JP7593572B2 - Three-dimensional DNA structure interaction analysis method - Google Patents

Description

The present invention relates to a method for analyzing interactions in the three-dimensional structure of DNA sequences and an analytical kit for implementing the method, and in particular to a three-dimensional DNA structure interaction analysis method that can rapidly and accurately analyze DNA sequences that interact with a target DNA sequence.

In recent years, the sequence of the human genome has been deciphered, revealing human genetic information, but this information has been provided simply as one-dimensional linear sequence information. However, actual genomic DNA is housed within the nucleus in a folded, complex three-dimensional structure, and it is difficult to completely predict the spatial arrangement (perspective) of each part of the sequence from the one-dimensional linear sequence. In other words, when focusing on a certain region of a DNA sequence, it is often the case that a DNA sequence located spatially close to that region is actually located far away in terms of the one-dimensional linear sequence.

In recent years, it has become clear that the expression of certain genes is controlled by interactions between sequences in three-dimensional space, and methods for analyzing sequence interactions, such as the chromosome conformation capture (3C) method and its improved version, the HiC method, have been proposed.

The 3C method is a technique in which the genome is fixed (cross-linked) while maintaining its higher-order structure, DNA is cut, and then spatially adjacent ends are linked by ligation (proximity ligation). Proximity ligation with the higher-order structure fixed results in the bonding of sequences that are spatially close to each other. For this reason, the DNA sequence resulting from the ligation is confirmed to be different from the one-dimensional sequence on the genome. The methods proposed to analyze the three-dimensional interactions of DNA sequences are based on this 3C method.

However, the 3C method can only analyze interactions between sequences that have been focused on in advance, i.e., sequences that have already been identified.

The HiC method compensates for this shortcoming and has been proposed as a method for analyzing DNA interactions genome-wide, and is designed to analyze interactions between unknown sequences. In the HiC method, the ends of DNA fragments generated by DNA cleavage are biotinylated and then proximity ligation is performed. A group of ligated DNA fragments is collected using the biotin label as a marker, and after further fragmentation, an adapter for PCR is added, and the base sequence is identified by amplification and sequencing. This is the basic method.

On the other hand, the HiC method theoretically detects all three-dimensional DNA structures, which means that the sequencing costs are enormous and very complicated computer analysis is required, leaving problems in versatility. Therefore, in order to reduce costs, a new method has been proposed that combines hybridization with a specific sequence (see Patent Document 1 and Non-Patent Document 1).

In addition, because DNA forms chromatin, a ChIA-PET method (see Patent Document 2) has been proposed in which the chromatin is fragmented, and then the chromatin fragments to be analyzed are recovered using chromatin immunoprecipitation (ChIP), and the interactions of DNA sequences are analyzed based on the chromatin fragments.

Special table 2016-537029 JP2007-289152

Stefan Schoenfelder, Biola-Maria Javierre, Mayra Furlan-Magaril, Steven W. Wingett, and Peter Fraser, Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions, Journal of Visualized Experiments.(136), e57320 ，June 2018

However, the HiC method requires at least 10 ⁶ cells as a sample number, and 10 ⁶ types of fragments are ligated per cell. The degree of freedom of the DNA pairs (DNA fragments in which spatially adjacent DNA sequences are aligned side by side by ligation) is estimated to be as high as 10 ^18. Here, it is known that in the PCR amplification performed prior to the determination of the base sequence of the DNA pair, the DNA pair generated by the amplification is biased due to various factors such as GC% and difference in the length of the restriction enzyme fragment. In addition, the sequence of the collection of DNA pairs obtained (HiC library) is overwhelmingly more complex than that of a normal sequence, so there is a large room for interpretation of the analysis results, and the reliability of the analysis results may be reduced in some cases.

The method described in Patent Document 1 and Non-Patent Document 1 proposes to improve the conventional comprehensive HiC method and to perform a sequence specific to a region of interest by hybridizing a HiC library with a DNA microarray. This reduces the load of sequencing and reduces the analysis load by compressing the amount of data. However, the chromatin cutting process for creating DNA pairs is performed genome-wide as in the conventional method. Therefore, a huge number of DNA pairs are generated overall, and a huge amount of impurities coexist in addition to the target substance. For this reason, mishybridization cannot be avoided. In addition, it is known that the probes used in the microarray are unintentionally biased at the sequence design stage, and there is a problem that the data finally obtained is not sufficiently reliable.

Furthermore, in the chromatin immunoprecipitation method, chromatin is fragmented by DNA cleavage using a restriction enzyme, and then immunoprecipitation is performed using an antibody targeting proteins such as histones. In the immunoprecipitation, the fragmented chromatin fragments are solubilized in a liquid phase by ultrasonication or restriction enzyme treatment, and antibody immunoprecipitation is performed to collect DNA fragments and form a library source for analysis. However, in this solubilization process (also called the nuclear fraction solubilization process), insoluble fractions are often generated. Since chromatin fragments that are not solubilized cannot be collected, they are not incorporated into the library source and are excluded from the analysis target. The resulting analysis data may be inaccurate from the actual data, which reduces the reliability of the results obtained.

The present invention has been made in consideration of the above-mentioned problems, and aims to provide a method for analyzing DNA sequence interactions that is simple, low-cost, has excellent reliability of analytical data, and is highly accurate, and an analytical kit for achieving this method.

[Item 1]
1. A method for analyzing interactions of DNA sequences in a three-dimensional DNA structure, comprising:
a DNA cleavage step of selecting an arbitrary protein adjacent to a DNA strand in the DNA sequence and specifically cleaving the DNA strand in the vicinity of the arbitrary protein;
a ligation step of ligating the DNA ends generated by the cleavage in the DNA cleavage step;
a selection step of selecting the ligated DNA fragments;
a sequencing step of determining a base sequence of the DNA fragment selected in the selection step;
and a defixation step of defixing the DNA structure of the DNA fragment prior to the sequencing step,
A method for analyzing three-dimensional DNA structure interactions, comprising analyzing the interactions of the DNA sequences.

[Item 2]
A labeling step of adding a label to the DNA end generated by the cleavage in the DNA cleavage step,
2. The method for three-dimensional DNA structure interaction analysis according to item 1, wherein the selection step selects the ligated DNA fragments based on the label.

[Item 3]
3. The method for three-dimensional DNA structure interaction analysis according to item 1 or 2, wherein the selection step is performed after fragmenting the DNA fragments ligated in the ligation step into shorter DNA fragments.

[Item 4]
The method for three-dimensional DNA structure interaction analysis according to any one of items 1 to 3, characterized in that the sequencing step comprises amplifying the selected DNA fragment and determining the base sequence of the amplified product.

[Item 5]
5. The method for analyzing three-dimensional DNA structure interaction according to item 4, wherein the selection step comprises adding a predetermined adaptor sequence to the cut end of the amplified DNA fragment.

[Item 6]
1. A method for analyzing interactions of DNA sequences in a three-dimensional DNA structure, comprising:
a DNA cleavage step of selecting an arbitrary protein adjacent to a DNA strand in the DNA sequence and specifically cleaving the DNA strand in the vicinity of the arbitrary protein;
a ligation step of ligating the DNA ends generated by the cleavage in the DNA cleavage step;
a capture step of capturing the ligated DNA fragments;
a fixation release step of releasing the fixation of the DNA structure of the DNA fragment captured in the capture step;
and a sequencing step of determining the base sequence of the DNA fragment after the fixation is released,
A method for analyzing three-dimensional DNA structure interactions, comprising analyzing the interactions of the DNA sequences.

[Item 7]
1. A method for analyzing interactions of DNA sequences in a three-dimensional DNA structure, comprising:
a DNA cleavage step of selecting an arbitrary protein adjacent to a DNA strand in the DNA sequence and specifically cleaving the DNA strand in the vicinity of the arbitrary protein;
a capture step of capturing DNA fragments generated by cleavage in the DNA cleavage step;
a ligation step of ligating the ends of the DNA fragments captured in the capture step;
a defixation step of defixing the DNA structure of the ligated DNA fragments;
and a sequencing step of determining the base sequence of the DNA fragment after the fixation is released,
A method for analyzing three-dimensional DNA structure interactions, comprising analyzing the interactions of the DNA sequences.

[Item 8]
8. The three-dimensional DNA structure interaction analysis method according to item 6 or 7, wherein the sequencing step comprises amplifying the DNA fragments after the immobilization is released and determining the base sequence of the amplified product.

[Item 9]
the DNA cleavage step includes a primary antibody binding step of binding a primary antibody to the arbitrary protein, and an enzyme binding step of binding an enzyme that cleaves DNA to the arbitrary protein via the primary antibody,
9. The method for three-dimensional DNA structure interaction analysis according to any one of items 1 to 8, characterized in that a DNA strand in the vicinity of the arbitrary protein is specifically cleaved by an enzyme that cleaves the DNA.

[Item 10]
10. The three-dimensional DNA structure interaction analysis method according to any one of items 1 to 9, further comprising a cross-linking step of immobilizing the DNA structure by cross-linking prior to the DNA cleavage step.

[Item 11]
1. An analytical kit for use in analyzing interactions of DNA sequences in a three-dimensional DNA structure, comprising:
A primary antibody that binds to a protein of interest;
A DNA cleaving enzyme that binds to the primary antibody;
a label for identifying a DNA end generated by cleavage with the DNA cleavage enzyme; and
and an enzyme for ligating labeled DNA ends.

According to the method of the present invention, an analysis sample is prepared by selectively and locally cutting (fragmenting) the DNA region to be analyzed, so that a high-quality analysis sample with few contaminants can be obtained by focusing on the DNA region to be analyzed. Therefore, the amount of sample to be prepared can be reduced, and in some cases, can be reduced drastically.
Furthermore, compared with the HiC method, which identifies interactions based on genome-wide analysis, the method of the present invention makes it possible to analyze interactions between sequences by focusing on a target DNA region, thereby significantly shortening the time required to obtain the required analysis results and enabling rapid and low-cost analysis.
Furthermore, even when amplification is performed, in samples containing a large amount of impurities as in conventional methods, other fragments present in large quantities are also amplified in the PCR amplification process, and a large amount of sample must be prepared in order to obtain sequence data for any of the fragments to be analyzed. However, in the present invention, since the amount of impurities is small, an increase in noise can be suppressed even when PCR amplification is performed, and highly reliable analysis results can be obtained.

In one embodiment, when the method of the present invention includes a labeling step in which the DNA cleavage step adds a label to select the DNA end generated by cleavage, and the selection step selects the ligated DNA fragment based on the label, the DNA fragment can be recovered without immunoprecipitation (chromatin immunoprecipitation) for proteins, making it possible to eliminate the need for the nuclear fraction solubilization step required in the chromatin immunoprecipitation method and eliminating the influence of the insoluble fraction. That is, the HiChIP method, in which the whole genome is analyzed, is a conventional analytical method that uses chromatin immunoprecipitation, but it is difficult to completely avoid the occurrence of regions that cannot be analyzed due to the influence of the insoluble fraction generated in the nuclear fraction solubilization step. For this reason, when analyzing interactions from the obtained sequence results, the reliability of the analytical results may decrease in some cases. However, when the present invention is configured with a labeling step, the influence of the insoluble fraction as in the chromatin immunoprecipitation method can be eliminated, and more reliable analytical results can be obtained.

FIG. 1 shows, by way of example, a flow chart of the method of the invention. Figure 2 shows a heat map of interactions of chromatin regions containing H3K4me3. The vertical and horizontal axes show chromosomes 1 to 23, respectively, with gene regions arranged in parallel. In Figure 2, the regions corresponding to each chromosome are divided by vertical and horizontal lines, and the same gene region of the same chromosome is shown by a diagonal line. The interactions of different gene regions detected are represented by dots, and the degree of interaction of chromatin regions is shown by grayscale shading based on the density of the dots on the heat map. FIG. 3 shows the localization of H3K4me3 on the genome and the interaction between chromatin regions where H3K4me3 is present, analyzed as an example. At the top, the position on the genome is indicated by numbers, and the top row is a distribution map showing the localization of H3K4me3 analyzed in the results obtained by carrying out the known CUT&RUN method (Skene et al., 2018). The middle and bottom rows are the results obtained by Example 1 of the present application, and the middle row is a distribution map showing the localization of H3K4me3, and the bottom row shows the interaction between chromatin regions where H3K4me3 is present in a loop shape. Below the bottom row, the positions of genes (FBXL18, MIR589, ACTB) in chromosome 7 are displayed, and the range marked to the right of the gene name indicates the location of the corresponding gene. FIG. 4 is a partially enlarged view of FIG. FIG. 5 is a partially enlarged view of FIG. Figure 6 shows a heat map of interactions of chromatin regions containing CTCF. The vertical and horizontal axes show chromosomes 1 to 23, respectively, with gene regions arranged in parallel. In Figure 6, the regions corresponding to each chromosome are divided by vertical and horizontal lines, and the same gene region on the same chromosome is shown by a diagonal line. The interactions of different gene regions detected are represented by dots, and the degree of interaction of chromatin regions is shown by the grayscale shading on the heat map. FIG. 7 shows the localization of CTCF on the genome and the interaction between chromatin regions where CTCF is present, analyzed as an example. The top part shows the position on the genome with numbers, and the top row is a distribution map showing the localization of CTCF obtained in Example 2 of the present application. The middle row shows the CTCF binding sites present on the genome identified by the Jaspar Transcription Factor Database, and those in one direction are underlined according to the direction of the sequence on the genome. The bottom row shows the interaction between chromatin regions where CTCF is present, obtained in Example 2 of the present application, in the form of a loop. Below the bottom row, the positions of genes (FBXL18, MIR589, ACTB) in chromosome 7 are displayed, and the range marked to the right of the gene name indicates the position of the corresponding gene. FIG. 8 is a partially enlarged view of FIG. FIG. 9 is a partially enlarged view of FIG. FIG. 10 shows the localization of CTCF on the genome and the interaction between chromatin regions in which CTCF is present, analyzed as an example. At the top, the position on the genome is indicated by numbers, and the top row is a distribution map showing the localization of CTCF analyzed in the results obtained by carrying out the known CUT&RUN method (Skene et al., 2018). The middle row shows the CTCF binding sites present on the genome identified by the Jaspar Transcription Factor Database, and those in one direction are underlined according to the direction of the sequence on the genome. The bottom row shows the interaction between chromatin regions in which CTCF is present obtained by Example 3 of the present application in a loop shape below the localization of CTCF, together with a distribution map showing the localization of CTCF obtained by Example 3 of the present application. Further below the bottom row, the positions of genes (FBXL18, MIR589, ACTB) in chromosome 7 are displayed, and the range marked to the right of the gene name indicates the position of the corresponding gene. FIG. 11 is a partially enlarged view of FIG. FIG. 12 is a partially enlarged view of FIG.

The present invention is a method for analyzing interactions of DNA sequences based on the three-dimensional structure of DNA and an analytical kit for achieving this method.

In the present invention, the three-dimensional structure of DNA refers to the higher-order structures formed by the DNA double helix, such as structures with loops and folds. These structures may consist of DNA alone or may contain other molecules such as proteins. The concept also includes the DNA-protein complex chromatin. The main function of chromatin is said to be to package DNA into a small volume that fits inside the cell, as well as to control gene expression, DNA replication and repair. The most abundant protein components of chromatin are histones, which condense DNA.

Recently, large megabase-sized local chromatin interaction domains, called topological domains (hereinafter referred to as "TDs"), have been identified. These domains correlate with regions of the genome that limit the spread of heterochromatin. These domains are stable across different cell types and are highly conserved across species. Topological domains also interact with each other. In the present invention, we analyze DNA sequence interactions not only within topological domains but also between topological domains.

Due to such a three-dimensional structure, it is difficult to completely predict the spatial arrangement (perspective) of each part of a genomic DNA sequence from a one-dimensional linear sequence. In other words, when focusing on a certain region of a DNA sequence, it is often the case that a DNA sequence located spatially close to that region is a sequence located far away in a one-dimensional linear sequence. It is presumed that gene expression is regulated by the interaction of DNA sequences, and the spatial perspective of DNA sequences is important information. DNA sequence interaction or three-dimensional DNA structure interaction represents, for example, the spatial arrangement (e.g., perspective) between DNA sequences in a three-dimensional structure.

The method of the present invention can include, in any order, a cleavage step for specifically cleaving a DNA strand (a target DNA region) near a preselected arbitrary protein among various proteins that bind, complex, or attach to a DNA strand, a ligation step for ligating the DNA ends resulting from the cleavage to create a sequence (DNA pair) in which spatially adjacent DNA sequences (although their loci on the genome are separated) are arranged side by side, a selection step for selecting the created DNA pair or a capture step for capturing (based on the protein molecules that bind) the ligated DNA fragments or the DNA fragments produced by the cleavage in the DNA cleavage step, a fixation release step, and a sequencing step for amplifying the DNA pair as desired and determining the base sequence. The target DNA region is a portion of the DNA strand near the arbitrary protein, and is the DNA region to be analyzed. Each step will be described below.

The present invention may also be configured to include a crosslinking step prior to the cleavage step, in which the higher-order structure within the cell is fixed by artificial manipulation.

(Cross-linking step)
The crosslinking step is a treatment for fixing the spatial arrangement of DNA sequences in the cell nucleus. For example, formaldehyde can be used to crosslink proteins and nucleic acids, or one protein and another nearby protein. Interacting base sequences, such as promoters and enhancers, are often in contact with specific proteins and are in close proximity through the proteins. In other words, the higher-order structure of DNA is often formed with the involvement of proteins. For this reason, the spatial structure of the DNA sequence is fixed by fixing (crosslinking) the DNA and the proteins in contact with it using formaldehyde.

Examples of agents that cross-link DNA and can be used in the present invention include, in addition to formaldehyde, EGS (ethylene glycol bis(succinimidyl succinate)) and DSG (disuccinimidyl glutarate).

Formaldehyde can be used to form cross-links that span relatively short distances, e.g., about 2 Å, thereby allowing the formation of DNA pairs that can be used to analyze close interactions between DNA sequences.

Formaldehyde cross-linking is a suitable method for detecting two sequences that directly interact with each other, but formaldehyde is a so-called zero-length cross-linking agent, and it can only create DNA pairs with sequences that are in very close contact. Therefore, by using a relatively long cross-linking agent such as EGS (16.1 Å) or DSG (7.7 Å) instead of formaldehyde, it is possible to capture large protein complexes with more complex higher-order structures and create DNA pairs for detecting interactions between DNA sequences located farther apart than can be cross-linked with formaldehyde.

This cross-linking step may be performed, for example, by incubating the cells in a 0.05% to 4% formaldehyde solution, more preferably in a 0.5% to 2.5% formaldehyde solution, and even more preferably in a 1% to 2% formaldehyde solution, in an environment ranging from ice cooling to 37°C. The incubation time is 30 seconds to 16 hours, preferably 5 minutes to 30 minutes, and more preferably 10 minutes to 15 minutes. When such a cross-linking step is performed, the higher-order structure is sufficiently fixed to accurately fix the positional relationship of the DNA sequence, thereby improving the accuracy of the DNA pair obtained in the subsequent steps.

(Cutting Step)
The cleavage step is a step of mainly cleaving a DNA strand near a preselected protein (target protein) in a DNA sequence to which the protein is in contact, using an enzyme. During this cleavage step, permeabilization of the nuclear membrane may be performed to increase the permeability of the enzyme and to facilitate cleavage of DNA present in the cell nucleus.

A surfactant is used for the permeabilization treatment. Preferably, a nonionic or zwitterionic surfactant is used. Examples of such surfactants include, but are not limited to, digitonin, octylphenol ethoxylates (trade names: Triton X100, Triton X114, Nonidet P40, etc.), n-dodecyl-β-D-maltoside (DDM), polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monooleate (Tween 80), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (trade name CHAPS), etc. Cells may be treated with liquid nitrogen before or after permeabilization using a surfactant.

In this cleavage step, proteins adjacent to the interacting DNA sequences are targeted, e.g., proteins that associate with, form complexes with, or are attached to one or both of the interacting DNA sequences and are spatially proximate to these DNA sequences.

Such proteins can be any of various proteins that come into contact with chromatin (DNA chains) and are appropriately selected depending on the subject of analysis. Examples of such proteins include CTCF, a protein known to be closely involved in the formation of the three-dimensional structure of DNA, and RNA polymerase II, which is involved in the transcription of RNA, and histone H3, histone H4, histone H2A, and histone H2B, which are essential for the nucleosome structure, but are not limited to these.

Then, a primary antibody that directly binds to the selected protein (target protein) is prepared. Such a primary antibody may be a monoclonal or polyclonal antibody that corresponds to the target protein. For example, monoclonal antibodies against CTCF, RNA polymerase II, histone H3, histone H4, histone H2A, histone H2B, etc., that correspond to the proteins exemplified above are used. Furthermore, the antibody does not necessarily have to be a complete antibody, and may be a functional fragment of an antibody that has a similar function, for example, a fragment that has the function of binding to the target protein.

In the cleavage step, DNA cleavage is performed by an enzyme that binds to the target protein via the primary antibody that has captured the target protein, in other words, an enzyme that binds to the DNA strand via the primary antibody. The enzyme binds to the DNA strand via the primary antibody means that the enzyme binds directly or indirectly to the primary antibody that directly binds to the target protein that is attached to and bound to the DNA strand. Any known enzyme can be appropriately selected and used as long as it has the ability to cleave the DNA strand in the vicinity of the target protein. Examples of such enzymes include, but are not limited to, various endonucleases such as micrococcal nuclease (MNase), DNAse I (DNaseI), and transposase, and various restriction enzymes such as HindIII, MboI, and DpnII. Preferably, an endonuclease that performs non-specific cleavage is used, and more preferably, MNase is used. Here, "vicinity" means within 1000 bp from the position of the target protein, for example, within 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, or 200 bp, preferably within 200 bp. For example, transposase, MNase, DNase I, HindIII, MboI, or DpnII can be used to cleave within 1000 bp from the position of the target protein, and MNase, DNase I, HindIII, MboI, or DpnII can be used to cleave within 200 bp from the position of the target protein.

This cleavage step allows the acquisition of DNA fragments with base lengths according to the characteristics of the cleavage enzyme; for example, when MNase is used, DNA fragments of about 200 bp (approximately mononucleosome size) can be obtained. The length of the DNA fragments obtained varies depending on the conditions under which the cleavage step is performed, and may include mononucleosome-sized fragments as well as multiples of mononucleosome-sized fragments.

If the enzyme has DNA cleavage ability but does not inherently have antibody binding ability, it is used in the cleavage step after being given antibody binding ability. Various methods can be used to give antibody binding ability to the enzyme, such as a fusion protein in which a protein that shows high affinity to the Fc region of an antibody is fused to the enzyme to be used to give antibody binding ability, or a method in which an enzyme is complexed with a secondary antibody that specifically binds to the above-mentioned primary antibody through various chemical reactions. Various known proteins can be used as proteins that show affinity to this antibody, such as protein A, protein G, and protein L, but are not limited to these.

For the fusion reaction between a protein such as protein A and an enzyme, any known method can be used as appropriate, including, but not limited to, the periodic acid method, glutaraldehyde method, maleimide method, and NHS (N-hydroxysuccinimide) method.

Fusion proteins can also be produced using bioengineering techniques. Specifically, a gene encoding the sequence of a cleavage enzyme can be introduced into a vector (plasmid) that produces a fusion protein to construct a vector, or an already constructed vector can be introduced into E. coli, and the transformed E. coli can be cultured while inducing expression of the fusion protein, to produce the desired fusion protein.

For example, a fusion protein of Protein A and MNase (pA-MN) can be prepared using a known vector such as pK19pA-MN. Alternatively, DNA encoding Protein A and MNase can be introduced into a known plasmid such as pET21a to construct an expression vector for pA-MN, which can then be used to prepare the protein. In this case, the expressed pA-MN can be made soluble, and when a suspension produced by disrupting E. coli is centrifuged, the pA-MN is contained in the resulting supernatant. The pA-MN can then be recovered from the supernatant by adsorption using IgG-Sepharose beads or the like, followed by washing and elution.

In this cleavage step, the procedure for binding the enzyme to the DNA strand via the primary antibody may be as follows: 1) After adding the primary antibody to the DNA fragment, an enzyme capable of binding to the primary antibody is sequentially added, and the enzyme is bound to the primary antibody. 2) A primary antibody and an enzyme that have been previously bound to each other are added to capture the target protein and bind the enzyme to the DNA strand via the primary antibody. 3) After adding the primary antibody, a secondary antibody is added, and an enzyme that has the ability to bind to this secondary antibody is added. 4) After adding the primary antibody, a secondary antibody and an enzyme that have been previously bound to each other are added. In the case of a method in which a primary antibody and an enzyme that have been previously bound to each other are added to the DNA fragment, the primary antibody binding step and the enzyme binding step described in item 9 are performed at the same time.

The above enzymes are preferably those that can easily adjust the timing of DNA strand cleavage (enzyme activity). Examples of methods for adjusting enzyme activity include reaction temperature, light irradiation, and metal ion concentration. Therefore, it is preferable to select an enzyme that uses a metal ion as an effector, which allows easy on-off switching of the reaction. Specifically, examples of calcium ion-dependent enzymes include MNase and DNase I, and an example of magnesium ion-dependent enzymes includes transposase. With such enzymes, DNA cleavage is performed when the corresponding ion is added.

This cleavage step cuts the DNA strand near any preselected target protein, and the adjacent cut ends are then joined in the subsequent ligation step to create a DNA pair with a different sequence than the sequence on the genome.

In the present invention, this cleavage step does not involve genome-wide random cleavage, but rather involves target protein-specific cleavage. Therefore, the resulting group of DNA pairs (DNA library) contains the desired DNA sequence to be analyzed at a high concentration (in principle, all of the resulting DNA pairs) compared to DNA libraries obtained by conventional genome-wide random cleavage. In other words, it is possible to provide a high-quality analysis sample with extremely little unnecessary DNA fragments (ligation by-products, impurities) that become noise.

In the conventional HiC method, the data analysis after sequencing removes information on unintended DNA pairs to improve the accuracy of the analysis data. In addition, in order to improve the accuracy of the final data, sequencing is performed with a huge number of reads, and a considerable number of cells is required to achieve this. On the other hand, the present invention improves the quality of the DNA library itself used for sequencing, thereby improving the reliability of the data. Therefore, it is possible to reduce the number of cells required for analysis and perform analysis with a small amount of sample. Even when amplification is performed, the proportion of the target DNA pair contained in the analysis sample is extremely high, so the influence of amplification bias during PCR due to impurities can be suppressed. Furthermore, it is possible to suppress the reading of unnecessary DNA fragments, and the labor and cost required for analysis can be significantly reduced.

In addition, in order to efficiently select and concentrate DNA pairs, a labeling step may be provided in which, for example, biotin is added as a label to the cut ends. In the present invention, it is more preferable to provide this labeling step in order to concentrate DNA pairs without using immunoprecipitation. This labeling step may be provided before the selection step described below. For this reason, for example, it may be provided following the cleavage step, or may be provided so as to be performed together with ligation in the ligation step. In this way, by providing a labeling step, the DNA pairs to be analyzed can be concentrated by a label such as biotin. In other words, since the necessary DNA pairs can be concentrated without using immunoprecipitation, there is no need to solubilize the nuclear fraction required in immunoprecipitation, and the quality of the formed DNA library can be further improved.

Biotin can be added to the cleaved DNA ends according to standard methods. For example, the protruding ends resulting from cleavage can be blunted by an extension reaction using deoxynucleotide triphosphate (dNTP) as a substrate, and if this dNTP is biotinylated, biotin can be added to the cleaved ends. Various commercially available biotinylation reagents can be used for biotinylation. If the cleaved ends are blunt, biotin can be added after first making them protruding ends.

In addition, the substance added to the cut end is not limited to biotin, as long as it allows the selection (concentration) of DNA pairs. However, since protease treatment is preferable for removing the fixation of the DNA structure, it is preferable to select a compound that is not digested by proteases (has protease resistance) even when selecting other substances. An example of such a compound is methylated dATP. If this methylated dATP is added to the cut end, DNA pairs can be concentrated with an anti-methylated dATP antibody. In addition, a compound that reacts with click chemistry, which is a cyclization reaction between a nitrogen triple bond and an alkyne, may be used as a label. An example of such a compound is a non-natural base with an alkyne (5-ethynyl-2'-deoxyuridine (EdU, etc.)). Such a compound added to the cut end can be bound to an azide compound by a click reaction, and can be bound to azidobiotin having an azide group or an azide group-modified flag tag, etc., thereby enabling enrichment based on these biotins or flag tags. Also, instead of biotinylated dNTPs, a short DNA (linker DNA) to which biotin has been added may be used as a label, and a ligation reaction may be performed via this linker DNA.

(Ligation step)
This step is a step of performing a ligation reaction using DNA ligase. Ligation means linking two DNA fragments by a phosphodiester bond. As the DNA ligase, a mesothermal DNA ligase such as T4 DNA ligase or E. coli DNA ligase can be used. A heat-resistant DNA ligase may also be used. This ligation reaction links the cut ends located in close proximity to each other in space, thereby constructing a DNA pair in which the sequence portions located far apart in the genome are arranged side by side.

In this ligation step, the dNTPs (biotinylated dNTPs) present at the ends may be digested using an exonuclease to remove those that were not ligated.

(Fixation removal step)
This step is a step of decomposing proteins contained in the sample by using protease for the sample after the ligation reaction. This decomposes proteins in the sample, including proteins that bind to DNA, nuclear membranes, etc. The interacting DNA sequences are located proximally as a result of the target protein being attached to the DNA chain, but after the DNA pair is formed by the ligation reaction, fixation by protein molecules is no longer necessary, so the protein is digested in this step. This eliminates the fixation of the DNA structure by the protein (fixation of the spatial structure of the DNA sequence). Along with this protein digestion, the fixation by crosslinking based on the crosslinking step is also eliminated. This results in a group of DNA pairs (ligation products) in which interacting DNA sequences are lined up and linked. Note that when a selection step (described later) is performed based on the label added in the labeling step (when chromatin immunoprecipitation is not performed), the target sample (DNA pair) can be taken out from inside the nucleus by digesting the nuclear membrane in this step. Therefore, the solubilization step of the nuclear fraction can be eliminated.
It is preferable that this step is performed following the ligation step, but it is performed after ligation and before the PCR amplification operation or the sequencing step. In addition, in the present invention, when a step of performing chromatin immunoprecipitation is performed, this step is not performed before the immunoprecipitation. In addition, when the fixation of the DNA structure is released as a result of fragmentation in the selection step, the fragmentation process in the selection step described below corresponds to the fixation release step. Even in such a case, if it is necessary to release the crosslinking based on the crosslinking step, this step constitutes a part of the fixation release step. In addition, even if it is not necessary to release the fixation of the DNA structure by this step because the fixation of the DNA structure is released by fragmentation, a step of digestion with a protease may be performed in the same manner as this step to digest the nuclear membrane, etc.

(Sorting step)
This is a step for selectively recovering DNA pairs. In this step, it is preferable to further fragment the DNA pairs obtained as ligation products, taking into consideration PCR efficiency and sequence efficiency. In addition, adapter sequences may be introduced into the ends of the fragments during fragmentation.

In one embodiment, the step of selecting the ligated DNA pair includes not only selecting the ligated DNA pair in the initial sequence state, but also selecting DNA fragments that have been processed or treated after ligation, DNA fragments that have been further fragmented by the treatment, and DNA fragments to which any DNA sequence has been added. For example, when a PCR step is performed, the DNA sequence added here may be a short adapter sequence. In this way, in the subsequent PCR step, a primer containing a complementary sequence of the short adapter sequence and an index sequence is hybridized to the short adapter sequence bound to the DNA pair (DNA fragment), and the index sequence can be added to the DNA pair while performing the amplification reaction. In addition, when the PCR step is not performed, a DNA sequence having both the index sequence and the short adapter sequence may be ligated to the DNA pair as an adapter sequence (full adapter), and a DNA to which the sequence selected in the selection step is added may be constructed. For example, the adapter sequence (full adapter) or the index sequence may include a DNA sequence complementary to a DNA fragment fixed to a flow cell used in a general known sequencer system.
In this selection step, for example, if a label (biotin) is added to the junction of each ligation product by the label addition step, the DNA pair (DNA fragment) is collected using the label as a target. For this collection, any protein that shows affinity to the label can be used, and various commercially available proteins can be used. For example, when the label is biotin, biotin-binding proteins such as avidin, streptavidin, and deglycosylated avidin that have extremely high affinity to biotin can be used. In addition, in this step, the selection operation may be performed using a support such as beads in which these proteins are immobilized. In this step, the DNA pair to be analyzed is selectively collected, and a DNA library is constructed. In addition, for example, the DNA library constructed of the DNA pairs collected based on the label may be hybridized with oligonucleotides to further select the DNA pairs.
In one embodiment, the step of selecting the DNA pairs (DNA fragments) includes selecting the DNA pairs by electrophoresis after ligation. For example, a label-free method is available in which the ligation products are electrophoresed using an agarose gel and selected by DNA strand length.

Sequencing Step
If desired, PCR is performed on the DNA library constructed as above according to a conventional method to amplify each DNA pair (DNA fragment). Then, the obtained PCR product is sequenced. When the DNA fragment is amplified, the amplification does not only mean amplifying the entire length of the DNA fragment, but also includes amplifying a part of it. The DNA pair (DNA fragment) concentrated by the PCR or the DNA pair in the DNA library constructed in the selection step is pre-treated, and the base sequence of both ends of the DNA pair is analyzed and sequenced by a massively parallel DNA sequence analyzer or the Sanger method. The length of the base sequence to be analyzed may be from one base to the entire length of the DNA fragment. The length of the base sequence to be analyzed may be set arbitrarily, and may be, for example, several bases or more, 10 bases or more, 15 bases or more, 20 bases or more, or 30 bases or more. Preferably, it is, for example, about 30-100 bases.

(Analysis method)
The obtained sequence data can be analyzed using the conventional HiC method. First, the sequences at both ends of the obtained base sequence are independently mapped to a reference genome sequence (mouse for mouse cells, human for human cells). In this method, adjacent DNA is artificially bonded to create DNA fragments (DNA pairs), so the ends can be independently mapped to the genome, but they may not be able to be mapped as DNA fragments. For example, if the DNA of chromosome 1 and the DNA of chromosome 2 are adjacent and a DNA fragment is created by this method, the obtained DNA fragment will have the sequence of chromosome 1 at one end and the sequence of chromosome 2 at the other end.

In this analysis, the same analytical techniques as those used in general HiC experiments can be applied, and such sequences (artificial sequences that cannot exist in the reference genome sequence) can be selectively extracted and further high-level analysis can be performed.

For example, by generating a contact map (contact probability matrix) as a heat map, the interaction between a DNA sequence at one location on the genome and another at another location can be visualized. A contact map is a symmetric matrix. The same genome sequence is arranged on the X and Y coordinates, and if there is a paired-end read from the sequence data that maps to positions Xi and Yj, the count for (Xi, Yj) is incremented by one. Therefore, a large value at each coordinate indicates a region pair with a high probability of contact. The coordinate values can be displayed on the heat map in a corresponding shade of color.

(sample amount)
The present method can obtain an analysis sample with extremely reduced impurities compared to conventional methods, and as a result, the number of cells required for analysis can be about 1 to 10 ¹⁰ , preferably about 1 to 10 ⁷ , and more preferably about 1 to 10 ⁶ . The upper limit of the number of cells is not necessarily limited, and may be 10 ¹⁰ or more.

Exemplary embodiments of the method of the present invention
In one embodiment, the method of the present invention can be used as an alternative to the conventional HiC method. In such a case, the above-mentioned crosslinking step is appropriately provided, followed by the cleavage step, labeling step, ligation step, fixation removal step, selection step, and sequencing step in sequence. According to this method, the nuclear membrane can also be digested in the fixation removal step as described above. In the chromatin immunoprecipitation method, the generation of an insoluble fraction is inevitable in principle because the nuclear fraction needs to be extracted. In the conventional immunoprecipitation method in which chromatin is cut genome-wide with a restriction enzyme to generate DNA fragments, it is important to solubilize all DNA fragments and take them out of the nucleus. This is because the remaining insoluble fraction in the nucleus will cause data errors. On the other hand, if the DNA sequence to be analyzed is selected based on the label added by the above-mentioned labeling step, the DNA pair can be selected using a protease after the ligation process, and a good analysis sample (without insoluble fractions that are not eluted from the nucleus) can be prepared. In one embodiment, when a chromatin immunoprecipitation step is provided to capture the target DNA fragment, only the target DNA fragment needs to be released into the supernatant, so that the undigested long-chain DNA fragments that are not the target of analysis do not affect the data obtained even if they remain in the nucleus as an insoluble fraction. Therefore, it is possible to suppress the decrease in reliability of data caused by the insoluble fraction in the nuclear solubilization step that occurs in the conventional immunoprecipitation method. In addition, unlike the conventional HiC method, DNA cleavage is performed specifically for a specific protein, so that the DNA sequence of the desired target of analysis is concentrated in the DNA library to be analyzed, and a high-quality analysis sample with extremely few unnecessary DNA fragments that become noise can be provided.
Furthermore, by independently analyzing the DNA cleavage site from the sequence results of the obtained DNA pair, the localization of the target protein can be detected. In other words, according to this method having a cleavage step for performing target protein-specific DNA cleavage, both the three-dimensional DNA structure in the nucleus mediated by the target protein and the localization of the target protein in the nucleus can be analyzed at the same time.
Furthermore, a protein biotinylation step for biotinylating a protein that binds to DNA may be provided before the cleavage step for cleaving DNA specifically in a target protein, and a solubilization step (dialysis process) for solubilizing the cleaved fragments by nuclear membrane permeabilization may be provided after the cleavage step to release the protein-DNA complex from within the nucleus into the supernatant, which is then pulled down using beads carrying streptavidin or the like, and then ligated onto the beads. Furthermore, a ligation step may be configured in which a fixation removal step, a selection step, and a sequencing step are sequentially performed to obtain sequence data.

In another embodiment, the method of the present invention may be configured to include a nuclear fraction solubilization step and a chromatin immunoprecipitation step, assuming that chromatin immunoprecipitation is performed. The nuclear fraction solubilization step and the chromatin immunoprecipitation step can be performed according to a conventional method. In such a case, as an example, after the above-mentioned crosslinking step is appropriately provided, a cleavage step, a labeling step, a ligation step, a nuclear fraction solubilization step, a chromatin immunoprecipitation step, a fixation removal step, a selection step, and a sequencing step are sequentially provided, thereby providing a method that can reduce impurities in the preparation stage of a DNA library to be analyzed more than conventional HiChIP as an alternative to conventional HiChIP. An example of the method of this embodiment corresponds to the three-dimensional DNA structure interaction analysis method described in item 6 above. The chromatin immunoprecipitation step included in the method of this embodiment is included in the capture step described in item 6 above.

In another embodiment, the method of the present invention includes the above-mentioned crosslinking step as appropriate, followed by the cleavage step, the nuclear fraction solubilization step, the chromatin immunoprecipitation step, the ligation step, the labeling step, the fixation removal step, the selection step, and the sequencing step in sequence to configure the present invention, thereby providing a method that can reduce impurities in the preparation stage of a DNA library to be analyzed more than ChIA-PET as an alternative to ChIA-PET. In this example, the selection step may be unnecessary because the DNA pair can be concentrated based on the chromatin immunoprecipitation step. In addition, in this embodiment, additional steps may be appropriately provided, for example, a step of appropriately refragmenting or adding a sequence to the ligated DNA fragments may be provided, and the DNA fragments after such a step may be amplified. An example of the method of this embodiment corresponds to the three-dimensional DNA structure interaction analysis method described in item 7 above. The chromatin immunoprecipitation step included in the method of this embodiment is included in the capture step described in item 7 above.

In one embodiment, the analytical kit of the present invention comprises a primary antibody that binds to any protein, a DNA cleavage enzyme that binds to the primary antibody, a label for identifying DNA ends generated by cleavage with the DNA cleavage enzyme, and an enzyme that ligates the labeled DNA ends. The primary antibody, DNA cleavage enzyme, label, and DNA end ligation enzyme contained in the analytical kit are materials used in the method of the present invention for analyzing interactions of DNA sequences based on the three-dimensional structure of DNA.
The primary antibody and the DNA cleaving enzyme may be contained separately, or the primary antibody and the DNA cleaving enzyme may be contained in a bound state.

The present invention will now be described in more detail with reference to examples. However, the following examples are merely examples of the present invention and are not intended to be limiting.

In the examples, the following reagents were used:
(reagent)
PBS (Phosphate Buffered Saline)
Sterile water (Nacalai Tesque)
1M CaCl ₂ (Calcium Chloride)/sterile water
1M DTT (Dithiothreitol)
1M Glycine/PBS
0.5% BSA/sterile water (0.5 wt.% bovine serum albumin (BSA) aqueous solution)
75% Ethanol/Sterile Water
Tween-20 (Chem Cruz)
DMSO (Dimethyl Sulfoxide)
5M NaCl
1M _MgCl2
1 M Tris-HCl (pH7.4) (Sigma-Aldrich)
0.5 M EDTA (pH8.0) (Ambion)
0.5 M EGTA (AG Scientific)
EGTA (Sigma-Aldrich)
Triton® X-100 (Nacalai Tesque)
IGEPAL® CA-630 (Sigma-Aldrich)
Spermidine Trihydrochloride (Sigma-Aldrich)
Digitonin (Nacalai Tesque)
cOmplete ^TM , EDTA-free Protease Inhibitor Cocktail Tablets (Roche)
1mol/l-HEPES buffer solution (Nacalai Tesque)

RSB (Resuspension Buffer)
composition:
10mM Tris-HCl (pH 7.4)
10 mM NaCl
3 mM _MgCl2
0.1% Tween-20
Sterile water
RSB was prepared with reference to Buenrostro JD, Wu B, Chang HY, Greenleaf WJ: ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr Protoc Mol Biol. 2015;109:21-9 and Corces MR, Trevino AE, Hamilton EG, Greenside PG, Sinnott-Armstrong NA, Vesuna S, Satpathy AT, Rubin AJ, Montine KS, Wu B, et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods. 2017;14:959-62.

5% Digitonin
composition:
50mg Digitonin (Nacalai Tesque)
500 μl DMSO
500 μl Sterile water

Lysis Buffer
composition:
0.1% IGEPAL CA-630
0.01% Digitonin
R.S.B.
Buenrostro JD, Wu B, Chang HY, Greenleaf WJ: ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr Protoc Mol Biol. 2015;109:21-9 and Corces MR, Trevino AE, Hamilton EG, Greenside PG , Sinnott-Armstrong NA, Vesuna S, Satpathy AT, Rubin AJ, Montine KS, Wu B, et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods. 2017;14:959-62 With reference to this, a lysis buffer was prepared.

Wash Buffer
composition:
1 ml of 1 M HEPES, 1.5 ml of 5 M NaCl, and 12.5 μl of 2 M spermidine were added, and sterile water was added up to 50 ml. Furthermore, one tablet of cOmplete ^™ , EDTA-free Protease Inhibitor Cocktail Tablets (Roche) was added to perform wash. A buffer was prepared.
Wash Buffer was prepared with reference to Skene et al., 2018. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nature Protocols. 2018. Vol. 13. No. 5.

Digitonin Buffer
composition:
0.02% Digitonin (0.0375% in Example 3)
Wash Buffer
Digitonin Buffer was prepared with reference to Skene et al., 2018. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nature Protocols. 2018. Vol. 13. No. 5.

Antibody Buffer
An antibody buffer was prepared by adding 2 ml of Digitonin Buffer and 8 μl of 0.5 M EDTA (pH 8.0).
Antibody buffer was prepared with reference to Skene et al., 2018. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nature Protocols. 2018. Vol. 13. No. 5.

Mbuffer #2
composition:
50 mM NaCl
10mM Tris-HCl (pH 7.4)
10 mM _MgCl2
Sterile water
Mbuffer #2 was prepared with reference to Rando et al., 2016. Micro-C XL. Protocol Exchange.

10x NEBuffer#2
composition:
1M NaCl
200mM Tris-HCl (pH 7.4)
200mM _MgCl2
Sterile water
10x NEBuffer #2 was prepared with reference to Rando et al., 2016. Micro-C XL. Protocol Exchange.

10X T4 DNA ligase buffer (no ATP)
composition:
500 mM NaCl
583mM Tris-HCl (pH 7.4)
200mM MgCl2
117mM DTT
10X T4 DNA ligase buffer was prepared with reference to Rando et al., 2016. Micro-C XL. Protocol Exchange.

1X Bind and Wash Buffer
composition:
5mM Tris-HCl, pH 7.4
0.5mM EDTA
1M NaCl
Sterile water
1X Bind and Wash Buffer was prepared with reference to Gabdank et al. 2016. A streamlined tethered chromosome conformation capture protocol. BMC Genomics. 2016. 17:274.

2X Bind and Wash Buffer
composition:
10mM Tris-HCl, pH 7.4
1mM EDTA
2M NaCl
Sterile water
2X Bind and Wash Buffer was prepared with reference to Gabdank et al. 2016. A streamlined tethered chromosome conformation capture protocol. BMC Genomics. 2016. 17:274.

Example 1
Expression and purification of fusion protein of protein A and MNase (pA-MN) E. coli strain One Shot® BL21(DE3) (Invitrogen) was transformed with plasmid pET11a-pA-MN (a vector constructed by introducing a DNA sequence expressing pA-MN into pET11a) and cultured at 37°C in LB medium (Lennox) (Funakoshi) containing 100 mg/L carbenicillin (Nacalai Tesque). Expression of pA-MN was induced by adding 0.1 mM isopropyl-β-thiogalactopyranoside (IPTG) to exponentially growing cells, changing the culture temperature to 18°C, and restarting the culture. After a 19-h induction period, bacteria were harvested from 500 ml of culture and collected in 30 ml TEN (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, and 150 mM NaCl) supplemented with 5 mM DTT. One mg chicken egg white lysozyme (Sigma) was added and incubated on ice for 10 min. Cells were disrupted by repeated sonication using an ultrasonic homogenizer (BIORUPTOR® II Type 6, Sonic Bio). The extract was clarified by centrifugation at 15,000 × G for 20 min in a centrifuge (MX-301, TOMY) at 4°C. The majority of pA-MN was recovered in the soluble supernatant (S1). pA-MN was purified from S1 using beads (IgG-Sepharose 6 Fast Flow, GE Healthcare). 30 ml of clarified S1 was incubated with 600 μl of IgG-Sepharose beads for 16 h with shaking. The beads were collected by passing through an Econopack chromatography column (BIO-RAD) and washed twice with TEN supplemented with 0.03% detergent (EMPIGEN® BB detergent, SIGMA), twice with TEN500 (TEN supplemented with 500 mM NaCl) containing 0.03% Empigen® BB, and once with 5 mM ammonium acetate (pH 5.0). pA-MN was eluted with 0.5 M acetic acid/ammonium acetate (pH 3.4). The eluate was neutralized by adding 1 mol/L NaOH aqueous solution. The same volume of glycerol was added to the neutralized eluate to adjust the final concentration to 50%. Approximately 1 mg of highly purified protein was obtained per 500 ml of starting culture. Long-term aliquots were stored at -80°C and short-term aliquots at -30°C.

HEK293T cells were suspended in PBS (FBS-PBS) containing 2 vol% fetal bovine serum (FBS), then poured into a tube so that the number of cells was 10 ⁷ , and centrifuged at room temperature for 5 minutes at a centrifugal force of 300 g. The separated cells were suspended in 1 ml of FBS-PBS containing 1 wt.% formaldehyde (trade name: Formaldehyde solution, model number F8775, Sigma-Aldrich), and gently stirred. Then, 340 μl of 1 M glycine PBS solution was added so that the final concentration was 250 mM, and a mixing operation was performed for 5 minutes at room temperature, followed by stirring by inversion as appropriate while holding on ice for 15 minutes. Then, washing was performed by performing a centrifugation operation at 4 °C, a centrifugal force of 800 g, for 5 minutes, and resuspension with 5 ml of PBS multiple times. In addition, during the centrifugation operation, the separated supernatant was appropriately removed. In the final washing, the supernatant (PBS) after centrifugation was removed as much as possible, and then the sample was rapidly cooled in liquid nitrogen.

The cell sample prepared by the above procedure was lysed, resuspended in 1 ml of Lysis Buffer, and kept on ice for 30 minutes while periodically stirring. After that, 1 ml of RSB was added, and the sample was centrifuged at 4°C for 5 minutes at 800 g, and then washed with 1.5 ml of Wash Buffer. After washing by adding Wash Buffer and centrifuging several times, the precipitate was collected by centrifugation at 4°C for 5 minutes at 800 g, and resuspended in 500 μl of Antibody Buffer solution containing 1 μg of anti-H3K4me3 antibody (model number ab8580, Abcam), and allowed to react overnight at 4°C. The anti-H3K4me3 antibody is an antibody against H3K4me3 (histone H3 with the fourth lysine trimethylated).

The sample after the reaction was centrifuged twice at 4° C. at a centrifugal force of 500 g for 5 minutes and resuspended in 1 ml of Digitonin Buffer, then centrifuged at 4° C. at a centrifugal force of 500 g for 5 minutes, and resuspended in 40 μl of Digitonin Buffer. pA-MN diluted 1000-fold with Digitonin Buffer (concentration appropriately adjusted) was added and reacted for 1 hour at 4° C. After that, centrifuged several times at 4° C. at a centrifugal force of 500 g for 5 minutes and resuspended in 1 ml of Digitonin buffer, and the precipitate centrifuged at 4° C. at a centrifugal force of 500 g for 5 minutes was resuspended in 150 μl of Digitonin buffer, and 3 μl of 100 mM CaCl ₂ solution was added and reacted at 37° C. for 20 minutes. Subsequently, ethylene glycol tetraacetic acid (EGTA) was added to a final concentration of 2 mM, and the mixture was treated at 65° C. for 10 minutes.

After that, centrifugation was performed at 4°C at a centrifugal force of 16,000 g for 5 minutes, and the pellet thus separated was suspended in 1 ml of Mbuffer #2. This was then repeated. The pellet was then resuspended in 30 μl of Mbuffer #2 (29 μl Mbuffer #2 + 1 μl 0.5% BSA/sterile water) containing a 0.5 wt.% aqueous solution of bovine serum albumin (BSA).

Next, 2.5 μl of a dephosphorylation enzyme (product name: Shrimp Alkaline Phosphatase (rSAP), New England BioLabs) was added to 30 μl of the sample prepared by the above procedure, and dephosphorylation treatment was carried out at 37°C for 45 minutes. At the end of the reaction, the dephosphorylation reaction was stopped by heat treatment at 65°C for 5 minutes.

Then, 3 μl of NEBuffer#2 adjusted to a 10x concentration (10x NEBuffer#2), 0.2 μl of an aqueous solution containing 0.5 wt.% BSA, 4.5 μl of 10 mM ATP solution (trade name Adenosine 5'-Triphosphate (ATP), New England BioLabs), 0.15 μl of 1 M DTT (trade name UltraPure ^™ Dithiothreitol (Clelands Reagent), Invitrogen/Life Technologies), 1.5 μl of T4 DNA polymerase (trade name T4 DNA Polymerase (5 U/μl), Thermo Scientific), and 3 μl of T4 polynucleotide kinase (10 U/μl) (trade name T4 Polynucleotide Kinase, New England BioLabs) were added and reacted at 37° C. for 7 minutes. Thereafter, 10x T4 DNA ligase buffer 6 μl, 0.3 μl of 0.5 wt. % BSA-containing aqueous solution, and biotinylated dNTP (biotinylated dATP (trade name: Biotin-14-dATP, Invitrogen), biotinylated dCTP (trade name: Biotin-14-dCTP, Invitrogen) at a final concentration of 0.1 mM, dGTP (trade name: dGTP, Toyobo), and dTTP (trade name: dTTP, Toyobo) at a final concentration of 0.05 mM were added to make a sample with a volume of about 100 μl, and the sample was reacted at 25° C. for 25 minutes, 12° C. for 15 minutes, and then maintained at 4° C. Then, ethylenediaminetetraacetic acid (EDTA) was added to a final concentration of 30 mM, and the sample was heat-treated at 65° C. to inactivate the enzyme activity. Through this series of treatments, biotin was added to the DNA cut ends.

Then, to about 110 μl of the heat-treated sample, 250 μl of ATP-added 10x concentration T4 Ligase buffer (trade name: 10X Buffer for T4 DNA ligase with 10 mM ATP; New England BioLabs), 12.5 μl of 0.5 wt.% BSA-containing aqueous solution, 3 μl of 1M _MgCl2 , and 12.5 μl of T4 DNA ligase (400 U/μl) (trade name T4 DNA Ligase, New England BioLabs) were added, and sterilized water was added to make the total volume 2500 μl, and the mixture was mixed at room temperature for 1 hour. Then, the mixture was centrifuged at 4°C, 16000 g for 10 minutes to obtain a pellet.

10 μl of NEBuffer#1 (product name NEBuffer 1, New England BioLabs) adjusted to a 10x concentration, 89 μl of sterile water, and 1 μl of Exonuclease III (100 U/μl, New England Biolabs) were added to the pellet to make 100 μl of sample solution. This was kept at 37°C for 5 minutes, and then 1/200 volume of protease (product name Proteinase K (Recombinant) Solution, Nacalai Tesque) was added and kept at 55°C for 3 hours to digest the proteins in the sample solution (decrosslinking).

Next, 100 μl of phenol-chloroform solution (trade name: phenol:chloroform:isoamyl alcohol 25:24:1, phenol content: approximately 33 w/w%, Nacalai Tesque) was added to the sample solution and mixed thoroughly, then centrifuged twice at room temperature, centrifugal force x 19,800 g, 10 minutes, and the supernatant was then collected. 1/10 volume of 3M sodium acetate solution (trade name: 3 mol/l Sodium Acetate Buffer Solution (pH 5.2), Nacalai Tesque) and 2.5 volumes of 100% ethanol were added to the collected supernatant, mixed, and left to stand at -80°C for 1 hour. The sample was centrifuged at 4°C, 19800g for 15 minutes to precipitate the DNA, and the supernatant was removed, washed with 1 ml of 75% ethanol, and centrifuged again at 4°C, 19800g for 15 minutes to remove the supernatant. The precipitate was air-dried at room temperature for 20 minutes, and 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) (product name Buffer EB Elution Buffer, Qiagen) was added and held at 37°C for 15 minutes to prepare a DNA solution. The prepared DNA solution was further purified, and the amount of DNA was confirmed using an ultramicrospectrophotometer (product name Qubit 3.0 Fluorometer, Invitrogen/Life Technologies).

The DNA solution was then dispensed into tubes so that the amount of DNA was 10 ng, and 25 μl of 2x concentrated TD buffer (trade name Tagment DNA Buffer (Nextera® DNA Library Prep Kit, Illumina) and 1 μl of transposome (Tagment DNA Enzyme 1 (TDE1), Illumina) were added, after which sterile water was added until the total solution volume reached 50 μl. This was kept at 55°C for 10 minutes to perform tagmentation. The post-reaction sample was purified to make 50 μl of DNA solution (Buffer EB solution).

Next, 10 μl of streptavidin bead dispersion (trade name Dynabeads ^TM (registered trademark) M-280 Streptavidin, Invitrogen/Thermo Fisher Scientific) was washed twice with 500 μl of 1x Bind and Wash buffer, and then suspended in 50 μl of 2x Bind and Wash Buffer. This 50 μl of bead dispersion was mixed with the above-purified DNA solution in a tube, and after keeping it at room temperature for 30 minutes, it was placed on a magnetic stand (for 1.5 ml), the supernatant was removed, and the streptavidin beads were recovered. The recovered beads were washed once with 500 μl of 1x Bind and wash buffer containing 0.1 vol% nonionic surfactant solution (trade name Triton X-100, Nacalai Tesque), and then further washed with 500 μl of Buffer EB, and then suspended in 40 μl of Buffer EB to obtain a dispersion.

10 μl of this dispersion was mixed with 10 μl of sterile water, 2.5 μl of 10 μM forward primer (N707, Nextera® XT Index Kit v.2, Illumina), 2.5 μl of reverse primer (S511, product name: Nextera® XT Index Kit v.2, Illumina), and 25 μl of DNA polymerase (KAPA HiFi HotStart ReadyMix (2X), Kapa Biosystems), and then PCR was performed. The PCR conditions were preheating at 72°C for 5 minutes, initial denaturation at 98°C for 3 minutes, thermal denaturation at 98°C for 10 seconds, annealing reaction at 63°C for 30 seconds, and extension reaction at 72°C for 1 minute for 13 cycles, and a final extension reaction at 72°C for 1 minute was performed, after which the amplified product was cooled to 4°C.

Prior to this PCR, real-time PCR was performed using a portion of the DNA solution, and the optimal number of cycles was determined from the plateau.

In order to recover DNA fragments of the desired DNA size of 200-1000 bp from the obtained PCR products, appropriate size selection was performed, and finally, 10 μl of a DNA library (DNA/Buffer EB solution) containing DNA fragments of 150 bp to 1300 bp was obtained.

Using the obtained DNA library, the base sequences at both ends of the DNA fragments (DNA pairs) were analyzed using a massively parallel DNA sequence analyzer (device name: NextSeq 500, Illumina). The length of the base sequence to be analyzed was 36 bases. The sequences at both ends of the obtained base sequences were independently mapped to the reference human genome sequence to analyze the interaction of the DNA sequences. The results are shown in Figures 2 and 3.

2 shows a heat map of interactions of chromatin regions where H3K4me3 exists. In order to establish the method of the present invention, interaction analysis of DNA sequences was performed using an anti-H3K4me3 antibody, and interactions of chromatin regions where H3K4me3 exists were analyzed using Juicebox.
FIG. 3 shows the localization of H3K4me3 on the genome and the interaction of chromatin regions containing H3K4me3, which were analyzed as an example. The interaction between H3K4me3 localized near the ACTB gene region (chromosome 7) and the chromatin region containing histone post-translational modifications was analyzed using WashU EpiGenome Browser. From the above results, the method of the present invention showed the same localization of H3K4me3 as CUT&RUN, which was performed separately, and further confirmed the interaction of chromatin regions containing H3K4me3. In addition, it was confirmed that the interaction of DNA sequences to which H3K4me3 binds occurs not only between the same gene and its neighboring regions, but also between distant gene regions.
FIG. 4 is a partially enlarged view of FIG.
FIG. 5 is a partially enlarged view of FIG.

Example 2
DNA sequence interaction analysis was performed in the same manner as in Example 1, except that the target protein was CTCF, 5 μg of anti-CTCF antibody (ab70303, Abcam) was used instead of the anti-H3K4me3 antibody in Example 1, the reverse primer was changed from S511 to S516 (product name: Nextera (registered trademark) XT Index Kit v.2, Illumina), and PCR was performed for 13 or 14 cycles.
The results are shown in Figures 6 and 7.

6 shows a heat map of interactions of chromatin regions where CTCF exists. In order to establish the method of the present invention, interaction analysis of DNA sequences was performed using an anti-CTCF antibody as in Example 1, and interactions of chromatin regions where CTCF exists were analyzed using Juicebox.
FIG. 7 shows the localization of CTCF on the genome and the interaction of the chromatin region where CTCF is present, analyzed as an example. The interaction of CTCF localized near the ACTB gene region (chromosome 7) with the chromatin region containing the transcription factor was analyzed using WashU EpiGenome Browser. As a result, the interaction of the chromatin region to which CTCF binds could be confirmed using the method of the present invention. It was also confirmed that the interaction of the DNA sequence to which CTCF binds occurs not only between the same gene and its neighboring region, but also between distant gene regions.
FIG. 8 is a partially enlarged view of FIG.
FIG. 9 is a partially enlarged view of FIG.

Example 3
The number of samples used in the analysis was 10 ⁶ cells, and the analysis of DNA sequence interactions was carried out according to Example 1. Specifically, the procedure is as follows.

HEK293T cells were suspended in PBS (FBS-PBS) containing 2 vol% fetal bovine serum (FBS), then poured into a tube so that the number of cells was 10 ⁶ , and centrifuged at room temperature for 5 minutes at a centrifugal force of 300 g. The separated cells were suspended in 1 ml of FBS-PBS containing 1 wt.% formaldehyde (trade name: Formaldehyde solution, model number F8775, Sigma-Aldrich), and gently stirred. Then, 340 μl of 1 M glycine PBS solution was added so that the final concentration was 250 mM, and a mixing operation was performed for 5 minutes at room temperature, followed by stirring by inversion as appropriate while holding on ice for 15 minutes. Then, washing was performed by performing a centrifugation operation at 4°C, a centrifugal force of 800 g, for 5 minutes, and resuspension with 5 ml of PBS multiple times. In addition, during the centrifugation operation, the separated supernatant was appropriately removed. In the final washing, the supernatant (PBS) after centrifugation was removed as much as possible, and then the sample was rapidly cooled in liquid nitrogen.

The cell sample prepared by the above procedure was lysed, resuspended in 1 ml of lysis buffer, and kept on ice for 30 minutes while periodically stirring. After that, 1 ml of RSB was added, and the sample was centrifuged at 4°C for 5 minutes at a centrifugal force of 800 g, and then washed with 1.5 ml of wash buffer. After performing the washing operation by adding wash buffer and centrifuging several times, the precipitate was collected after centrifugation at 4°C for 5 minutes at a centrifugal force of 800 g, and resuspended in 500 μl of antibody buffer solution containing 1 μg of anti-CTCF antibody (model number ab70303, Abcam), and reacted overnight at 4°C.

The sample after the reaction was centrifuged twice at 4° C. at a centrifugal force of 500 g for 5 minutes and resuspended in 1 ml of Digitonin Buffer, then centrifuged at 4° C. at a centrifugal force of 500 g for 5 minutes, and resuspended in 45 μl of Digitonin Buffer. pA-MN diluted 1000-fold with Digitonin Buffer (concentration appropriately adjusted) was added and reacted for 1 hour at 4° C. After that, centrifuged several times at 4° C. at a centrifugal force of 500 g for 5 minutes and resuspended in 1 ml of Digitonin buffer, and the precipitate centrifuged at 4° C. at a centrifugal force of 500 g for 5 minutes was resuspended in 150 μl of Digitonin buffer, and 3 μl of 100 mM CaCl ₂ solution was added and reacted at 37° C. for 20 minutes. Subsequently, ethylene glycol tetraacetic acid (EGTA) was added to a final concentration of 2 mM, and the mixture was treated at 65° C. for 10 minutes.

After that, centrifugation was performed at 4°C at a centrifugal force of 16,000 g for 5 minutes, and the pellet thus separated was suspended in 1 ml of Mbuffer #2. This was then repeated. The pellet was then resuspended in 30 μl of Mbuffer #2 (29 μl Mbuffer #2 + 1 μl 0.5% BSA/sterile water) containing a 0.5 wt.% aqueous solution of bovine serum albumin (BSA).

Next, 2.5 μl of a dephosphorylation enzyme (product name: Shrimp Alkaline Phosphatase (rSAP), New England BioLabs) was added to 30 μl of the sample prepared by the above procedure, and dephosphorylation treatment was carried out at 37°C for 45 minutes. At the end of the reaction, the dephosphorylation reaction was stopped by heat treatment at 65°C for 5 minutes.

Then, 3 μl of NEBuffer#2 (10x NEBuffer#2) adjusted to a 10x concentration, 0.2 μl of an aqueous solution containing 0.5 wt.% BSA, 4.5 μl of 10 mM ATP solution (trade name Adenosine 5'-Triphosphate (ATP), New England BioLabs), 0.15 μl of 1 M DTT (trade name UltraPure ^™ Dithiothreitol (Clelands Reagent), Invitrogen/Life Technologies), 1.5 μl of T4 DNA polymerase (trade name T4 DNA Polymerase (5 U/μl), Thermo Scientific), and 3 μl of T4 polynucleotide kinase (10 U/μl) (trade name T4 Polynucleotide Kinase, New England BioLabs) were added and reacted at 37° C. for 7 minutes, followed by addition of 10x T4 DNA ligase buffer. 6 μl, 0.3 μl of 0.5 wt. % BSA-containing aqueous solution, and biotinylated dNTP (biotinylated dATP (trade name: Biotin-14-dATP, Invitrogen), biotinylated dCTP (trade name: Biotin-14-dCTP, Invitrogen) at a final concentration of 0.1 mM, dGTP (trade name: dGTP, Toyobo), and dTTP (trade name: dTTP, Toyobo) at a final concentration of 0.05 mM were added to make a sample with a volume of about 100 μl, and the sample was reacted at 25° C. for 25 minutes, 12° C. for 15 minutes, and then maintained at 4° C. Then, ethylenediaminetetraacetic acid (EDTA) was added to a final concentration of 30 mM, and the sample was heat-treated at 65° C. to inactivate the enzyme activity. Through this series of treatments, biotin was added to the DNA cut ends.

Then, to about 110 μl of the heat-treated sample, 250 μl of ATP-added 10x concentration T4 Ligase buffer (trade name: 10X Buffer for T4 DNA ligase with 10 mM ATP; New England BioLabs), 12.5 μl of 0.5 wt.% BSA-containing aqueous solution, 3 μl of 1M _MgCl2 , and 12.5 μl of T4 DNA ligase (400 U/μl) (trade name T4 DNA Ligase, New England BioLabs) were added, and sterilized water was added to make the total volume 2500 μl, and the mixture was mixed overnight at 16° C. Then, the mixture was centrifuged at 4° C. and a centrifugal force of 16000 g for 10 minutes to obtain a pellet.

10 μl of NEBuffer#1 (product name NEBuffer 1, New England BioLabs) adjusted to a 10x concentration, 89 μl of sterile water, and 1 μl of Exonuclease III (100 U/μl, New England Biolabs) were added to the pellet to make 100 μl of sample solution. This was kept at 37°C for 5 minutes, and then 1/200 volume of protease (product name Proteinase K (Recombinant) Solution, Nacalai Tesque) was added and kept at 55°C for 3 hours to digest the proteins in the sample solution (decrosslinking).

The DNA solution was further purified and the amount of DNA was confirmed using an ultra-microspectrophotometer (Qubit 3.0 Fluorometer, Invitrogen/Life Technologies).

The DNA solution was then dispensed into tubes so that the amount of DNA was 10 ng, and 25 μl of 2x concentrated TD buffer (trade name Tagment DNA Buffer (Nextera® DNA Library Prep Kit, Illumina) and 0.5 μl of transposome (Tagment DNA Enzyme 1 (TDE1), Illumina) were added, after which sterile water was added until the total solution volume reached 50 μl. This was kept at 55°C for 10 minutes to perform tagmentation. The post-reaction sample was purified to make 50 μl of DNA solution (Buffer EB solution).

Next, 10 μl of streptavidin bead dispersion (trade name Dynabeads ^TM (registered trademark) M-280 Streptavidin, Invitrogen/Thermo Fisher Scientific) was washed twice with 500 μl of 1x Bind and Wash buffer, and then suspended in 50 μl of 2x Bind and Wash Buffer. This 50 μl of bead dispersion was mixed with the above-purified DNA solution in a tube, and after keeping it at room temperature for 30 minutes, it was placed on a magnetic stand (for 1.5 ml), the supernatant was removed, and the streptavidin beads were recovered. The recovered beads were washed once with 500 μl of 1x Bind and wash buffer containing 0.1 vol% nonionic surfactant solution (trade name Triton X-100, Nacalai Tesque), further washed with 500 μl of Buffer EB, and then suspended in 10 μl of Buffer EB.

10 μl of sterile water, 10 μl of 10 μM forward primer (N704, Nextera® XT Index Kit v.2, Illumina), 2.5 μl of reverse primer (S521, product name: Nextera® XT Index Kit v.2, Illumina), and 25 μl of DNA polymerase (KAPA HiFi HotStart ReadyMix (2X), Kapa Biosystems) were added to 10 μl of the mixture, and PCR was performed. The PCR conditions were preheating at 72°C for 5 minutes, initial denaturation at 98°C for 3 minutes, thermal denaturation at 98°C for 10 seconds, annealing reaction at 63°C for 30 seconds, and extension reaction at 72°C for 1 minute for 11 cycles, and a final extension reaction at 72°C for 1 minute, after which the amplified product was cooled to 4°C.

Prior to this PCR, real-time PCR was performed using a portion of the DNA solution, and the optimal number of cycles was determined from the plateau.

In order to recover DNA fragments of the desired DNA size of 200-1000 bp from the obtained PCR products, appropriate size selection was performed, and finally, 10 μl of a DNA library (DNA/Buffer EB solution) containing DNA fragments of 150 bp to 1300 bp was obtained.

Using this obtained DNA library, the base sequences at both ends of the DNA fragments (DNA pairs) were analyzed using a massively parallel DNA sequence analyzer (device name: NextSeq 500, Illumina). The length of the base sequence to be analyzed was 36 bases. The sequences at both ends of the obtained base sequence were independently mapped to a reference human genome sequence to analyze the interaction of the DNA sequences. The results are shown in Figure 10.

FIG. 10 shows the localization of CTCF on the genome and the interaction between chromatin regions where CTCF is present, analyzed as an example. At the top, the position on the genome is indicated by numbers, and in the upper row, a distribution map showing the localization of CTCF analyzed in the results obtained by separately performing the known CUT&RUN method (Skene et al., 2018) is shown as reference information. The middle row shows the CTCF binding sites present on the genome identified by the Jaspar Transcription Factor Database, and those in one direction are underlined according to the direction of the sequence on the genome. The lower row shows the interaction between chromatin regions where CTCF is present obtained by Example 3 of the present application in a loop shape below the localization of CTCF, together with the distribution map showing the localization of CTCF obtained by Example 3 of the present application. Further below the lower row, the positions of genes (FBXL18, MIR589, ACTB) in chromosome 7 are displayed, and the range marked to the right of the gene name indicates the position of the corresponding gene.
FIG. 11 is a partially enlarged view of FIG.
FIG. 12 is a partially enlarged view of FIG.

Claims (12)

1. A method for analyzing interactions of DNA sequences in a three-dimensional DNA structure, comprising:
(1) a cross-linking step for fixing the spatial arrangement of DNA sequences in the cell nucleus prior to the DNA cleavage step;
(2) a primary antibody binding step of selecting an arbitrary protein adjacent to a DNA strand in the DNA sequence and binding a primary antibody to the arbitrary protein; and an enzyme binding step of binding an enzyme that cleaves DNA to the arbitrary protein via the primary antibody, wherein the DNA cleaving step is characterized in that the DNA strand in the vicinity of the arbitrary protein is specifically cleaved by the enzyme that cleaves DNA;
(3) a ligation step of ligating the DNA ends generated by the cleavage in the DNA cleavage step;
(4) a selection step of selecting the ligated DNA fragments;
(5) a sequencing step of determining the base sequence of the DNA fragment selected in the selection step;
(6) a step of releasing the fixation of the DNA structure of the DNA fragment prior to the sequencing step,
A method for analyzing three-dimensional DNA structure interactions, comprising analyzing the interactions of the DNA sequences. The three-dimensional DNA structure interaction analysis method according to claim 1, characterized in that the interaction of the DNA sequences is an interaction between a DNA sequence adjacent to the given protein and a DNA sequence that is spatially adjacent to the adjacent DNA sequence but at a distant genomic locus. The three-dimensional DNA structure interaction analysis method according to claim 1 or 2, characterized in that the crosslinking step is performed by fixing with a drug selected from the group consisting of formaldehyde, EGS (ethylene glycol bis(succinimidyl succinate)) and DSG (disuccinimidyl glutarate). A labeling step of adding a label to the DNA end generated by the cleavage in the DNA cleavage step,
4. The method for analyzing three-dimensional DNA structure interaction according to claim 1, wherein the selection step selects the ligated DNA fragments based on the label. The three-dimensional DNA structure interaction analysis method according to any one of claims 1 to 4, characterized in that the selection step involves fragmenting the DNA fragments ligated in the ligation step into shorter DNA fragments and then selecting them. The three-dimensional DNA structure interaction analysis method according to any one of claims 1 to 5, characterized in that the sequencing step involves amplifying the selected DNA fragments and determining the base sequence of the amplified product. The three-dimensional DNA structure interaction analysis method according to claim 6, characterized in that the selection step adds a predetermined adapter sequence to the cut end of the amplified DNA fragment. 1. A method for analyzing interactions of DNA sequences in a three-dimensional DNA structure, comprising:
(1) a cross-linking step for fixing the spatial arrangement of DNA sequences in the cell nucleus prior to the DNA cleavage step;
(2) a primary antibody binding step of selecting an arbitrary protein adjacent to a DNA strand in the DNA sequence and binding a primary antibody to the arbitrary protein; and an enzyme binding step of binding an enzyme that cleaves DNA to the arbitrary protein via the primary antibody, wherein the DNA cleaving step is characterized in that the DNA strand in the vicinity of the arbitrary protein is specifically cleaved by the enzyme that cleaves DNA;
(3) a ligation step of ligating the DNA ends generated by the cleavage in the DNA cleavage step;
(4) a capture step of capturing the ligated DNA fragments;
(5) a fixation release step of releasing the fixation of the DNA structure of the DNA fragment captured in the capture step;
(6) a sequencing step of determining the base sequence of the DNA fragment after the fixation is released,
A method for analyzing three-dimensional DNA structure interactions, comprising analyzing the interactions of the DNA sequences. 1. A method for analyzing interactions of DNA sequences in a three-dimensional DNA structure, comprising:
(1) a cross-linking step for fixing the spatial arrangement of DNA sequences in the cell nucleus prior to the DNA cleavage step;
(2) a primary antibody binding step of selecting an arbitrary protein adjacent to a DNA strand in the DNA sequence and binding a primary antibody to the arbitrary protein; and an enzyme binding step of binding an enzyme that cleaves DNA to the arbitrary protein via the primary antibody, wherein the DNA cleaving step is characterized in that the DNA strand in the vicinity of the arbitrary protein is specifically cleaved by the enzyme that cleaves DNA;
(3) a capture step of capturing DNA fragments generated by the cleavage in the DNA cleavage step;
(4) a ligation step of ligating the ends of the DNA fragments captured in the capture step;
(5) a defixation step of defixing the DNA structure of the ligated DNA fragments;
(6) a sequencing step of determining the base sequence of the DNA fragment after the fixation is released,
A method for analyzing three-dimensional DNA structure interactions, comprising analyzing the interactions of the DNA sequences. The three-dimensional DNA structure interaction analysis method according to claim 8 or 9, characterized in that the interaction of the DNA sequences is an interaction between a DNA sequence adjacent to the arbitrary protein and a DNA sequence that is spatially adjacent to the adjacent DNA sequence but at a distant genomic locus. The three-dimensional DNA structure interaction analysis method according to any one of claims 8 to 10, characterized in that the crosslinking step is performed by fixing with a drug selected from the group consisting of formaldehyde, EGS (ethylene glycol bis(succinimidyl succinate)) and DSG (disuccinimidyl glutarate). The three-dimensional DNA structure interaction analysis method according to any one of claims 8 to 11, characterized in that the sequencing step involves amplifying the DNA fragments after the fixation is released and sequencing the amplified product to determine the base sequence.