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WO2025066559A1 - Procédé de test d'interactions protéine-adn sur la base de désaminase - Google Patents

Procédé de test d'interactions protéine-adn sur la base de désaminase Download PDF

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Publication number
WO2025066559A1
WO2025066559A1 PCT/CN2024/110080 CN2024110080W WO2025066559A1 WO 2025066559 A1 WO2025066559 A1 WO 2025066559A1 CN 2024110080 W CN2024110080 W CN 2024110080W WO 2025066559 A1 WO2025066559 A1 WO 2025066559A1
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cell
dna
cells
protein
target protein
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Chinese (zh)
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高绍荣
刘晓雨
张家民
师知非
陈西洋
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Tongji University
Shanghai East Hospital
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Tongji University
Shanghai East Hospital
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • 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.
  • 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.
  • 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.
  • ChIP seq chromatin immunoprecipitation sequencing
  • CUT&RUN CUT&Tag
  • CUT&Tag CUT&RUN and CUT&Tag
  • 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.
  • DamID and DiMeLo-seq based on third-generation sequencing. These methods have become important tools for studying gene regulation and protein-DNA interactions.
  • the present invention relates to a qDEPI-seq (Quantitative DNA Deaminase Based Protein-DNA Interaction Mapping Assay Using Sequencing) method, by fusing a protein A/G antibody binding domain to a highly active double-stranded DNA dCTP deaminase DddA, the protein A/G functional domain can bind to an antibody targeting a target protein, so that the double-stranded DNA dCTP deaminase is anchored in situ at the binding or action site of the target protein, and a deamination reaction is performed on DNA near the site to complete the conversion of dCTP to dUTP. Subsequently, the extracted DNA is used to prepare a library for sequencing.
  • qDEPI-seq Quantitative DNA Deaminase Based Protein-DNA Interaction Mapping Assay Using Sequencing
  • the in situ dUTP formed by the deamination reaction of dCTP on the genome will be paired with dATP, thereby finally realizing the conversion of the sequence from C-G to T-A.
  • a bioinformatics method is used to find and statistically calculate the number, position, conversion efficiency and other information of the above-mentioned conversion sites, so as to obtain information such as the binding site 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.
  • 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.
  • 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.
  • Embodiment 1 A method for detecting the interaction between a target protein and genomic DNA in a cell, the method comprising:
  • step 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;
  • step b) contacting a fusion protein comprising a DNA deaminase and protein A or protein G with the product of step b), allowing The fusion protein is fully combined with the complex, and the activity of the DNA deaminase is inhibited;
  • step c) 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;
  • 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.
  • Embodiment 3 The method of embodiment 1 or 2, wherein the permeabilization buffer contains a detergent such as Digitonin, for example, the permeabilization buffer contains approximately 0.02% Digitonin.
  • Embodiment 4 The method of embodiment 3, wherein the permeabilization buffer comprises approximately 20 mM HEPES pH 7.5, approximately 150 mM NaCl, approximately 0.5 mM Spermidine, approximately 0.1% BSA, approximately 1X PIC, approximately 2 mM EDTA and approximately 0.02% Digitonin.
  • 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).
  • 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.
  • 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,
  • 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.
  • 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.
  • SsdA single-stranded DNA deaminase A
  • DddA double-stranded DNA deaminase A
  • 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 with SEQ ID NO:1.
  • 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%, or at least 99.9% sequence identity with 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%, or at least 99.9% sequence identity with SEQ ID NO:3.
  • Embodiment 11 The method of any one of embodiments 1-10, wherein the DNA deaminase is combined with protein A or protein White G was fused directly or via a peptide linker.
  • 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.
  • Embodiment 13 The method of any one of Embodiments 1-12, wherein the fusion protein of DNA deaminase and protein A comprises the amino acid sequence shown in SEQ ID NO: 6 or 7.
  • 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.
  • Embodiment 15 The method of any one of embodiments 1-14, wherein step b) is carried out at low temperature.
  • step b) is carried out at about 4°C to about 10°C, preferably about 4°C.
  • 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 contains a detergent selected from Ttriton x 100, Digitonin or sodium deoxycholate, preferably, step b) the first binding buffer contains approximately 0.02% Digitonin.
  • the first binding buffer contains a detergent selected from Ttriton x 100, Digitonin or sodium deoxycholate, preferably, step b) the first binding buffer contains approximately 0.02% Digitonin.
  • Embodiment 17 The method of embodiment 16, wherein the first binding buffer
  • i) further comprises a protease inhibitor
  • ii) does not contain serum albumin such as BSA;
  • iii) contains about 90 mM to about 200 mM, preferably about 150 mM, of a metal salt such as NaCl.
  • 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.
  • 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.
  • 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.
  • the second binding buffer comprises about 90 mM to about 200 mM, preferably about 150 mM, of a salt such as NaCl.
  • Embodiment 21 The method of embodiment 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;
  • iii) contains a protease inhibitor.
  • 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 ⁇ M or higher, preferably about 0.02 to about 0.08 ⁇ M.
  • 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.
  • Embodiment 24 The method of any one of embodiments 1-23, wherein step d) comprises treating the product of step c)
  • the buffer is exchanged to a deamination reaction buffer
  • the deamination reaction buffer contains 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 MgCl 2 , preferably, the deamination reaction buffer contains about 50 mM NaCl and/or 2 mM MgCl 2 .
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • Q6U Biyuntian, D7239M
  • 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.
  • Implementation Option 30 The method of Implementation Option 29, wherein a U-tolerant DNA polymerase is used for DNA amplification in library establishment, for example, the U-tolerant DNA polymerase is selected from: HiFi V3, Phusion U, PhantaUc, Q5U and Q6U.
  • Embodiment 31 A method according to 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 C-G to T-A 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.
  • C-G to T-A transitions i.e., C to T and G to A
  • 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.
  • the target protein is a DNA binding protein
  • the target protein is a transcription factor, preferably CTCF; or preferably, the target protein is a histone.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 1 Schematic diagram of the qDEPI-seq workflow.
  • FIG. 1 The library fragment size distribution shown by the library quality inspection results obtained by the Agilent 2100 biochip analysis system for the library constructed in Example 1.
  • FIG. 7 Next-generation sequencing results of a library obtained from a single MII oocyte sample.
  • the C-G to T-A ratios upstream and downstream of the peak center in the public ChIP-seq data show the location of the target protein on the genome.
  • 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.
  • “A and/or B” encompasses “A,” “A and B,” and “B.”
  • “A, B, and/or C” encompasses “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”
  • 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. Acid, but still has the activity described in the present invention.
  • methionine encoded by the start codon at the N-terminus of the polypeptide will 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.
  • 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.
  • Gene 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).
  • the present invention provides a method for detecting the interaction between a target protein and genomic DNA in a cell, the method comprising:
  • step 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;
  • step b) 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;
  • the at least one cell is immobilized on a solid support, such as magnetic beads.
  • 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.
  • 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.
  • the cells are treated with formaldehyde at a final concentration of about 0.1% to cross-link the proteins and DNA in the cells.
  • the permeabilization buffer comprises a detergent.
  • the detergent is Digitonin.
  • the permeabilization buffer comprises approximately 0.02% Digitonin.
  • the permeabilization buffer comprises approximately 20 mM HEPES pH 7.5, approximately 150 mM NaCl, approximately 0.5 mM Spermidine, approximately 0.1% BSA, approximately 1X PIC, approximately 2 mM EDTA, and approximately 0.02% Digitonin.
  • 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.).
  • 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).
  • 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.
  • Deaminase refers to an enzyme that catalyzes a deamination reaction.
  • 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 that in the subsequent amplification reaction for sequencing, the C-G of the original genomic DNA is converted to T-A. By detecting the conversion of C-G to T-A, the deamination site can be detected.
  • 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.
  • telomere length 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.
  • the DNA deaminase is DddA or a functional fragment thereof, preferably DddA tox.
  • 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.
  • Protein A or Protein G is well known in the art, which 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 covered by the Protein A or Protein G of the present invention.
  • 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.
  • 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.
  • the DNA deaminase can be fused directly to protein A or protein G, or fused via a peptide linker.
  • the peptide linker is a rigid peptide linker.
  • the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 4 or 5, preferably the amino acid sequence shown in SEQ ID NO: 4.
  • the fusion protein also contains a tag, such as a tag for separation and/or purification of the fusion protein.
  • a tag for separation and/or purification of the fusion protein.
  • Methods for recombinant protein production are known in the art.
  • 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.
  • the fusion protein comprises a His tag.
  • 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.
  • 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.
  • the second antibody specifically binds to the constant region 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.
  • step b) is performed at low temperature.
  • step b) is performed at about 4°C to about 10°C, preferably about 4°C.
  • step b) is performed in a first binding buffer.
  • the first binding buffer comprises a detergent.
  • the detergent is, for example, Ttriton x 100, Digitonin, or sodium deoxycholate.
  • step b) the first binding buffer comprises approximately 0.02% Digitonin.
  • step b) the first binding buffer further comprises a protease inhibitor such as commercially available PIC (protease inhibitor cocktail).
  • a protease inhibitor such as commercially available PIC (protease inhibitor cocktail).
  • the first binding buffer does not contain serum albumin such as BSA (bovine serum albumin).
  • the first binding buffer comprises about 90 mM to about 200 mM, preferably about 150 mM of a metal salt such as NaCl.
  • the first binding buffer comprises approximately 20 mM HEPES pH 7.5, approximately 150 mM NaCl, approximately 0.5 mM Spermidine, approximately 0.1% BSA (bovine serum albumin), approximately 1X PIC (protease inhibitor cocktail), and approximately 0.02% Digitonin.
  • step b) the first binding buffer comprises approximately 20 mM HEPES pH 7.5, approximately 150 mM NaCl, approximately 0.5 mM Spermidine, approximately 1X PIC (protease inhibitor cocktail), and approximately 0.02% Digitonin.
  • 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.
  • step b) further comprises the step of removing unbound first antibody.
  • step c) 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.
  • step c) is performed at low temperature.
  • step c) is performed at about 4°C to about 10°C, preferably about 4°C.
  • step c) is performed in a second binding buffer.
  • the second binding buffer comprises a high concentration of salt.
  • the second binding buffer comprises about 90 mM to about 200 mM, preferably about 150 mM of a metal salt such as NaCl.
  • 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.
  • the second binding buffer of step c) contains a detergent.
  • the detergent is, for example, Ttriton x 100, Digitonin, or sodium deoxycholate.
  • the buffer of step c) contains approximately 0.02% Digitonin.
  • step c) the second binding buffer further comprises a protease inhibitor such as commercially available PIC (protease inhibitor cocktail).
  • a protease inhibitor such as commercially available PIC (protease inhibitor cocktail).
  • step c) the second binding buffer comprises approximately 20 mM HEPES pH 7.5, approximately 150 mM NaCl, approximately 0.5 mM Spermidine, approximately 0.1% BSA, approximately 1X PIC, and approximately 0.02% Digitonin. In some embodiments, step c) the second binding buffer comprises approximately 20 mM HEPES pH 7.5, approximately 150 mM NaCl, approximately 0.5 mM Spermidine, approximately 1X PIC, and approximately 0.02% Digitonin.
  • the second binding buffer in step c) comprises approximately 10 mM Tris-HCl pH 8.0, approximately 90 mM NaCl, 0.04% Triton X100, 0.02% DOC (sodium deoxycholate), 0.1 mM PMSF (phenylmethylsulfonyl fluoride) and 1X PIC.
  • the concentration of the fusion protein in step c) is about 0.02 to about 0.24 ⁇ M or higher, preferably about 0.02 to about 0.08 ⁇ M.
  • 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.
  • step c) further comprises the step of removing unbound fusion protein.
  • step d) comprises exchanging the buffer of the product of step c) with a deamination reaction buffer.
  • the deamination reaction buffer comprises a low concentration of salt.
  • 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 MgCl 2 .
  • the deamination reaction buffer comprises about 50 mM NaCl and/or 2 mM MgCl 2 .
  • the deamination reaction buffer of step d) comprises about 20 mM MES pH 6.4, about 1 mM DTT, about 2 mM MgCl 2 , 1X PIC.
  • 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.
  • 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 degrade the region or vicinity of the genomic DNA that interacts with the target protein.
  • 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.
  • step d) further comprises adding a uracil glycosylase inhibitor (UGI) to the reaction.
  • UGI uracil glycosylase inhibitor
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • Q6U Biyuntian, D7239M
  • 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.
  • 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.
  • 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.
  • the DNA library was constructed using the DNA Library Prep Kit V2 for Illumina TD502.
  • a U-tolerant DNA polymerase is used for DNA amplification in the library establishment, for example, the U-tolerant DNA polymerase is selected from: HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U.
  • Sequencing described herein can be first generation sequencing such as Sanger sequencing, second generation sequencing (NGS) or other high throughput sequencing.
  • first generation sequencing such as Sanger sequencing, second generation sequencing (NGS) or other high throughput sequencing.
  • second generation sequencing of Illumina can be used.
  • the target protein can interact (bind) with a specific region of genomic DNA, such as a specific sequence (motif)
  • 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 nearby (upstream and/or downstream) can be converted to 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 existence and/or distribution of deamination sites, the existence and/or distribution of the interaction between the target protein and the genomic DNA can be determined. For example, if there is a deamination site in a certain region, the target protein can be identified. The downstream DNA all showed significant CG to TA conversion, and the intermediate sequence without CG to TA conversion may be the sequence (such as binding motif) where the target protein interacts (binds) with the genomic DNA.
  • the interaction of the target protein with the cellular genomic DNA or a portion thereof is determined by analyzing the presence of C-G to T-A 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.
  • the interaction of the target protein with the cellular genomic DNA or a portion thereof is determined by analyzing the position and/or density and/or conversion rate of C-G to T-A transitions in the cellular genomic DNA or a portion thereof relative to a control genomic DNA or a portion thereof.
  • 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.
  • the target protein described herein is, for example, a DNA binding protein, and examples of DNA binding proteins may be transcription factors, histones, etc.
  • the target protein is a histone.
  • the transcription factors include, but are not limited to, CTCF, Klf4, Thap11, SP1, Nrf1, etc., preferably CTCF.
  • 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.
  • the cells described herein can be animal cells, plant cells, or microbial cells.
  • 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.
  • the cell may be a cell line cell.
  • the cell may be a primary cell from a different organ or tissue.
  • 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.
  • the cell is a cell treated with a specific substance (compound) or condition or a cell at a specific developmental stage.
  • 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.
  • the specific substance can be a drug.
  • 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.
  • 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.
  • 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.
  • 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.
  • the kit further comprises the first binding buffer, the second binding buffer and/or the deamination reaction buffer described above.
  • the kit further includes reagents for amplifying the genomic DNA portion of interest, such as specific primers.
  • the kit further comprises reagents for establishing a genomic library.
  • the kit further comprises reagents for sequencing, such as Sanger sequencing or next generation sequencing.
  • 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 1 qDEPI-seq detection of a small amount of mouse embryonic fibroblasts
  • 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.
  • Binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl 2 , 1 mM MnCl 2 ).
  • NEB Library amplification was completed using HF dUTP-tolerant high-fidelity DNA polymerase and Novizan TruePrep Index Kit 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.
  • the constructed library was sent to the company for high-throughput sequencing.
  • the sequencing machine used was Illumina X TEN, and the sequencing mode was double-end 150bp.
  • 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.
  • 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.
  • 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.
  • 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.
  • step 2) Transfer the eggs from step 2) to 1 mL of cold Dig-Wash Buffer (Washing buffer supplemented with 0.02% Digitonin) and wash twice.
  • 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 ⁇ M and incubate at 4°C for 1 hour.
  • META Tn5 Library construction was performed based on the published META-CS seq technology.
  • META Tn5 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 ⁇ L 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’.
  • First strand labeling was performed by adding 13 ⁇ L strand tagging Mix 1 (containing 5 ⁇ L PhU-HF reaction buffer, 0.8 ⁇ L 50 ⁇ M Adp1 primer mix, 0.6 ⁇ L 100 mM MgCl 2 , 0.6 ⁇ L water, 0.5 ⁇ L 10 mM dNTP mix, 0.25 ⁇ L 20 mg/mL BSA (NEB) and 0.25 ⁇ L 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.
  • Adp1 primer was removed by adding 1 ⁇ L exonuclease I (NEB; M0293) and incubating at 37°C for 30 min and 80°C for 20 min.
  • Second strand labeling by adding 4 ⁇ L of strand tagging Mix 2 (containing 1 ⁇ L of PhU-HF reaction buffer, 1 ⁇ L of 50 ⁇ M Adp2 primer mix, 0.855 ⁇ L of water, 0.1 ⁇ L of 10 mM dNTP mix, and 0.05 ⁇ L of PhU DNA polymerase) and incubate at 72 °C for 3 min, 98 °C for 30 s, 62 °C for 5 min, and 72 °C for 1 min.
  • Remove the Adp2 primer by adding 1 ⁇ L of exonuclease I (NEB; M0293) and incubate at 37 °C for 30 min and 80 °C for 20 min.
  • the constructed library was sent to the company for high-throughput sequencing.
  • the sequencing machine used was Illumina X TEN.
  • 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.
  • the coverage of the test 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.
  • the method of the present invention is also very effective in detecting CTCF binding sites in single cells.
  • Figure 8 shows the CTCF peak information and motif sites obtained from the ChIP seq data in the public database.
  • 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 some TC sites upstream and downstream of the CTCF motif is close to 100%, indicating the feasibility of quasi-quantification.
  • 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.
  • 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.
  • the buffer system during the in situ binding of antibodies was tested, including NaCl concentrations (90mM and 150mM), surfactant types (Triton X100, digitonin, sodium deoxycholate, etc.) and concentrations.
  • NaCl concentrations 90mM and 150mM
  • surfactant types Triton X100, digitonin, sodium deoxycholate, etc.
  • concentrations concentrations. The results are shown in Figure 10, indicating that the experiment is more efficient at 150mM NaCl, and the type of surfactant has no significant effect.
  • the addition of BSA may have a negative impact.
  • the fifth IgG group is the control group.
  • 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.
  • the best reaction time is about 30 minutes.
  • the reaction of the group with a reaction time of less than 30 minutes is not complete, and the group with a reaction time of 60 minutes does not significantly improve the average conversion efficiency.
  • Deamination-modified DNA may produce amplification preference during PCR amplification, and this preference may affect the final sequencing quality of this method. Therefore, in the experiment shown in Figure 13, DNA fragments containing 2, 4, 6, and 8 dUTPs and original fragments without dUTP were obtained by artificial synthesis to simulate the DNA after the deamination reaction in the method. These artificially synthesized DNA fragments with different dUTP contents are mixed in a certain proportion and PCR amplified to complete the library establishment. After performing second-generation sequencing, the results are shown in Figure 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 preference.

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Abstract

La présente invention appartient au domaine technique de la biologie, et concerne en particulier un procédé de test d'interactions protéine-ADN sur la base d'une désaminase.
PCT/CN2024/110080 2023-09-28 2024-08-06 Procédé de test d'interactions protéine-adn sur la base de désaminase Pending WO2025066559A1 (fr)

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