a variety of tags that can be used for separation and / or purification of proteins are known in the art, including but not limited to His tags, smt3 tags, GST tags, etc. Generally speaking, these tags do not change the activity of the target protein. In some preferred embodiments, the fusion protein comprises a His tag.

[0107] In some embodiments, the fusion protein of the DNA deaminase and protein A or protein G comprises the amino acid sequence shown in SEQ ID NO:6 or 7.

[0108] In some embodiments, step b) can also add a second antibody, which specifically binds to the first antibody, or specifically binds to a different epitope of the target protein. In some embodiments, the second antibody specifically binds to the constant region of the first antibody. Depending on the source of the first antibody, a suitable commercial second antibody can be selected. For example, if the first antibody is a mouse antibody, a commercial rabbit anti-mouse or goat anti-mouse IgG second antibody can be selected.

[0109] In some embodiments, step b) is performed at low temperature. For example, step b) is performed at about 4°C to about 10°C, preferably about 4°C.

[0110] In some embodiments, step b) is performed in a first binding buffer. In some embodiments, the first binding buffer comprises a detergent. The detergent is, for example, Ttriton x 100, Digitonin, or sodium deoxycholate. In some embodiments, step b) the first binding buffer comprises approximately 0.02% Digitonin.

[0111] In some embodiments, step b) the first binding buffer further comprises a protease inhibitor such as commercially available PIC (protease inhibitor cocktail).

[0112] In some embodiments, the first binding buffer does not contain serum albumin such as BSA (bovine serum albumin).

[0113] In some embodiments, the first binding buffer comprises about 90 mM to about 200 mM, preferably about 150 mM of a metal salt such as NaCl.

[0114] In some embodiments, the first binding buffer comprises about 20 mM HEPES pH 7.5, about 150 mM NaCl, about 0.5 mM Spermidine, about 0.1% BSA (bovine serum albumin), about 1X PIC (protease inhibitor cocktail), and about 0.02% Digitonin. In some embodiments, step b) the first binding buffer comprises about 20 mM HEPES pH 7.5, about 150 mM NaCl, about 0.5 mM Spermidine, about 1X PIC (protease inhibitor cocktail), and about 0.02% Digitonin.

[0115] In some embodiments, step b) is performed for about 1 hour to about 12 hours or longer, such as about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours or longer.

[0116] In some embodiments, step b) further comprises the step of removing unbound first antibody.

[0117] The present inventors surprisingly found that carrying out step c) at low temperature and / or in the presence of high salt can allow the fusion protein to fully bind to the first antibody-target protein complex and can significantly inhibit the activity of DNA deaminase, thereby avoiding undesired deamination.

[0118] In some embodiments, step c) is performed at low temperature. For example, step c) is performed at about 4°C to about 10°C, preferably about 4°C.

[0119] In some embodiments, step c) is performed in a second binding buffer. In some embodiments, the second binding buffer comprises a high concentration of salt. For example, step c) the second binding buffer comprises about 90 mM to about 200 mM, preferably about 150 mM of a metal salt such as NaCl.

[0120] In some embodiments, step c) the second binding buffer comprises about 0.05% to about 0.2%, such as about 0.1%, of serum albumin such as BSA. In some preferred embodiments, step c) the second binding buffer does not comprise serum albumin such as BSA.

[0121] In some embodiments, the second binding buffer of step c) contains a detergent. The detergent is, for example, Ttriton x 100, Digitonin or sodium deoxycholate. In some embodiments, the buffer of step c) contains about 0.02% Digitonin.

[0122] In some embodiments, step c) the second binding buffer further comprises a protease inhibitor such as commercially available PIC (protease inhibitor cocktail).

[0123] In some embodiments, step c) the second binding buffer comprises about 20 mM HEPES pH 7.5, about 150 mM NaCl, about 0.5 mM Spermidine, about 0.1% BSA, about 1X PIC, and about 0.02% Digitonin. In some embodiments, step c) the second binding buffer comprises about 20 mM HEPES pH 7.5, about 150 mM NaCl, about 0.5 mM Spermidine, about 1X PIC, and about 0.02% Digitonin.

[0124] In some embodiments, the second binding buffer in step c) comprises about 10 mM Tris-HCl pH 8.0, about 90 mM NaCl, 0.04% Triton X100, 0.02% DOC (sodium deoxycholate), 0.1 mM PMSF (phenylmethylsulfonyl fluoride) and 1X PIC.

[0125] In some embodiments, the concentration of the fusion protein in step c) is about 0.02 to about 0.24 μM or higher, preferably about 0.02 to about 0.08 μM.

[0126] In some embodiments, step c) is performed for about 0.5 hours to about 2 hours or longer, such as for about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours or longer.

[0127] In some embodiments, step c) further comprises the step of removing unbound fusion protein.

[0128] In some embodiments, step d) comprises exchanging the buffer of the product of step c) with a deamination reaction buffer. In some embodiments, the deamination reaction buffer comprises a low concentration of salt. For example, the deamination reaction buffer of step d) comprises no more than about 50 mM, about 10 mM, no more than about 5 mM, preferably no more than about 2 mM of a metal salt such as NaCl or MgCl2. In some preferred embodiments, the deamination reaction buffer comprises about 50 mM NaCl and / or 2 mM MgCl2.

[0129] In some embodiments, the deamination reaction buffer of step d) comprises about 20 mM MES pH 6.4, about 1 mM DTT, about 2 mM MgCl2, 1X PIC.

[0130] In some embodiments, the reaction of step d) is carried out at about 20° C. to about 40° C. In some preferred embodiments, the reaction of step d) is carried out at about 37° C.

[0131] Replacing the deamination reaction buffer and reacting at an elevated temperature in step d) can restore the activity of the DNA deaminase, thereby allowing the DNA deaminase to deaminize one or more cytidines (C) in or near the region of the genomic DNA that interacts with the target protein.

[0132] In some embodiments, the reaction of step d) is carried out for about 10 minutes to about 2 hours or longer, for example, for about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours or longer. In some preferred embodiments, the reaction of step d) is carried out for at least 30 minutes.

[0133] In some embodiments, step d) further comprises adding a uracil glycosylase inhibitor (UGI) to the reaction. Adding UGI can inhibit the activity of the enzyme present in the reaction system that may remove U, thereby avoiding the effects caused by the removal of U produced by the deamination reaction. UGI that can be used in the method of the present invention can be commercially available.

[0134] The isolation of genomic DNA in step e) can be performed by conventional methods known in the art, for example, by using a commercial genomic DNA extraction kit.

[0135] The method of the present invention can be used to detect the interaction between certain specific regions of the cell genome and the target protein. In this case, only these specific regions need to be sequenced.

[0136] Therefore, in some embodiments, step f) comprises amplifying a specific portion of the genomic DNA and sequencing it to determine the interaction between the specific portion of the cell genome and the target protein.

[0137] In some embodiments, the amplification is PCR amplification, such as PCR amplification performed using a U-tolerant DNA polymerase, for example, the U-tolerant DNA polymerase is selected from: HiFi V3 (component in the Novozymes single-stranded library construction kit, NE103), Phusion U (Thermo Scientific F555L), Phanta Uc (vazyme, P507-01), Q5U (NEB, M0515L) and Q6U (Biyuntian, D7239M).

[0138] The method of the present invention can also be used to detect the interaction between the target protein and genomic DNA at the whole genome level.

[0139] Therefore, in some embodiments, step f) comprises establishing a whole genome DNA library from the isolated genomic DNA, and performing sequencing based on the whole genome library, thereby determining the interaction between the target protein and genomic DNA at the whole genome level.

[0140] Various methods known in the art for establishing a whole genome sequencing library from isolated genomic DNA, such as a double-stranded DNA library construction method, can be used. A whole genome DNA library can be established by a commercial kit, such as a double-stranded library construction using Tn5, such as the Novozyme TD502 kit.

[0141] In some specific embodiments, by Novozyme The DNA library was constructed using DNA Library Prep Kit V2forIllumina TD502.

[0142] In some embodiments, a U-tolerant DNA polymerase is used for DNA amplification in the library establishment, for example, the U-tolerant DNA polymerase is selected from the group consisting of HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U.

[0143] Sequencing described herein can be first generation sequencing such as Sanger sequencing, second generation sequencing (NGS) or other high throughput sequencing. For example, the second generation sequencing of Illumina can be used.

[0144] If the target protein can interact (bind) with a specific region of genomic DNA, such as a specific sequence (motif), the specific DNA region of the interaction will be protected by the target protein and will not be deaminated, while the C in the DNA region near it (upstream and / or downstream) can be converted into U by DNA deaminase reaction, resulting in a C to T conversion at this site in the final sequencing result (G to A conversion for the relative chain). Therefore, by analyzing the presence and / or distribution of deamination sites, the presence and / or distribution of the interaction between the target protein and the genomic DNA can be determined. For example, if there is a significant CG to TA conversion in both the upstream and downstream DNA of a certain region, the sequence in the middle that does not show a CG to TA conversion may be the sequence (such as a binding motif) where the target protein interacts (binds) with the genomic DNA.

[0145] Thus, in some embodiments, the interaction of a target protein with the genomic DNA or a portion thereof of the cell is determined by analyzing the presence of CG to TA transitions (i.e., C to T and G to A) in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

[0146] In some embodiments, the interaction of the target protein with the genomic DNA or a portion thereof of the cell is determined by analyzing the position and / or density and / or rate of CG to TA transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

[0147] The sequence of the control genomic DNA or its part can be the sequence of the genomic DNA or its part from a public database. Alternatively, the sequence of the control genomic DNA or its part can also be the sequence of the genomic DNA or its part of the same type of cell that has not been treated with the DNA deaminase.

[0148] The target protein described herein is, for example, a DNA binding protein, and examples of DNA binding proteins may be transcription factors, histones, etc. Preferably, the target protein is a histone. The transcription factors include, but are not limited to, CTCF, Klf4, Thap11, SP1, Nrf1, etc., preferably CTCF.

[0149] The antibodies described herein can be prepared by methods known in the art or can be commercially obtained. The antibody should be an antibody that can be bound by protein A or protein G, for example, it is an IgG antibody or it contains an Fc region derived from IgG.

[0150] The cells described herein can be animal cells, plant cells, or microbial cells. For example, the cells are mammalian cells, including but not limited to cells of humans, mice, rats, cats, dogs, pigs, and cattle; or, the cells are monocotyledonous or dicotyledonous plant cells, such as cells of rice, corn, wheat, sorghum, soybean, potato, tomato, etc.; or, the cells are fungi such as yeast cells.

[0151] The cell may be a cell line cell. Alternatively, the cell may be a primary cell from a different organ or tissue. For example, the cell may be from blood, cerebrospinal fluid, or a biopsy. The cell may be a hepatocyte, a cardiomyocyte, a neuron, a fibroblast, an epithelial cell, or the like. The cell may also be a tumor cell, a stem cell such as an embryonic stem cell, or an induced pluripotent stem cell.

[0152] In some embodiments, the cell is a cell treated with a specific substance (compound) or condition or a cell at a specific developmental stage. Thus, the method of the present invention can measure the effect of the specific substance (compound) or condition or specific developmental stage on the interaction between a specific target protein and genomic DNA. For example, the specific substance can be a drug.

[0153] 3. Test kit

[0154] In another aspect, the present invention provides a kit for detecting the interaction between a target protein and genomic DNA in a cell, which comprises at least the fusion protein comprising a DNA deaminase and protein A or protein G as described herein above.

[0155] In some embodiments, the kit is used to detect the interaction between the target protein and genomic DNA in a cell by the method of the present invention.

[0156] In some embodiments, the kit further comprises a first antibody that specifically binds to the target protein, and optionally a second antibody that specifically binds to the first antibody.

[0157] In some embodiments, the kit further comprises a permeabilization buffer comprising a detergent and / or a reagent for isolating cell nuclei. The permeabilization buffer is as defined above.

[0158] In some embodiments, the kit further comprises the first binding buffer, the second binding buffer and / or the deamination reaction buffer described above.

[0159] In some embodiments, the kit further includes reagents for amplifying the genomic DNA portion of interest, such as specific primers.

[0160] In some embodiments, the kit further comprises reagents for establishing a genomic library.

[0161] In some embodiments, the kit further comprises reagents for sequencing, such as Sanger sequencing or next generation sequencing.

[0162] The kit generally also includes a label indicating the intended use and / or method of use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit. Example

[0163] The techniques used in the following examples, including sequencing library establishment and high-throughput sequencing technology, as well as molecular biology techniques such as cell fixation and DNA purification, and cell culture and detection techniques, are conventional techniques known to those skilled in the art, unless otherwise specified. The instruments, equipment, reagents, and cell lines used, unless otherwise specified in this specification, are generally available to researchers and technicians in the field through public channels. In the examples listed in the specific implementation methods, the cell collection, processing, and single cell separation schemes and some buffer systems represent only a few of the many feasible treatment schemes.

[0164] Example 1: qDEPI-seq detection of a small amount of mouse embryonic fibroblasts

[0165] Purpose:

[0166] The method of the present invention is used to detect the CTCF-DNA interaction of a small amount of mouse embryonic fibroblasts, and a DNA library that can be used for Illumina detection is constructed.

[0167] 1. Obtaining Mouse Embryonic Fibroblasts

[0168] 1) Remove the culture medium of the cultured mouse fibroblasts and wash them once in PBS buffer.

[0169] 2) Add to TE solution and place at 37°C for 5 minutes to digest into free single cells.

[0170] 3) Add five times the amount of culture medium to terminate the enzyme digestion reaction, collect the cells, remove the culture medium after centrifugation, resuspend in PBS and wash once.

[0171] 2. Magnetic Bead Capture of Mouse Embryonic Fibroblasts

[0172] 1) Wash the cells with 1 ml of room temperature Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Permidine, 0.1% BSA, 1X PIC) and centrifuge at 1000 rpm for 3 minutes at room temperature, then discard the liquid. Wash twice in total

[0173] 2) Pipette 600 μl of room temperature wash buffer and gently pipette to resuspend.

[0174] 3) Transfer 20 μl CoA beads into an EP tube containing 3X volume of pre-cooled Binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2).

[0175] 4) Wash the beads twice in 1 mL of cold Binding buffer and resuspend in 300 μL of binding buffer.

[0176] 5) Add beads to cells and vortex gently, then incubate at room temperature for 10 minutes.

[0177] 3 Antibody Immunoconjugate

[0178] 1) Place the EP tube containing beads on a magnetic stand to wash and discard the liquid.

[0179] 2) Use 1 mL of cold Blocking buffer (Washing buffer supplemented with 2 mM EDTA and 0.02% Digitonin) to gently permeabilize the cells and block the beads adsorbed with cells, and incubate at room temperature for 5 minutes.

[0180] 3) Discard the supernatant and wash the complex of cells and beads in 1 mL of cold Dig-Wash Buffer (Washing buffer supplemented with 0.02% Digitonin), and then resuspend it in 300 μL of cold Dig-Wash Buffer.

[0181] 4) Add 2 μL rabbit anti-mouse CTCF antibody (1:150 dilution) and gently shake to mix.

[0182] 5) Incubate the samples with rotation at 4°C for 2 hours or overnight.

[0183] 4. DddA-protein A / G binding to antibodies

[0184] 1) Discard the supernatant and wash the sample twice in 1 mL of cold Dig-Wash Buffer. Discard the supernatant and resuspend the sample in 300 μL of cold Dig-Wash Buffer. Wash the EP tube containing the beads on a magnetic stand and discard the liquid.

[0185] 2) Add DddA-protein A / G to a final concentration of 0.08 μM and incubate at low temperature (4°C) for 1 hour.

[0186] 3) Discard the supernatant and wash the cell-beads complex in 1 mL cold Dig-Wash Buffer (Washing buffer supplemented with 0.02% Digitonin), and resuspend the sample in 300 μL cold reaction buffer (20 mM MES pH 6.4, 1 mM DTT, 2 mM MgCl2, 1X PIC).

[0187] 5. Deamination reaction and DNA collection

[0188] 1) Incubate at 37°C for 30 minutes.

[0189] 2) Add 10 mM NaHCO3, 1% SDS, 1 μL proteinase K, and incubate at 65°C with shaking for 1 hour.

[0190] 3) Add an equal volume of PCI and vortex at full speed for 2 seconds to mix. Transfer to a phase-lock tube and centrifuge at 16,000g for 5 minutes at room temperature.

[0191] 4) Transfer the upper layer to a new tube containing 0.5 μL glycoblue and 1 / 10 volume 3M NaAc pH 5.2 and mix well. Add an equal volume of isopropanol and shake to mix well. Place it at -80℃ for 2 hours or overnight.

[0192] 5) Centrifuge at 16,000 x g for 15 min at 4°C and discard the supernatant.

[0193] 6) Wash the pellet twice with 1 ml of 80% ethanol, incubate at room temperature for 5 min each time, and then centrifuge at 16,000 x g for 5 min at 4°C. Carefully discard the liquid and air dry, and re-dissolve the pellet with water.

[0194] 6. Library establishment

[0195] 1) Select the total amount of starting DNA according to the measured DNA concentration and experimental requirements. Take 5 ng starting amount as an example and use Novizan DNA Library Prep Kit V2for Illumina TD502 was used for Tn5-based DNA fragmentation (the system was prepared according to the kit instructions with 5×TTBL, TTE Mix V5, and 5 ng DNA supplemented with ddH2O to 20 μL).

[0196] 2) Incubate in PCR instrument at 55°C for 10 minutes.

[0197] 3) Add 5X Stop Buffer to terminate the reaction. Then repeat steps 3-6 in "5 Deamination reaction and DNA collection" to purify the fragmented DNA.

[0198] 4) Using NEB Library amplification was completed using HF dUTP-tolerant high-fidelity DNA polymerase and Novizan TruePrep IndexKit V2 for Illumina (Vazyme#TD202) adapter kit. The PCR program was 72°C for 3 min, 98°C for 30 s, followed by 98°C for 10 s, 61.5°C for 30 s, and 72°C for 30 s for 8-12 cycles, and finally 72°C for 5 min.

[0199] 5) Add 0.6X XP reagent, for DNA purification, the supernatant was transferred to the original system 0.2X After purification, elute the DNA in a new PCR tube containing XP reagent and at least 11 μl of Elution Buffer.

[0200] 6) Take out 1 μl of the eluted DNA and use Qubit dsDNA HS Assay Kit (purchased from Invitrogen, catalog number Q32851) to detect the concentration.

[0201] 7) DNA fragments were analyzed using the Agilent 2100 biochip analysis system.

[0202] 8) The constructed library was sent to the company for high-throughput sequencing. The sequencing machine used was IlluminaXTEN, and the sequencing mode was double-end 150bp.

[0203] Experimental results:

[0204] 1) 500 and 5000 cells were used for detection respectively, and the coverage of the detection data on the genome could reach about 95% of the whole genome, without obvious decrease, which shows that the method of the present invention is very effective for detecting CTCF binding sites in a small number of cells.

[0205] 2) If Figure 2 As shown, the fragment lengths of the detected data are mostly concentrated in the range of 300-600 bp, without a large number of fragments that are too long or too short.

[0206] 3) If Figure 4 As shown, after testing different surfactant digitonin concentrations (0.012%, 0.02%, 0.04%), it was found that the 0.02% concentration was optimal.

[0207] 4) If Figure 3 As shown, using a small number of cells to detect the binding site of CTCF, a specific high CG to TA conversion efficiency region can be seen, which shows that the experimental scheme, sequencing and analysis of the present invention are feasible. And the verification results of this scheme did not find significant differences when the number of cells was reduced from 5000 to 500, and the reproducibility between samples was excellent.

[0208] Therefore, from the above experimental results, it can be seen that the experimental data quality obtained by using the experimental method of the present invention to detect CTCF binding sites in a small number of cells is not significantly different from that of a large number of cells, and both can show high conversion results in the CTCF motif region. This shows that the experimental method of the present invention can well meet the needs of protein-DNA interaction site detection in a small number of cell samples, and can also be performed at the level of transcription factors such as CTCF that are difficult to measure in previous methods.

[0209] Example 2: qDEPI-seq detection of mouse MII oocytes

[0210] Purpose:

[0211] The method of the present invention is used to detect the CTCF-DNA interaction of a single mouse MII oocyte, and to construct a DNA library that can be used for Illumina detection.

[0212] 1. Obtaining mouse MII oocytes

[0213] 1) Take 6-8 week old C57 female mice and inject 5U pregnant mare serum gonadotropin (PMSG) intraperitoneally. 48 hours later, inject 6U human chorionic gonadotropin (hCG) intraperitoneally.

[0214] 2) After 16-18 hours, the mother mouse was killed and the MII eggs were taken out. The granulosa cells were removed by hyaluronidase and the zona pellucida was removed by pronase to obtain the naked MII oocytes.

[0215] 3) The eggs were washed three times in PBS solution to remove the enzymes, and then transferred to fresh PBS solution.

[0216] 2 Microfixation of Mouse MII Eggs

[0217] 1) Prepare a micro-fixation system, add 37% formaldehyde solution to 500 μl PBS solution to make the final concentration of formaldehyde 0.1%, transfer the MII eggs temporarily stored in PBS to the solution, and incubate at room temperature for 2 minutes.

[0218] 2) Add 15 μl of 2.5 M glycine solution to the system and incubate at room temperature for 5 minutes to stop cross-linking.

[0219] 3) Wash the oocytes treated above twice with fresh PBS.

[0220] 3. Permeabilization of mouse MII oocytes

[0221] 1) Transfer the eggs to 1 ml of room temperature Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Permidine, 0.1% BSA, 1X PIC) and wash the cells twice in total.

[0222] 2) Transfer the eggs to 1 mL of cold Blocking buffer (Wash buffer supplemented with 2 mM EDTA and 0.02% Digitonin) for membrane permeabilization and blocking, and incubate at room temperature for 5 minutes.

[0223] 3) The eggs from step 2) were transferred to 1 mL of cold Dig-Wash Buffer (Washing buffer supplemented with 0.02% Digitonin) and washed twice.

[0224] 4) Transfer the eggs to 300 μl cold Dig-Wash Buffer with 2 μl rabbit anti-mouse CTCF antibody (1:150 dilution) and incubate on a shaker at 4°C for 2 hours or overnight.

[0225] 4. DddA-protein A / G binding to antibodies

[0226] 1) Transfer the eggs to 1 mL of cold Dig-Wash Buffer and wash the sample twice.

[0227] 2) Prepare D-ChIP Buffer (10 mM Tris-HCl pH 8.0, 90 mM NaCl, 0.04% Triton X100, 0.02% DOC, 0.1 mM PMSF, 1X PIC) and add DddA-protein A / G to a final concentration of 0.08 μM, and incubate at 4°C for 1 hour.

[0228] 3) Transfer the eggs to 1 mL cold D-ChIP Buffer and wash twice, and transfer the sample to 300 μL cold reaction buffer (20 mM MES pH 6.4, 1 mM DTT, 2 mM MgCl2, 1X PIC).

[0229] 5. Deamination reaction and DNA collection

[0230] 1) Incubate at 37°C for 30 minutes.

[0231] 2) Library construction was performed based on the published META-CS seq technology. For details on the construction of META Tn5, please refer to the literature Accurate SNV detection in single cells by transposon-based whole-genome amplification of complementary strands. Individual eggs were transferred to 2 μL META lysis buffer (20 mM Tris-HCl pH 8.0, 20 mM NaCl, 0.15% Triton X-100, 25 mM DTT, 1 mM EDTA, 1.5 mg / mL Qiagen protease [1919155], 500 nM vector ssDNA) and lysed at 50°C for 1 hour and 70°C for 15 minutes. The sequence of the vector ssDNA is 5'-TCAGGTTTTCCTGAA-3'.

[0232] 3) Single cell lysates were transposed by adding 8 μL of transposition mix (10 mM TAPS pH 8.5, 5 mM MgCl2, 8% PEG 8000 and 0.38 nM META Tn5) and incubating for 10 minutes at 55° C. The reaction was terminated by adding 2 μL of stop buffer containing 300 nM NaCl, 45 mM EDTA, 0.01% Triton X-100, 1 mg / mL Qiagen protease and incubating at 50° C. for 30 minutes and 70° C. for 15 minutes.

[0233] 4) First strand labeling was performed by adding 13 μL strand tagging Mix 1 (containing 5 μL PhU-HF reaction buffer, 0.8 μL 50 μM Adp1 primer mix, 0.6 μL 100 mM MgCl2, 0.6 μL water, 0.5 μL 10 mM dNTP mix, 0.25 μL 20 mg / mL BSA (NEB) and 0.25 μL PhU DNA polymerase) and incubating at 72°C for 3 min, 98°C for 30 s, 62°C for 5 min, and 72°C for 1 min. The Adp1 primer was removed by adding 1 μL exonuclease I (NEB; M0293) and incubating at 37°C for 30 min and 80°C for 20 min.

[0234] 5) Second strand labeling was performed by adding 4 μL strand tagging Mix 2 (containing 1 μL PhU-HF reaction buffer, 1 μL 50 μM Adp2 primer mix, 0.855 μL water, 0.1 μL 10 mM dNTP mix, and 0.05 μL PhU DNA polymerase) and incubating at 72°C for 3 min, 98°C for 30 s, 62°C for 5 min, and 72°C for 1 min. The Adp2 primer was removed by adding 1 μL exonuclease I (NEB; M0293) and incubating at 37°C for 30 min and 80°C for 20 min.

[0235] 6) Amplify by adding 19 μL PCR Mix (containing 5 μL NEBNext Multiplex Oligos Universal Primer, 5 μL NEB Index Primers (NEB; E7335S, E7500S, E7710S, E7730S), 4 μL PhU-HF reaction buffer, 0.4 μL water, 0.4 μL 10 mM dNTP mixture and 0.2 μL PhU DNA polymerase).

[0236] 7) Add 0.6X XP reagent, DNA purification, this step takes the supernatant and transfers it to the original system 0.6X After purification, use at least 11 μl Elution Buffer to elute the purified DNA on the beads twice. The first elution is recorded as the long fragment library, and the second elution is recorded as the short fragment library.

[0237] 8) Take out 1 μl of the eluted DNA and use Qubit dsDNA HS Assay Kit (purchased from Invitrogen, catalog number Q32851) to detect the concentration.

[0238] 9) DNA fragments were analyzed using the Agilent 2100 biochip analysis system.

[0239] 10) The constructed library was sent to the company for high-throughput sequencing. The sequencing machine used was IlluminaXTEN. The sequencing mode of the long fragment library was double-end 150bp, and the sequencing mode of the short fragment library was double-end 150bp.

[0240] Experimental results:

[0241] 1) Using a single MII oocyte for detection, the coverage of the detection data on the genome can reach about 70% of the whole genome, which is a breakthrough in the detection of single-cell protein-DNA interactions. This shows that the method of the present invention is also very effective in detecting CTCF binding sites in single cells.

[0242] 2) If Figure 5 and Figure 6 As shown, the fragment lengths of the short fragment library of the detected data are mostly concentrated in the range of 300-600bp, and the fragment lengths of the long fragment library are mostly concentrated in the range of 300-600bp, both of which are in line with the expectations of library construction.

[0243] 3) If Figure 7 The results show that, when tested with a single oocyte, a specific region with high CG to TA conversion efficiency can be seen at the CTCF binding site, which is similar to that of a large number of cell samples. This indicates that the method, sequencing and analysis of the present invention are also feasible at the single cell level.

[0244] Figure 8 The CTCF peak information and motif sites obtained from ChIP seq data in the public database are shown in the figure. The method of the present invention can also be used to find that there are specific high-conversion rate sites nearby. The editing efficiency of TC sites upstream and downstream of some CTCF motifs is close to 100%, indicating the feasibility of quasi-quantification.

[0245] Therefore, from the above experimental results, it can be seen that the experimental data obtained by using the method of the present invention to detect CTCF binding sites in single oocytes is of qualified quality, and can show high conversion in the CTCF motif region similar to the results of a large number of cell samples. It shows that the method of the present invention can meet the needs of protein-DNA interaction site detection in single cell samples, and completes the detection of protein-DNA interaction information at the single cell starting level that has not been completed in previous methods.

[0246] Example 3. Optimization of qDEPI-seq detection

[0247] 1. Optimization of Protein A-DddA Fusion Protein

[0248] Different Protein A-DddA fusion proteins were tested using different linkers: flexible (F) and rigid (R). Fig. 9 As shown, rigid joints are better than flexible joints.

[0249] 2. Optimize the buffer system for in situ binding of antibodies to target proteins

[0250] The buffer system during the in situ binding of antibodies was tested, including NaCl concentration (90mM and 150mM), surfactant types (Triton X100, digitonin, sodium deoxycholate, etc.) and concentrations. Fig.10 As shown, the experiment is more efficient at 150 mM NaCl, and the type of surfactant has no significant effect. The addition of BSA may have a negative impact.

[0251] 3. Optimizing the binding of fusion protein and antibody

[0252] The buffer system and enzyme concentration during the binding process between the antibody and protein A-DddA were tested. Fig.11 It can be seen that a final concentration of 0.02-0.08 μM is sufficient to implement the present invention. The addition of BSA may have a negative effect.

[0253] 4. pA-D deamination reaction buffer optimization and reaction time optimization

[0254] Eight sets of tests were conducted according to the following table:

[0255]

[0256]

[0257] This experiment aims to explore the effects of NaCl concentration in the buffer system and reaction time on the experimental system during the deamination reaction. The fifth IgG group is the control group. After the protein A-DddA and antibody binding process, the samples were collected directly without deamination reaction (the other groups collected samples after deamination reaction). This is to reflect that the enzyme activity is well controlled during the immune binding reaction and the binding of protein A-DddA to the antibody in the experimental process.

[0258] Depend on Fig.12 It can be seen that the best reaction time is about 30 minutes. The reaction of the group with less than 30 minutes is not complete, and the group with 60 minutes has no significant improvement in average conversion efficiency.

[0259] At the same time, it can be seen that in the 50mM NaCl group, the activity of the enzyme tethered to the designated site (CTCF in the figure), that is, the conversion efficiency, was significantly improved, while when the NaCl concentration approached 100, there was a significant inhibitory effect.

[0260] From the results of the IgG-IP group, it can be seen that the activity of the enzyme is well controlled in the binding step of protein A-DddA and the antibody, indicating that the signal does not come from the background signal brought by this step.

[0261] 4. Impact of amplification bias

[0262] Deamination-modified DNA may produce amplification bias during PCR amplification, and this bias may affect the final sequencing quality of this method. Fig.13 In the experiment shown, DNA fragments containing 2, 4, 6, and 8 dUTPs and original fragments without dUTP were obtained by artificial synthesis to simulate the DNA after deamination reaction in the method. These artificially synthesized DNA fragments with different dUTP contents were mixed in a certain proportion and amplified by PCR to complete the library establishment. After the second-generation sequencing, the results are as follows Fig.13 The sequencing results show that the PCR amplification and library establishment process in the method of the present invention does not introduce significant amplification bias.

[0263] Sequence information:

[0264] SEQ ID NO:1 DddA tox

[0265] IGLNGGANVYHYAPNPVGWVDPWGLAGSYALGPYQISAPQLPAYNGQTVGTFYYVN

[0266] DAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFC

[0267] VNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0268] SEQ ID NO:2 Protein A

[0269] MTMITPSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEE

[0270] QRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTE

[0271] EQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK

[0272] SEQ ID NO:3ZZ domin Protein A

[0273] VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAP

[0274] KVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNGAQA

[0275] P

[0276] SEQ ID NO:4 Rigid joint

[0277] AEAAAKEAAAKEAAAKA

[0278] SEQ ID NO:5 Flexible joint

[0279] GGGGSGGGGSGGGGSGGGG

[0280] SEQ ID NO:6 Fusion protein

[0281] IGLNGGANVYHYAPNPVGWVDPWGLAGSYALGPYQISAPQLPAYNGQTVGTFYYVN

[0282] DAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFC

[0283] VNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGCASAEAA

[0284] AKEAAAKEAAAKAVDPAGLMTMITPSLKDDPSQSANLLSEAKKLNESQAPKADNKF

[0285] NKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNK

[0286] FNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK

[0287] SEQ ID NO:7 Fusion protein

[0288] HHHHHHSQDPLEVLFQGPEASIGLNGGANVYHYAPNPVGWVDPWGLAGSYALGPY

[0289] QISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFM

[0290] RDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKV

[0291] FTGNSNSPKSPTKGGCASAEAAAKEAAAKEAAAKAVDPAGLMTMITPSLKDDPSQSA

[0292] NLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSA

[0293] NLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVS

[0294] KEILAEAKKLNDAQAPK。

Claims

1. A method for detecting the interaction between a target protein and genomic DNA in a cell, the method comprising: a) treating at least one of the cells with a permeabilization buffer, or isolating a cell nucleus from at least one of the cells; b) contacting the permeabilized cells or isolated cell nuclei obtained in step a) with a first antibody that specifically binds to the target protein, thereby allowing the first antibody to fully bind to the target protein in the cell and form a complex; c) contacting a fusion protein comprising a DNA deaminase and protein A or protein G with the product of step b), allowing the fusion protein to fully bind to the complex while the activity of the DNA deaminase is inhibited; d) subjecting the product of step c) to a reaction under conditions that restore the activity of the DNA deaminase, thereby allowing the DNA deaminase to deaminize one or more cytidines (C) in or near the region of the genomic DNA that interacts with the target protein; e) isolating genomic DNA from the product of step d); and f) sequencing the isolated genomic DNA or a portion thereof, and determining the interaction between the target protein and the genomic DNA based on the sequencing result.

2. The method of claim 1, wherein in step a) the proteins and DNA in the cells are cross-linked, for example by UV cross-linking, by formaldehyde or paraformaldehyde fixation.

3. The method of claim 1 or 2, wherein the permeabilization buffer comprises a detergent such as Digitonin, for example, the permeabilization buffer comprises approximately 0.02% Digitonin.

4. The method of claim 3, wherein the permeabilization buffer comprises about 20 mM HEPES pH 7.5, about 150 mM NaCl, about 0.5 mM Spermidine, about 0.1% BSA, about IX PIC, about 2 mM EDTA, and about 0.02% Digitonin.

5. The method of any one of claims 1 to 4, wherein step a) of "treating at least one of the cells with a permeabilization buffer comprising a detergent" is performed at about 20°C to about 37°C, preferably at room temperature (e.g., about 25°C).

6. The method of any one of claims 1 to 5, wherein in step a) at least one of the cells is treated with a permeabilization buffer comprising a detergent for about 5 minutes to about 10 minutes.

7. The method of any one of claims 1-6, wherein the at least one cell is from about 1 cell to about 5000 cells or more, Preferably, the at least one cell is no more than 5000 cells, no more than 2500 cells, no more than 1000 cells, no more than 500 cells, no more than 100 cells, no more than 50 cells, no more than 10 cells, more preferably, the at least one cell is 1 cell.

8. The method of any one of claims 1 to 7, wherein the DNA deaminase can be single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA) or a functional fragment thereof, for example, the DNA deaminase is DddA or a functional fragment thereof, preferably DddA tox.

9. The method of claim 8, wherein the DddA tox comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:

1.

10. The method of any one of claims 1 to 9, wherein protein A comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:2; or protein A comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:

3.

11. The method of any one of claims 1 to 10, wherein the DNA deaminase is fused to Protein A or Protein G directly or via a peptide linker.

12. The method of claim 11, wherein the peptide linker is a flexible peptide linker or a rigid peptide linker, preferably a rigid peptide linker, for example, the rigid peptide linker comprises the amino acid sequence shown in SEQ ID NO: 4, and the flexible peptide linker comprises the amino acid sequence shown in SEQ ID NO:

5.

13. The method according to any one of claims 1 to 12, wherein the fusion protein of DNA deaminase and protein A comprises the amino acid sequence shown in SEQ ID NO: 6 or 7.

14. The method according to any one of claims 1 to 13, wherein a second antibody may be further added in step b), wherein the second antibody specifically binds to the first antibody, or specifically binds to a different epitope of the target protein.

15. The method of any one of claims 1 to 14, wherein step b) is performed at low temperature. For example, step b) is performed at about 4°C to about 10°C, preferably about 4°C.

16. The method of any one of claims 1-15, wherein step b) is performed in a first binding buffer, for example, the first binding buffer comprises a detergent selected from Ttriton x 100, Digitonin or sodium deoxycholate, preferably, the first binding buffer of step b) comprises about 0.02% Digitonin.

17. The method of claim 16, wherein the first binding buffer i) further comprises a protease inhibitor; ii) does not contain serum albumin such as BSA; and / or iii) contains about 90 mM to about 200 mM, preferably about 150 mM, of a metal salt such as NaCl.

18. The method of any one of claims 1-17, wherein step b) is performed for about 1 hour to about 12 hours or longer, such as for about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours or longer.

19. The method of any one of claims 1 to 18, wherein step c) is performed at low temperature, for example, step c) is performed at about 4°C to about 10°C, preferably about 4°C.

20. The method of any one of claims 1 to 19, wherein step c) is performed in a second binding buffer, for example comprising about 90 mM to about 200 mM, preferably about 150 mM, of a salt such as NaCl.

21. The method of claim 20, wherein the second binding buffer i) does not contain serum albumin such as BSA; ii) comprising a detergent selected from Ttriton x 100, Digitonin or sodium deoxycholate, for example comprising about 0.02% Digitonin; and / or iii) contains a protease inhibitor.

22. The method of any one of claims 1 to 21, wherein the concentration of the fusion protein in step c) is about 0.02 to about 0.24 μM or higher, preferably about 0.02 to about 0.08 μM.

23. The method of any one of claims 1-21, wherein step c) is performed for about 0.5 hour to about 2 hours or more, such as for about 0.5 hour, about 1 hour, about 1.5 hours, about 2 hours or more.

24. The method of any one of claims 1 to 23, wherein step d) comprises exchanging the buffer of the product of step c) with a deamination reaction buffer, for example, the deamination reaction buffer comprises no more than about 50 mM, about 10 mM, no more than about 5 mM, preferably no more than about 2 mM of a metal salt such as NaCl or MgCl2, preferably, the deamination reaction buffer comprises about 50 mM NaCl and / or 2 mM MgCl2.

25. The method of any one of claims 1 to 24, wherein the reaction in step d) is carried out at about 20°C to about 40°C, preferably, the reaction in step d) is carried out at about 37°C.

26. The method of any one of claims 1 to 25, wherein the reaction of step d) is carried out for about 10 minutes to about 2 hours or longer, preferably, the reaction of step d) is carried out for at least 30 minutes.

27. The method of any one of claims 1 to 26, wherein step f) comprises amplifying a specific portion of the genomic DNA and sequencing it, thereby determining the interaction of the specific portion of the cell genome with the target protein.

28. The method of claim 27, wherein the amplification is PCR amplification, such as PCR amplification performed using a U-tolerant DNA polymerase, for example, the U-tolerant DNA polymerase is selected from: HiFi V3 (component in the Novozymes single-stranded library construction kit, NE103), Phusion U (Thermo Scientific F555L), Phanta Uc (vazyme, P507-01), Q5U (NEB, M0515L) and Q6U (Biyuntian, D7239M).

29. The method of any one of claims 1 to 26, wherein step f) comprises establishing a whole genome DNA library from the isolated genomic DNA, and performing sequencing based on the whole genome library, thereby determining the interaction between the target protein and genomic DNA at the whole genome level.

30. The method of claim 29, wherein a U-tolerant DNA polymerase is used for DNA amplification in the library establishment, for example, the U-tolerant DNA polymerase is selected from the group consisting of HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U.

31. The method of any one of claims 1-30, wherein the interaction of the target protein with the cellular genomic DNA or a portion thereof is determined by analyzing the presence of CG to TA transitions (i.e., C to T and G to A) in the cellular genomic DNA or a portion thereof relative to a control genomic DNA or a portion thereof.

32. The method of any one of claims 1-31, wherein the target protein is a DNA binding protein, such as the target protein is a transcription factor, preferably CTCF; or preferably the target protein is a histone.

33. The method of any one of claims 1-32, wherein the cell is an animal cell, a plant cell or a microbial cell.

34. The method of claim 33, wherein the cell is a mammalian cell, including but not limited to cells of humans, mice, rats, cats, dogs, pigs, and cows; or, the cell is a monocotyledonous or dicotyledonous plant cell, such as cells of rice, corn, wheat, sorghum, soybean, potato, tomato, etc.; or, the cell is a fungus such as a yeast cell.

35. The method of claim 33, wherein the cell is a cell line cell; or, the cell is a primary cell from a different organ or tissue, for example, the cell is from blood, cerebrospinal fluid, biopsy tissue. The cell can be a hepatocyte, a cardiomyocyte, a neuronal cell, a fibroblast, an epithelial cell; or, the cell is a tumor cell, a stem cell such as an embryonic stem cell or an induced pluripotent stem cell.

36. The method of claim 33, wherein the cell is a cell treated with a specific substance or condition or a cell at a specific developmental stage, for example, the specific substance is a drug.

37. A kit for detecting the interaction between a target protein and genomic DNA in a cell, comprising at least a fusion protein comprising DNA deaminase and protein A or protein G, a first antibody that specifically binds to the target protein, and optionally a second antibody that specifically binds to the first antibody.

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