Method for Mapping the Binding Site of a Compound

Abstract

The present invention relates to mapping the binding sites of test compounds within nucleic acids. The nucleic acid is contacted with a tagged test compound that binds to the nucleic acid or a protein associated with the nucleic acid at one or more locations. The tagged test compound is contacted with a first binding member that specifically binds to the tag and a second binding member that specifically binds to the first binding member and is accompanied by an activatable nuclease, such that the second binding member binds to the first binding member bound to the tagged test compound at one or more binding sites. The nuclease is then activated to cleave the nucleic acid at the binding sites to generate fragments. The sequence of the generated fragments indicates the binding sites of the test compound.

Description

Technical Field

[0001] The present invention relates to a method and kit for mapping the location of a site within a nucleic acid sequence where a chemical compound binds to a nucleic acid or a protein associated with the nucleic acid.

Background Art

[0002] The development of early anti-cancer drugs and antibiotics, which have come to be widely used, has been based on small molecules that directly target DNA in cells (1). In the past 20 years, the understanding of the structure and function of the genome, including interacting chromatin proteins, has increased significantly, thereby creating more opportunities to intervene in biology and pathology using small molecules. An essential aspect of the development of small molecule probes or therapeutic agents is the ability to verify target engagement at the molecular level (2). When the genome itself or chromatin structure serves as a target, the development of small molecule probes or therapeutic agents requires mapping at the molecular level where drug molecules bind throughout the genome.

[0003] Mapping inhibitors against chromatin has been found to be difficult and is mainly limited to a few high-affinity ligands against chromatin-binding proteins, including the bromodomain inhibitor JQ1 and the CDK9 inhibitor AT7519 (3-5). Genome-wide mapping involves immobilizing small molecules using an affinity tag, followed by pull-down of sheared chromatin and DNA sequencing. These techniques require high binding affinity and low dissociation rate, and generally are not applicable to many probes due to the potential for low signal, high background, and epitope masking caused by formaldehyde cross-linking. Also, a relatively low DNA recovery rate has to be overcome by a large amount of input material (4), thereby precluding application to rare cell populations.

[0004] The binding preference of DNA minor groove-binding molecules has been biophysically mapped using a randomized synthetic DNA oligonucleotide pool (6, 7), but the differences in accessibility to native chromatin have not been described. Alternatively, the DNA-binding agent psoralen can be UV cross-linked to DNA and its binding sites mapped, or similarly, the binding sites of small molecules with a psoralen moiety can be mapped (3, 8). A practical challenge to overcome with conventional methods is that the strength of the non-covalent interaction needs to overcome any dissociation between the DNA and the small molecule during subsequent DNA processing. Additionally, chemotherapeutic drugs that target DNA, such as doxorubicin, have been widely used clinically for decades. However, it has been impossible to measure where and to what extent these chemotherapeutic drugs bind to the human genome. Therefore, a general method for mapping small molecule-DNA interactions in intact cells in situ provides useful insights into the pharmacogenetics of DNA-binding agent action and enhances the ability to utilize the genome as a therapeutic target.

Summary of the Invention

[0005] The inventors have developed a method that enables the efficient and high-resolution mapping of the location of the binding sites of compounds such as drugs within nucleic acid sequences such as the genome.

[0006] A first aspect of the invention is a method for mapping the location of one or more binding sites of a test compound within a nucleic acid, comprising: (i) contacting the nucleic acid with a tagged test compound comprising a test compound covalently linked to a tag, wherein the tagged test compound binds to the nucleic acid or a protein associated with the nucleic acid at one or more locations within the nucleic acid; (ii) contacting the tagged test compound with a first binding member that specifically binds to the tag, such that the first binding member binds to the tagged test compound; (iii) A step of contacting a nucleic acid with a second binding member that specifically binds to a first binding member and is accompanied by an activatable nuclease, such that as a result, the second binding member binds to the first binding member that is bound to the tagged test compound at one or more binding sites. (iv) A step of activating the nuclease, such that as a result, the nucleic acid is cleaved by the nuclease at one or more binding sites to generate fragments. (v) A step of determining the sequence of the generated fragments. Provided is a method comprising the above.

[0007] The sequence of the nucleic acid fragment can indicate the location of one or more binding sites of the test compound within the nucleic acid.

[0008] The steps (i) and (ii) of the method of the first aspect may be carried out simultaneously or sequentially in any order. In some embodiments, the method of the first aspect comprises a step of contacting a tagged test compound with a first binding member that specifically binds to the tag, such that as a result, the first binding member binds to the tagged test compound to form a complex composed of the first binding member and the tagged test compound, and a step of contacting the nucleic acid with the complex. In other embodiments, the method of the first aspect may comprise a step of contacting the nucleic acid with the tagged test compound and then contacting the nucleic acid with a first binding member that specifically binds to the tag, such that as a result, the first binding member binds to the tagged test compound bound to the nucleic acid.

[0009] In some preferred embodiments of the first aspect, the nuclease may be a transposase. For example, the method of the first aspect may comprise (i) A step of contacting a nucleic acid with a tagged test compound comprising a test compound linked to a tag by a covalent bond, wherein the tagged test compound binds to the nucleic acid or a protein associated with the nucleic acid at one or more locations within the nucleic acid. (ii) A step of contacting a tagged test compound with a first binding member that specifically binds to the tag, whereby the first binding member binds to the tagged test compound. (iii) A step of contacting a nucleic acid with a second binding member that specifically binds to the first binding member and is accompanied by an activatable transposase loaded with an oligonucleotide adapter, whereby the second binding member binds to the first binding member that has bound to the tagged test compound at one or more binding sites. (iv) A step of activating the transposase, whereby the nucleic acid is cleaved by the transposase at one or more binding sites to generate fragments each of which has an end labeled with an oligonucleotide adapter. (v) A step of determining the sequence of the labeled fragment. It may include.

[0010] In some embodiments of the first aspect, the sequence of the generated fragment or the labeled fragment may be determined by sequencing. For example, the method of the first aspect (i) A step of contacting a nucleic acid with a tagged test compound containing a test compound linked to a tag by a covalent bond, wherein the tagged test compound binds to the nucleic acid or a protein associated with the nucleic acid at one or more locations within the nucleic acid. (ii) A step of contacting the tagged test compound with a first binding member that specifically binds to the tag, whereby the first binding member binds to the tagged test compound. (iii) A step of contacting a nucleic acid with a second binding member that specifically binds to the first binding member and is accompanied by an activatable transposase loaded with a sequencing adapter, whereby the second binding member binds to the first binding member that has bound to the tagged test compound at one or more binding sites. (iv) A step of activating a transposase, as a result of which the nucleic acid is cleaved at one or more binding sites by the transposase, generating fragments each of whose ends is labeled with a sequencing adapter. (v) A step of sequencing the labeled fragments. It may include.

[0011] In other embodiments of the first aspect, the sequence of the generated fragments or the labeled fragments can be determined by a hybridization-based technique, preferably sequence-specific amplification.

[0012] The test compound can bind to the nucleic acid or a protein associated with the nucleic acid at multiple locations within the nucleic acid. The method of the first aspect may be useful for identifying or mapping these multiple locations.

[0013] The method of the first aspect may include mapping the locations of the binding sites of a plurality of test compounds. For example, a method of mapping the locations of the binding sites of a population of test compounds within a nucleic acid is as follows: (i) A step of contacting the nucleic acid with a population of tagged test compounds, wherein each tagged test compound in the population contains a test compound linked by a covalent bond to a tag, and the tagged test compounds in the population bind to one or more sites within the nucleic acid or a protein associated with the nucleic acid. (ii) A step of contacting the tagged test compounds with a population of first binding members, as a result of which each first binding member in the population of first binding members specifically binds to a different tagged test compound in the population of tagged test compounds. (iii) A step of contacting the nucleic acid with a population of second binding members associated with an activatable nuclease, as a result of which each second binding member in the population specifically binds to a different first binding member that has bound to a tagged test compound at a binding site within the nucleic acid. (iv) A step of activating a nuclease, as a result of which the nucleic acid is cleaved at one or more binding sites by the nuclease to generate fragments, and (v) A step of determining the sequence of the generated fragments, and may be included.

[0014] The sequence of the nucleic acid fragments generated by the nuclease, which is bound to the tagged test compound in the population via the second binding member and the first binding member, may indicate the location of the binding site(s) of one or more test compounds within the nucleic acid.

[0015] The method of the first aspect may include mapping the location of the binding site(s) of the test compound within the first nucleic acid and the second nucleic acid having the same or different nucleotide sequences, and identifying a location that is present in the first nucleic acid and not in the second nucleic acid, or present in the second nucleic acid and not in the first nucleic acid. One of the first nucleic acid and the second nucleic acid can be subjected to treatment. The effect on the location of the binding site of the treatment can be determined.

[0016] In some embodiments of the first aspect, the location of one or more binding sites of the test compound within the nucleic acid, or the effect on the binding of one or more binding sites of the test compound, of a second test compound without a tag can be determined. For example, step (i) of the method may further include contacting the nucleic acid with a second test compound without a tag, and optionally, the second test compound without a tag binds to the nucleic acid or a protein associated with the nucleic acid at one or more locations within the nucleic acid.

[0017] A second aspect of the present invention is a kit for mapping the location of one or more binding sites of a test compound within a nucleic acid, comprising a tag covalently linked or linkable to the test compound, and a first binding member that specifically binds to the tag, and a second binding member that specifically binds to the first antibody, and a nuclease associated with or associable with the second binding member, and Provided is a kit including

[0018] Other aspects and embodiments of the present invention will be described in more detail below.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] The present invention relates to a method and kit for mapping the location of a site in a nucleic acid to which a test compound, such as a chemotherapeutic agent, binds to the nucleic acid or a protein associated with the nucleic acid (i.e., a protein associated with the nucleic acid). The nucleic acid is contacted with a test compound linked covalently to a tag (i.e., a tagged test compound). The tagged test compound binds to the nucleic acid or a protein associated with the nucleic acid at one or more locations within the nucleic acid. The nucleic acid is also contacted with a first or primary binding member that binds specifically to the tag, such that the first binding member binds to the tagged test compound. The nucleic acid is then contacted with a second or secondary binding member that binds specifically to the first binding member. The second binding member binds to the first binding member that has bound to the tagged test compound at one or more binding sites. A nuclease associated with the second binding member is then activated to cleave the nucleic acid to produce nucleic acid fragments containing one or more binding sites. The sequence of the generated nucleic acid fragments can then be determined, for example, by sequencing or site-specific amplification. The location of one or more binding sites within the nucleic acid in the sample can be identified or mapped from the sequence of the generated fragments.

[0021] The nucleic acid may be RNA. For example, the location of the binding site of a tagged test compound within a population of RNA molecules, such as a cellular transcriptome or a portion or fraction thereof, can be determined. Suitable RNA molecules include nuclear RNA molecules, such as pre-mRNA and miRNA.

[0022] The nucleic acid may be DNA. For example, the location of the binding site of a tagged test compound within a cell genome or a portion or fraction thereof can be determined. In some embodiments, the nucleic acid may be a product generated by amplification of a population of DNA molecules, such as all or part of a genome, such as one or more regions or loci of interest.

[0023] In some embodiments, the nucleic acid may be in the form of chromatin. Chromatin is a complex of DNA and proteins that forms the chromosomes of eukaryotic cells. As used herein, chromatin can include part of a chromosome, an entire chromosome, or more than one chromosome. In preferred embodiments, chromatin as used herein can include part or all of the nuclear genome and / or mitochondrial genome of a eukaryotic cell, e.g., preferably the entire genome of a eukaryotic cell. Chromatin can include heterochromatin and euchromatin.

[0024] In some embodiments, the nucleic acid may be present in a sample. Suitable samples can include tissues, organoids, cells, cell extracts or cell fractions, e.g., subcellular organelles such as nuclei or mitochondria.

[0025] In some preferred embodiments, the nucleic acid may be present in a cell or cell extract. For example, a sample containing one or more cells can be contacted with a tagged test compound as described herein, such that the tagged test compound contacts nucleic acids within the cell. In some embodiments, the nucleic acid may be present in a single cell or an extract derived from a single cell. In other embodiments, the nucleic acid may be present in a population of cells or an extract derived from a population of cells.

[0026] This can enable mapping the location of binding sites of test compounds within the genome or transcriptome of the cell. For example, a method for mapping the location of one or more binding sites of a test compound within a cell genome or transcriptome is (i) contacting the cell with a tagged test compound comprising a test compound linked to a tag by a covalent bond, wherein the tagged test compound binds to a nucleic acid or a protein associated with the nucleic acid at one or more locations within the genome or transcriptome of the cell; (ii) contacting the tagged test compound with a first binding member that specifically binds to the tag, such that the first binding member binds to the tagged test compound; (iii) contacting the cells with a second binding member that specifically binds to the first binding member and is associated with an activatable nuclease, such that the second binding member binds to the first binding member that is bound to the tagged test compound at one or more binding sites; (iv) activating the nuclease, such that the nucleic acid is cleaved by the nuclease at one or more binding sites, generating fragments; (v) determining the sequence of the generated fragments; may be included.

[0027] The cells can be permeabilized before, after, or during step (i).

[0028] The sequence of the nucleic acid fragments can indicate the location of the binding sites of one or more test compounds within the cell genome or transcriptome.

[0029] In some embodiments, the cell can be a prokaryotic cell. For example, the prokaryotic cell can be contacted as described herein with an antibacterial agent linked to the tag by a covalent bond. Suitable antibacterial agents include compounds that bind to proteins associated with nucleic acids, such as DNA gyrase.

[0030] In other embodiments, the cell can be a eukaryotic cell. The eukaryotic cell can be, for example, an immortalized cell line or a primary cell isolated from an individual, or can be in the form of a tissue or an organoid. For example, the eukaryotic cell can be present in a sample obtained from an individual, such as a biopsy sample or a xenograft sample.

[0031] Suitable eukaryotic cells include mammalian cells, preferably human cells. For example, eukaryotic cells can include somatic cells and germ line cells, and can be fully or partially differentiated cells, or undifferentiated cells or pluripotent cells including stem cells such as adult stem cells or somatic stem cells, fetal stem cells or embryonic stem cells, and can be at any stage of development. Suitable eukaryotic cells also include induced pluripotent stem cells (iPSCs), which can be derived from any type of somatic cell according to standard techniques. Eukaryotic cells also include nervous system cells including neurons and glial cells, contractile muscle cells, smooth muscle cells, hepatocytes, hormone-synthesizing cells, sebaceous gland cells, pancreatic islet cells, adrenal cortical cells, fibroblasts, mesenchymal cells, epithelial cells, keratinocytes, endothelial cells, urothelial cells, bone cells, chondrocytes, immune cells such as white blood cells, mesothelial cells, and adipocytes.

[0032] Suitable eukaryotic cells also include normal cells or cells associated with disease states, such as cancer cells, such as carcinoma cells, sarcoma cells, lymphoma cells, blastoma cells, or germ line tumor cells, and cells having the genotype of genetic disorders such as Huntington's disease, cystic fibrosis, sickle cell anemia, phenylketonuria, Down syndrome, or Marfan syndrome.

[0033] In order for the first antibody and the second antibody to enter the cell and be able to access the nucleic acid, the cell can be permeabilized. Suitable methods for permeabilizing cells are known in the art and include contacting the cell with a surfactant such as 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., Triton X-100 (trademark)), nonylphenoxypolyethoxyethanol (e.g., NP-40 (trademark)), polyoxyethylene sorbitan monolaurate (e.g., Tween (trademark)), saponin, or digitonin. For example, the cell can be permeabilized by exposing it to digitonin, such as 0.05% digitonin.

[0034] In some embodiments, the cells may be viable cells or living cells. For example, step (i) of the methods described herein may include contacting viable cells with a tagged test compound. The viable cells can be treated or exposed to the tagged test compound, for example, in a culture medium for a defined period of time, such as 1 hour to 48 hours, 2 hours to 36 hours, or 3 hours to 24 hours, such that the tagged test compound binds to nucleic acids or proteins associated with nucleic acids within the cells. After treatment with the tagged test compound, the cells can be permeabilized prior to contacting with the first binding member in step (ii).

[0035] In some embodiments, the nucleic acids or cells containing nucleic acids may be fixed prior to contacting with the test compound. Suitable methods for fixing cells are known in the art and include contacting the cells with an aldehyde fixative, such as formaldehyde, formalin, or glutaraldehyde, or an alcohol fixative, such as methanol, ethanol, or acetone. For example, the cells can be fixed by exposing them to 0.1% formaldehyde.

[0036] In some embodiments, in the methods described herein, the nucleic acids or cells containing nucleic acids may be in solution. For example, the nucleic acids or cells containing nucleic acids can be contacted with the tagged test compound, the first binding member, and / or the second binding member in solution. The nucleic acids or cells containing nucleic acids can be washed between steps, for example, by centrifugation and resuspension.

[0037] In other embodiments, the nucleic acid or the cell containing the nucleic acid may be immobilized on a solid support. The solid support is an insoluble non-gelatinous body that provides a surface on which a capture molecule for capturing eukaryotic cells can be immobilized. Examples of suitable supports include glass slides, microwells, membranes, or microbeads. The support may be in particulate form or in solid form, including, for example, plates, test tubes, beads, balls, filters, fabrics, polymers, or membranes. The capture molecule can bind to a protein, glycoprotein, or other molecule on the surface of the eukaryotic cell. Capture molecules suitable for eukaryotic cells are known in the art, and examples of such capture molecules include lectins that bind to extracellular glycoproteins on the cell, such as concanavalin A. In a preferred embodiment, the solid support is magnetic beads, such as magnetic beads coated with a lectin such as concanavalin A.

[0038] The sequence of the nucleic acid fragment can indicate the nucleic acid sequence at the location of the binding site of the test compound. In some embodiments, the test compound can bind to the nucleic acid or a protein associated with the nucleic acid at multiple locations within the nucleic acid. The sequence of the nucleic acid fragment can indicate the nucleic acid sequence at the location of the binding site of the test compound.

[0039] The test compound may be a compound or molecule that binds to the nucleic acid or a protein associated with the nucleic acid.

[0040] In some embodiments, the test compound can bind to a nucleic acid such as RNA or DNA. The nucleic acid may be present in or extracted from a cell, organelle, or tissue, or may be a product of amplification of one or more regions or loci of interest within the nucleic acid.

[0041] In some embodiments, the test compound can bind to DNA, i.e., the test compound may be a DNA-binding compound. Suitable DNA-binding compounds include compounds that intercalate between the base pairs of chromatin DNA, compounds that bind to the major or minor groove of chromatin DNA, guanine quadruplexes, Z-DNA, H-DNA, i-motifs, and compounds that bind to specific secondary structures of chromatin DNA such as higher-order structures such as loop-forming interactions between enhancers and promoters, as well as compounds that bind to other nucleic acid features such as repetitive elements, DNA mismatches, and DNA damage sites. In other embodiments, the test compound can bind to RNA, i.e., the test compound may be an RNA-binding compound. Suitable RNA-binding compounds include RNA splicing modifiers such as risdiplam and its analogs.

[0042] In other embodiments, the test compound may bind to a protein associated with a nucleic acid, e.g., a protein associated with DNA or a protein associated with RNA. The location of the protein associated with the nucleic acid to which the test compound binds within the nucleic acid can be determined by the methods described herein. Proteins associated with nucleic acids include histones; transcription factors such as Sox2 and c-Myc; nucleases such as deoxyribonucleases and ribonucleases (RNAse); polymerases; helicases; gyrases; DNA damage repair enzymes such as PARP, ATM, and Rad51; epigenetic modulators such as histone deacetylases (HDAC), histone acetyltransferases, histone acetyltransferases, histone demethylases, histone methyltransferases, EZH2, DOT1L, protein arginine deiminase, and epigenetic reader domains including bromodomain (BRD) such as BRD2 and BRD4; DNA methyltransferases; kinases such as CDK4, CDK6, CDK7, CDK9, AMP-activated protein kinase (AMPK), aurora kinases; Janus kinases (JAK) and protein kinase C; nuclear receptors such as retinoic acid receptor, thyroid hormone receptor, progesterone receptor, glucocorticoid receptor, androgen receptor, and estrogen receptor; transcriptional cofactors, epigenetic transcriptional cofactors, and sirtuins.

[0043] In some preferred embodiments, the test compound can bind to histones. Examples of histones include histone H2A, H2B, H3, H4 (so-called core histones), and H1 / H5 (so-called linker histones). The core histones associate to form an octamer that associates with nucleosomal DNA to form a nucleosome, and the linker histone H1 binds to the nucleosome at the entry and exit sites of the DNA. The histone-binding compound can bind to unmodified histones or histones modified by histone marks, such as methylated histones, which may be monomethylated histones, dimethylated histones, or trimethylated histones, glycosylated histones, phosphorylated histones, ADP-ribosylated histones, acetylated histones, ubiquitinated histones, SUMOylated or citrullinated histones (Luger, K. et al (1997) Nature 389, 251-260, (Ausio J (2001) Biochem Cell Bio 79, 693).

[0044] The test compound can bind to nucleic acids or proteins associated with nucleic acids by covalent or non-covalent bonds.

[0045] In some preferred embodiments, the test compound binds by non-covalent bonds. For example, the test compound can bind by non-covalent bonds to chromatin with a Kd of 0.001 nM or more, 0.01 nM or more, 0.1 nM or more, 1 nM or more, 5 nM or more, 10 nM or more, 15 nM or more, or 20 nM or more. In some embodiments, the test compound can bind with an affinity of 0.1 nM to 20 μM, such as 1 nM to 10 μM or 5 nM to 1 μM.

[0046] In the methods described herein, the test compound can bind to nucleic acids or proteins associated with nucleic acids by non-covalent bonds, and is then not accompanied by covalent cross-linking to the nucleic acids or proteins associated with nucleic acids.

[0047] Suitable test compounds include peptides, e.g., peptides of 50 amino acids or less, and organic small molecules of less than 5 KDa or less than 1 kDa, e.g., drugs such as anti-cancer drugs. For example, as test compounds, nuclear receptor inhibitors such as estradiol, tamoxifen, raloxifene, dihydrotestosterone, bicalutamide, dexamethasone, retinoic acid, triiodothyronine, progesterone, mifepristone, and rosiglitazone; pyrrole-imidazole polyamides such as PIP1, PIP2 and PIP2 (Figure 12), kinase inhibitors such as PD0332991, LEE011, THZ, AT7519, flavopiridol, and genistein; BET bromodomain inhibitors such as JQ1 and iBET151; CDK9 inhibitors such as AT7519; mechlorethamine, doxorubicin, actinomycin, bleomycin, etoposide, thalidomide, carboplatin, oxaliplatin, mitomycin C, intercalating agents such as cisplatin, bleomycin and adriamycin; guanine quadruplex-binding compounds such as PDS and PhenDC; HDAC inhibitors such as FK228, SAHA, LBH589 and valproic acid; Dot1L inhibitors such as EPZ004777; PARP inhibitors such as olaparib, iniparib, rucaparib and veliparib; and DNA polymerase inhibitors such as aphidicolin can be mentioned.

[0048] The test compound can bind to the nucleic acid or the protein associated with the nucleic acid at one or more locations within the nucleic acid. The test compound can bind directly to the nucleic acid at one or more locations within the sequence of the nucleic acid, or the test compound can bind to the protein associated with the nucleic acid within the sequence of the nucleic acid at one or more locations. For example, the protein associated with the nucleic acid containing the binding site of the test compound can associate with the nucleic acid at one or more locations within the sequence of the nucleic acid.

[0049] The test compound is linked to the tag by a covalent bond to form a tagged test compound. The tag may be any label, molecule or group that enables specific binding of the binding member to the test compound to which the tag is attached. The tag can enable covalent or non-covalent binding of the binding member. Suitable tags include immunogens such as digoxigenin; short-chain peptides such as glutathione and FLAG™; or small organic compounds such as biotin and trimethoprim (TMP).

[0050] In some embodiments, a suitable tag can enable covalent binding of the binding member. Suitable tags can include click-based tags that react with the binding member by a click chemistry reaction. For example, a click-based tag can include a first click chemistry group. Suitable click chemistry groups are known in the art and include, among others, an azide group or an alkyne group. This tag can be reacted with a first binding member that includes a second click chemistry group that reacts with the first click chemistry group, such as the other of an azide group or an alkyne group, to covalently link the binding member and the tag. Other suitable tags can include HaloTag™ ligand, SNAP™ ligand, or CLIP™ ligand, which can be reacted covalently with a first binding member that is a HaloTag™, SNAP™ tag, or CLIP™ tag, respectively.

[0051] Preferably, the tag is biotin and the tagged test compound can be a biotinylated test compound.

[0052] The tag may be directly connected to the test compound or may be connected to the test compound via a linker. Suitable linkers include alkyl chains, PEG chains, and combinations thereof. Cleavable linkers such as disulfide can also be mentioned as suitable linkers. Linkers and methods suitable for linking the test compound and the tag by covalent bonds are known in the art.

[0053] The first binding member (also referred to as the primary binding member) specifically binds to the tag portion of the tagged test compound. For example, when the tag is biotin, the first binding member can specifically bind to biotin. Examples of the first binding member suitable for binding to the biotin tag include anti-biotin antibody, avidin, and streptavidin.

[0054] The binding member is a target molecule or ligand, for example, a molecule that specifically binds to a tag. The binding member can bind to the tag of the tagged test compound by covalent bonds or, more preferably, by non-covalent bonds. The binding member that specifically binds to the tag may not show any significant binding to molecules other than the tag. In particular, the binding member may not show any significant binding to proteins, DNA, or other antigens that may be present in cells or cell extracts. Generally, an antibody or other binding member that specifically binds to a tag has a binding affinity (Ka) of more than about 10 5 mol / liter (for example, 10 6 mol / liter or more, 10 7 mol / liter or more, 10 8 mol / liter or more, 10 9 mol / liter or more, 10 10 mol / liter or more, 10 11 mol / liter or more, or 10 12 mol / liter or more).

[0055] Suitable binding members can include antibodies, aptamers, enzymes, peptides, or other binding modalities. In some preferred embodiments, the binding member can be an antibody, for example, the whole antibody (e.g., IgG such as IgG4) or an antibody fragment (e.g., single-chain variable fragment (scFv), Fab, dAb, nanobody, single-chain antibody, F(ab)2 fragment, VHH fragment, VNAR fragment, or single-chain Fab fragment (scFab)).

[0056] Antibodies suitable for use as the first binding member or the second binding member (also referred to as the primary binding member and the secondary binding member, respectively) in this method can be monoclonal or polyclonal and can be produced using conventional techniques. For example, an antibody can be produced by immunizing a mammal (e.g., mouse, rat, rabbit, horse, goat, sheep, or monkey) with a tag. The antibody can be obtained from the immunized animal using any of a variety of techniques known in the art and can preferably be screened using binding to the tag of the antibody. For example, Western blotting techniques or immunoprecipitation can be used (Armitage et al. (1992) Nature 357: 80-82). Isolation of the antibody and / or antibody-producing cells from the animal can involve the step of sacrificing the animal.

[0057] Monoclonal antibodies can be produced by isolating antibody-producing cells from immunized mammals, fusing those antibody-producing cells with immortalized cells to generate a population of antibody-producing hybridoma cells. This population can then be screened to identify hybridoma cells that produce antibodies with optimal binding characteristics. Methods for producing hybridoma cells and monoclonal antibodies are known in the art (see, e.g., Harlow et al Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) pp. 353-355). As an alternative or supplement to immunizing a mammal with a peptide, antibodies specific for a tag can be obtained, for example, from a recombinantly produced library of expressed immunoglobulin variable domains using a lambda bacteriophage or filamentous bacteriophage that displays a functional immunoglobulin-binding domain on its surface. See, e.g., WO 92 / 01047. The library may be a naive library constructed from sequences obtained from organisms that have not been immunized with a peptide containing the epitope, or it may be constructed using sequences obtained from organisms that have been exposed to the antigen of interest.

[0058] Binding members suitable for use as the first binding member or the second binding member in the present method are known in the art and can be prepared using standard techniques or obtained from commercial sources.

[0059] The first binding member and the tagged test compound can be contacted sequentially or simultaneously with the nucleic acid.

[0060] In some embodiments, a tagged test compound can be contacted with a nucleic acid, such that binding of the tagged test compound occurs at one or more locations within the nucleic acid sequence. This can be useful, for example, when the nucleic acid is present in a living cell as described above. The first binding member can then be contacted with the nucleic acid, such that the first binding member binds to the tagged test compound that is bound at one or more locations.

[0061] In other embodiments, the tagged test compound and the first binding member can be contacted with the nucleic acid simultaneously. For example, a complex composed of the tagged test compound and the first binding member can be contacted with the nucleic acid. The methods described herein can further include contacting the tagged test compound with the first binding member to form a complex and then contacting the complex with chromatin. A method for mapping the location of one or more binding sites of a test compound within a nucleic acid is contacting the nucleic acid with a complex composed of a tagged test compound and a first binding member, wherein the tagged test compound includes a test compound linked by a covalent bond to a tag, and the tagged test compound binds to the nucleic acid or a protein associated with the nucleic acid at one or more locations within the nucleic acid, and the first binding member specifically binds to the tag; contacting the nucleic acid with a second binding member that specifically binds to the first binding member and is associated with an activatable nuclease, such that the second binding member binds to the first binding member that is bound to the tagged test compound at one or more binding sites; activating the nuclease, such that the nucleic acid is cleaved by the nuclease at one or more binding sites to generate fragments; determining the sequences of the generated fragments; and may include.

[0062] Optionally, a washing step can be carried out to remove unbound reagents. For example, test compounds that are not bound to chromatin after the contacting step can be removed by washing. Similarly, after the contacting step, the first binding member that is not bound to chromatin via the test compound can be removed by washing. Suitable washing methods are known in the art. For example, washing can be carried out using the buffers described herein, such as 20 mM HEPES, pH 7.5, 150 mM NaCl and 0.5 mM spermidine.

[0063] The second binding member (also referred to as the secondary binding member) specifically binds to the first binding member. For example, the second binding member may be an antibody. Suitable anti-immunoglobulin (Ig) antibodies are known in the art. For example, the first antibody may be derived from a first species, such as a mammalian species like rabbit, and the second antibody may bind to the antibody derived from the first species. For example, the second antibody may be derived from a second species, such as a different mammalian species like guinea pig.

[0064] The second binding member can be associated with the nuclease either by covalent bond or by non-covalent bond. In some embodiments, the second binding member and the transposase can be bound by non-covalent bond. Methods suitable for non-covalent binding between the nuclease and the binding member are known in the art. For example, the second binding member may be an antibody, and the nuclease can be fused with an immunoglobulin-binding moiety to form a fusion protein. The fusion protein can be bound to a second antibody by non-covalent bond through the immunoglobulin-binding moiety. Suitable Ig-binding moieties are known in the art, and these include protein A, protein G, protein L, protein Y, or any binding domain thereof, anti-Ig antibodies or antibody molecules, such as single-chain antibodies, Fab fragments, F(ab)2 fragments, VHH fragments, VNAR fragments, nanobodies, single-chain variable fragments (scFv), or single-chain Fab fragments (scFab). In other embodiments, the second binding member can be covalently bound to the nuclease. For example, the second binding member and the transposase can be contained in a single fusion protein.

[0065] A nuclease is an enzyme capable of cleaving the phosphodiester bond between nucleotides of nucleic acids. Suitable nucleases include transposases. The nuclease may be an activatable nuclease. An activatable nuclease can be converted from an inactive state to an active state by changing conditions. For example, a transposase activated by magnesium can be converted to an active state by increasing the concentration of magnesium ions (Mg 2+ ) or manganese ions (Mn 2+ ) to a concentration within the range of, for example, about 0.1 mM to about 10 mM. Micrococcal nuclease activated by calcium can be activated by increasing the concentration of calcium ions (Ca 2+) can be converted to the active state by raising it. Suitable activatable nucleases are available in the art (see, for example, U.S. Patent No. 9,005,935 and International Publication No. 2022 / 056309).

[0066] Suitable activatable nucleases are known in the art and include micrococcal nuclease (MNase) and transposases such as Tn5, Tn7, Mu, IS5, and IS91 (e.g., U.S. Patent No. 9,005,935, Mizuuchi, K., Cell, 35: 785, 1983, Savilahti, H, et al, EMBO J., 14: 4893, 1995, Goryshin and Reznikoff, J. Biol. Chem, 273:7367 (1998), and International Publication No. 2022 / 056309). In some preferred embodiments, the nuclease may be a transposase such as Tn5 transposase. One or more oligonucleotide adapters can be loaded onto the transposase. For example, an oligonucleotide adapter can be non-covalently bound to the transposase to form a complex composed of the transposase and one or more oligonucleotide adapters. Oligonucleotide adapters are described below. Methods suitable for loading oligonucleotide adapters onto transposases are known in the art. For example, the oligonucleotide adapter can be incubated with the transposase at room temperature for 1 hour. In some embodiments, the sequence of the labeled fragment can be determined by sequencing, and the oligonucleotide adapter may be a sequencing adapter. One or more sequencing adapters can be loaded onto the transposase. For example, a sequencing adapter can be non-covalently bound to the transposase to form a complex composed of the transposase and one or more sequencing adapters. Sequencing adapters are described below. Methods suitable for loading sequencing adapters onto transposases are known in the art. For example, the sequencing adapter can be incubated with the transposase at room temperature for 1 hour.

[0067] After the contacting step, the second binding member that is not bound to the first binding member can be removed by washing.

[0068] After the second binding member binds to the first binding member, the nuclease can be activated. Methods suitable for activating nucleases are known in the art. For example, for appropriate ions, such as magnesium or manganese ions for transposase and calcium for micrococcal nuclease, the nuclease can be activated by increasing the concentration. The activated nuclease cleaves the nucleic acid at the location where the tagged test compound in the nucleic acid binds to the nucleic acid or a protein associated with the nucleic acid, generating nucleic acid fragments. For example, the nuclease can cleave the nucleic acid at a location within 10 bp to 1000 bp, preferably within 50 bp to 500 bp, of the binding site. The nucleic acid fragment contains the sequence at the location where the tagged test compound of the nucleic acid binds to the nucleic acid or a protein associated with the nucleic acid.

[0069] Specific cleavage of the nucleic acid by the nuclease at the location where the tagged test compound binds generates a specific population of fragments containing the nucleic acid sequences at these locations. This enables the determination of the nucleic acid sequences at these locations by subsequent analysis. The sequences at these locations may be indistinguishable from other sequences (low signal-to-noise) if the nucleic acid is fragmented randomly.

[0070] In some embodiments, an oligonucleotide adapter, such as a sequencing adapter, can be appended to the ends of the nucleic acid fragments, for example, by ligation, to generate nucleic acid fragments with each end labeled with an oligonucleotide adapter. Suitable ligation methods are known in the art.

[0071] An oligonucleotide adapter may be a double-stranded oligonucleotide, a partially double-stranded oligonucleotide, or a single-stranded oligonucleotide that is attached to a nucleic acid molecule to enable determination of the sequence of the nucleic acid molecule. Examples of oligonucleotide adapters include amplification adapters and sequencing adapters. An amplification adapter may be, for example, a double-stranded oligonucleotide, a partially double-stranded oligonucleotide, or a single-stranded oligonucleotide that is attached to a nucleic acid molecule to enable amplification of the molecule by PCR. Suitable amplification adapters can specifically hybridize to amplification primers and are readily available in the art. A sequencing adapter is a double-stranded oligonucleotide, a partially double-stranded oligonucleotide, or a single-stranded oligonucleotide that is attached to a nucleic acid molecule to enable sequencing. Sequence adapters suitable for any sequencing method are available in the art. In some embodiments, the sequencing adapter may include a primer recognition sequence. The primer recognition sequence is a nucleotide sequence complementary to the sequence of an amplification primer. The presence of the primer recognition sequence enables amplification of a labeled fragment, for example, by PCR using an amplification primer that targets the primer recognition sequence. The primer recognition sequence may be a heterologous sequence that is not naturally present in the chromatin DNA sequence. Suitable primer recognition sequences are known in the art and include, for example, Illumina-compatible barcoded i7 / i5 primers.

[0072] In some embodiments, the sequencing adapter may include a first sequencing adapter and a second sequencing adapter. The first sequencing adapter may include a first primer recognition sequence. The second sequencing adapter may include a second primer recognition sequence.

[0073] In some embodiments, the sequencing adapter may further comprise a barcode. The barcode is a nucleotide sequence unique to the sample from which the nucleic acid population was obtained. Suitable barcode sequences can be 6 to 10 nucleotides. The barcode enables the unambiguous identification of sequence reads from a particular sample in a pooled multiplex sequencing reaction. Each sample may have a unique barcode, and thus all of the nucleic acids from the same sample receive the same barcode. Once prepared, nucleic acid populations from different samples can be mixed into a single pool and sequenced. The sample that is the origin of the sequence reads from the pool can then be identified from the barcode. For example, barcodes suitable for multiplex sequencing of 24 samples (24-plex reaction) can consist of at least 6 nucleotides, preferably 6 nucleotides (Craig DW et al. 2008. Nat Methods 5, 887, Cronn R et al. 2008. Nucleic Acids Res, 36, e122). The use of barcodes in sequencing reactions is known in the art. For example, Illumina sequencing 8mer barcodes i5 and i7 can be used.

[0074] In embodiments where the nuclease is an endonuclease such as MNase, an oligonucleotide adapter such as a sequencing adapter can be appended to the ends of the nucleic acid fragments, for example, by ligation.

[0075] In an embodiment where the nuclease is a transposase, an oligonucleotide adapter such as a sequencing adapter can be loaded onto the transposase. The transposase can cleave the nucleic acid and insert the oligonucleotide adapter at the ends of the nucleic acid fragments. Thereby, nucleic acid fragments labeled with oligonucleotide adapters at each end are generated, which may also be referred to herein as "tagmentation". An oligonucleotide adapter suitable for attachment to the transposase can be a double-stranded oligonucleotide containing a transposase recognition sequence and a primer recognition sequence. The transposase recognition sequence is the sequence targeted for transposition by the transposase. For example, with the reverse transposase recognition sequence, the transposon sequence inserted by the transposase can be flanked. The sequence of the transposase recognition sequence can be transposase-dependent. Transposase recognition sequences suitable for the transposase are known in the art. For example, transposase recognition sequences suitable for Tn5 include 19-mer terminal sequences, such as the outer end (OE) sequence of 5'-CTG ACT CTT ATA CAC AAG T-3' and the inner end (IE) sequence of 5'-CTG TCT CTT GAT CAG ATC T-3'; and mosaic end (ME) sequences such as 5'-CTG TCT CTT ATA CAC ATC T-3'. In some preferred embodiments, the 19-mer ME sequence can be used. In some embodiments, the oligonucleotide adapter may include a first oligonucleotide adapter and a second oligonucleotide adapter. The first oligonucleotide adapter may include a first transposase recognition site and a first primer recognition sequence. The second oligonucleotide adapter may include a second transposase recognition site and a second primer recognition sequence. For example, the sequencing adapter may include a first sequencing adapter and a second sequencing adapter. The first sequencing adapter may include a first transposase recognition site and a first primer recognition sequence.The second sequencing adapter may include a second transposase recognition site and a second primer recognition sequence.

[0076] In some embodiments, the labeled nucleic acid fragment prepared as described above can be amplified to directly determine the sequence of the fragment. The labeled nucleic acid fragment can be amplified using one or more primers that hybridize to a nucleic acid sequence known to contain the binding site of the test compound. Amplifying the labeled nucleic acid fragment with a primer to generate an amplified fragment indicates that the labeled nucleic acid fragment contains a nucleic acid sequence. For example, the labeled nucleic acid fragment can contain a nucleotide sequence within the nucleic acid to which the compound directly binds, or a nucleotide sequence that associates with a protein that associates with the nucleic acid to which the compound binds (or is contained within a protein or adjacent nucleosome that associates with the nucleic acid to which the compound binds).

[0077] Suitable nucleic acid sequences include RNA, such as nuclear RNAs like pre-mRNA and miRNA, and genomic DNA, such as chromosomal DNA or mitochondrial DNA sequences. The nucleic acid sequences can include binding sites, genes, and transcripts related to the biological response to the test compound and / or off-target toxicity. Suitable binding sites, genes, and transcripts can be, for example, those known in the art or previously determined by the methods described herein. It is preferred to perform the amplification of the labeled nucleic acid fragment using multiple sets of primers that each hybridize to different nucleic acid sequences known to contain the binding site of the test compound. This makes it possible to determine the presence of multiple nucleic acid sequences within the labeled nucleic acid fragment. A panel can be formed by multiple nucleic acid sequences to which the sets of primers hybridize. For example, the panel targeted by the sets of primers can include two, three, four, five, six, seven, eight, nine, ten or more, 100 or more, or 1000 or more nucleic acid sequences. The panel can be useful, for example, as a "fingerprint" or "signature" that enables the rapid identification of test compounds with desirable characteristics and can also reduce off-target toxicity. It is preferred to quantitatively amplify the labeled nucleic acid fragment so that the amount or number of copies of the nucleic acid sequences within the labeled nucleic acid fragment can be determined. This can, for example, make it possible to determine the amount of binding of the test compound to each nucleic acid sequence. Suitable amplification methods are well established in the art and include quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RT-PCR), and isothermal amplification methods.

[0078] A sample binding profile can be generated from the distribution of nucleic acid sequences amplified by multiple sets of primers. The sample binding profile can include a score or value indicating the number or density of each amplified nucleic acid sequence. The number or density of nucleic acid sequences containing the binding site can indicate the amount or degree of binding of the test compound at that site, or the occupancy of that site by the test compound. Thus, the sample binding profile can reflect the binding, occupancy, or distribution of the test compound at various binding sites defined by a panel of nucleic acid sequences amplified by multiple sets of primers. The sample binding profile can be represented in any convenient format, for example, numerically or graphically.

[0079] In other embodiments, the labeled nucleic acid fragments prepared as described above can be amplified to generate amplified fragments for sequencing. Amplification of the labeled nucleic acid fragments can be performed using primers that hybridize to the primer recognition sequences of the sequencing adapters. Suitable amplification methods are well established in the art and include, for example, polymerase chain reaction (PCR) (e.g., reviewed in "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, 1990, Academic Press, New York, Mullis et al, Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR technology, Stockton Press, NY, 1989, and Ehrlich et al, Science, 252:1643-1650, (1991)).

[0080] In some embodiments, nucleic acid fragments or amplified nucleic acid fragments can be extracted, for example, from a sample. Suitable methods for extracting and isolating nucleic acids from samples of biological fluids are known in the art and include phenol / chloroform extraction and alcohol precipitation, cesium chloride density gradient centrifugation, solid phase anion exchange chromatography, and silica membrane-based techniques (e.g., Quick-cfDNA™, Zymo Research Corp). Many of the known techniques and protocols for nucleic acid extraction, amplification, and sequencing described herein are known in the art. See, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992), Next-generation Sequencing: Current Technologies and Applications; ed Jianping Xu Caister Academic Press (2014).

[0081] Amplification can be used to generate a population or library of amplified fragments for sequencing. Each amplified fragment within the population or library can contain a nucleic acid sequence at the location of the binding site of a test compound. A population or library of amplified fragments generated as described herein can be purified using standard techniques including spin column chromatography (e.g., Ampure XP™ beads) prior to sequencing.

[0082] Sequencing of the identified or amplified fragment can be performed using standard sequencing techniques. Suitable techniques include Sanger sequencing, Solexa-Illumina sequencing, ligation-based sequencing (SOLiD™), pyrosequencing; single molecule real-time sequencing (SMRT™); PacBioscience sequencing; and semiconductor array sequencing (Ion Torrent™), and the use of any convenient low-throughput or high-throughput sequencing technique or platform, including but not limited to these. Sequencing is preferably carried out by next-generation sequencing techniques. Suitable protocols, reagents and instruments for nucleic acid sequencing are known in the art and are commercially available.

[0083] The sequence of the nucleic acid fragment can indicate the nucleic acid sequence at the location of the binding site of the test compound. For example, the sequence of the amplified fragment can include the nucleotide sequence to which the compound binds directly within the nucleic acid, or the nucleotide sequence that associates with a protein that associates with the nucleic acid to which the compound binds (or is contained within the same or adjacent nucleosomes as the protein that associates with the nucleic acid to which the compound binds). The position of one or more binding sites within the nucleic acid can be identified or mapped. For example, the position of one or more binding sites within the genomic DNA of a cell such as a eukaryotic cell, mammalian cell or human cell can be identified or mapped by the methods described herein.

[0084] In some embodiments, a set of sequence reads of the nucleic acid fragment can be generated by sequencing, for example, 1000 or more, 10,000 or more, 100,000 or more, 1,000,000, 10,000,000 or more, or 100,000,000 or more, or 1,000,000,000 or more sequence reads can be generated. The sequence reads can be analyzed by conventional bioinformatics techniques. Suitable techniques are known in the art. For example, overlapping reads, low-quality sequence reads and reads resulting from sequencing adapters only can be removed.

[0085] In some embodiments, nucleic acid fragment sequence reads within a population can be analyzed for the presence and / or pattern of binding sites. Nucleic acid fragment sequence reads within a population can be further analyzed for other features associated with binding sites and / or patterns of binding sites, such as the presence of mutations, epigenetic modifications, or sequence motifs.

[0086] Nucleic acid fragment sequence reads within a population can be mapped to one or more locations within a reference genome. Suitable reference genomes are available in the art. For example, nucleic acid fragment sequence reads within a human population can be mapped to locations within the sequence of the human genome. In some embodiments, the reference genome can be matched to the gender, ethnicity, and / or other characteristics of the individual from whom the sample was obtained.

[0087] Mapping of nucleic acid fragment sequence reads within a population can be performed by aligning the sequence reads within the population to a reference genome, such as the sequence of the human genome. The location of the sequence reads within the reference genome can be identified. Suitable software tools for mapping a population of sequence reads within a genome are readily available in the art.

[0088] The distribution of nucleic acid fragment sequence reads within a population within a genome or within a set of genomic sites or loci (i.e., the number of sequence reads mapped to each location within the genome or set of sites or loci) can be determined from the locations of the sequence reads within the population. Optionally, the distribution of amplified fragment sequence reads can be subjected to a mathematical transformation. Suitable transformations include Fourier transforms.

[0089] A sample binding profile can be generated from the distribution of nucleic acid fragment sequence reads or a transformed distribution. The sample binding profile can include a set of scores or values indicating the number or density of nucleic acid fragment sequence reads mapped to each location or position within the genome (i.e., a plot across the entire genome) or each location or position within a set of sites or loci within the genome. The number or density of nucleic acid fragment sequence reads mapped to a location or position within the genome can indicate the amount or degree of binding of a test compound at that location or position, or the occupancy by the test compound at that location or position. Thus, the sample binding profile can reflect the binding, occupancy, or distribution of a test compound in the genome or at a target locus within the genome. The sample binding profile can be represented in any convenient format, for example, numerically or graphically.

[0090] In some embodiments, the sample binding profile can be used to identify the location of binding sites that are associated with a biological response to a test compound and can be useful for determining or predicting an individual's response to treatment with the test compound. The sample binding profile can be used for therapeutic stratification, optimization of combination therapies, diagnosis of disease states, or determination of side effects of treatment with a test compound.

[0091] A marker location is a location within the genome where the binding of a test compound varies between different sources, i.e., a location where the binding of a test compound at the marker location is specific to a source derived from, for example, different individuals, tissues, or cell types. Binding of a compound in a set of marker locations within the sample binding profile can result in a signature that is characteristic of the source.

[0092] In some embodiments, suitable marker locations can be identified by determining binding at a plurality of candidate locations within a reference binding profile derived from a set of reference sources. The reference binding profile can include a set of scores or values indicating binding at a set of marker locations within genomic DNA from a known source, e.g., a particular known tissue or cell type. The reference binding profile can reflect binding at locations within genomic DNA from a known source. A suitable reference binding profile can also be obtained or generated by routine experiments using known tissues or cell types, or can be created from publicly available data sources such as databases of genomic information.

[0093] A candidate location can be identified as a marker binding location if the binding occupancy at that site is higher or lower for one reference source in the set than for other sources in the set. For example, the binding at a location can be higher or lower than the average binding at that location in other reference sources in the set. In some embodiments, the binding at a location in one reference source can be above or below a predetermined threshold relative to the average binding at locations in other reference sources in the set.

[0094] In other embodiments, suitable marker locations may be identified by providing a first set of sample binding profiles from control individuals, e.g., healthy individuals, and a second set of sample binding profiles from test individuals, e.g., individuals having a disease state such as a particular cancer, or individuals known to be responsive or non-responsive to a test compound. The binding or occupancy of a compound at multiple candidate locations within the first and second sets of sample binding profiles can be compared. A candidate location can be identified as a marker location if the binding or occupancy of the compound at that location is higher or lower in the first set of sample binding profiles than in the second set. For example, the average binding or occupancy of the compound at that location can be higher or lower in the first set than in the second set. In some embodiments, the average binding or occupancy of the test compound at a location within the first set can be above or below a predetermined threshold relative to the average binding or occupancy of the compound at that location within the second set.

[0095] A candidate location can also be identified as a marker location if it is found to be in linkage disequilibrium with a site where the binding or occupancy of the test compound is higher or lower for one reference source within the set than for other sources within the set.

[0096] The sample binding profile from an individual can be used to determine the binding or occupancy of a test compound at a set of marker locations in the individual. This can be useful for determining the effect of a test compound on an individual, or the responsiveness of an individual to treatment with a test compound, for example, to determine the efficacy of treatment of an individual with a test compound or the suitability of an individual for treatment with a test compound. The sample binding profile from an individual can also be used to demonstrate the mechanism of action of a test compound, as part of a clinical trial or as evidence of off-target toxicity if the toxicity is caused by binding to additional / alternative binding sites within the nucleic acid.

[0097] In some embodiments, the above method can be used in a multiplex assay to map the location of the binding sites of more than one test compound. For example, a method for mapping the location of the binding sites of a population of test compounds within a nucleic acid is (i) contacting the nucleic acid with a population of tagged test compounds, wherein each tagged test compound within the population comprises a test compound linked by a covalent bond to a tag, wherein the tagged test compounds within the population bind to one or more sites within the nucleic acid to the nucleic acid or a protein associated with the nucleic acid, (ii) contacting the tagged test compounds with a population of first binding members, such that each first binding member within the population specifically binds to a different tagged test compound, (iii) contacting the nucleic acid with a population of second binding members associated with an activatable nuclease, such that each second binding member within the population specifically binds to a different first binding member that has bound to a tagged test compound at a binding site within the nucleic acid, (iv) activating the nuclease, such that as a result, the nucleic acid is cleaved at one or more binding sites by the nuclease to generate fragments, (v) determining the sequences of the generated fragments, and may include.

[0098] The sequence of the nucleic acid fragment generated by the nuclease that binds to the tagged test compounds within the population via the second binding member and the first binding member may indicate the location of one or more binding sites of the test compound within the nucleic acid.

[0099] The population may comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more members.

[0100] In some embodiments, each tagged test compound within the population can be individually contacted with a first binding member to form a complex, and the complexes can be combined to create a population of complexes composed of the first binding member and the tagged test compound. Different first binding members can be associated with each tagged test compound within the population. The population of complexes can then be contacted with a nucleic acid.

[0101] In other embodiments, each tagged test compound within the population may have a different tag. The population of first binding members may have first binding members that specifically bind to the different tags of the tagged test compounds within the population, and thus, different first binding members are associated with each tagged test compound within the population.

[0102] The population of second binding members may include second binding members that specifically bind to each of the different first binding members within the population of first binding members, and thus, different second binding members are associated with each tagged test compound within the population via the first binding member.

[0103] In some embodiments, different nucleases can be associated with each of the different second binding members, and thus, the fragments generated by each nuclease can be distinguished.

[0104] In other embodiments, different oligonucleotide adapters can be loaded onto nucleases each associated with a different second binding member, such that the fragments generated by each nuclease are labeled with different adapters and can be distinguished. For example, the nuclease can be a transposase, and different oligonucleotide adapters can be loaded onto transposases each associated with a different second binding member. The transposase associated with the tagged test compound via the first binding member and the second binding member can cleave the nucleic acid at the binding site of the tagged test compound and insert an oligonucleotide adapter at the end of the nucleic acid fragment. Thereby, nucleic acid fragments are generated with each end labeled with an oligonucleotide adapter. Since different oligonucleotide adapters are loaded onto each different second binding member, the tagged test compound whose binding site is indicated by the sequence of the nucleic acid fragment can be identified using the sequence of the oligonucleotide adapter associated with each nucleic acid fragment.

[0105] In some embodiments, the methods described above can be used in competitive assays. An example of a competitive assay described herein is shown in FIG. 8. Suitable assays can include contacting a nucleic acid with a tagged test compound and a second test compound without a tag. For example, a method for mapping the location of one or more binding sites of a test compound within a nucleic acid is (i) contacting a nucleic acid with a tagged test compound comprising a test compound covalently linked to a tag and a second test compound without a tag, wherein the tagged test compound and optionally the second test compound without a tag bind to the nucleic acid or a protein associated with the nucleic acid at one or more locations within the nucleic acid; (ii) contacting the nucleic acid with a first binding member that specifically binds to the tag, such that the first binding member binds to the tagged test compound; (iii) A step of contacting a nucleic acid with a second binding member that specifically binds to a first binding member and is accompanied by an activatable nuclease, such that as a result, the second binding member binds to the first binding member that is bound to the tagged test compound at one or more binding sites. (iv) A step of activating the nuclease, such that as a result, the nucleic acid is cleaved at one or more binding sites by the nuclease to generate fragments. (v) A step of determining the sequence of the generated fragments. may include.

[0106] The tagged test compound and the second untagged test compound may be contacted with the nucleic acid simultaneously or sequentially. For example, after contacting the nucleic acid with the second untagged test compound, it may be contacted with the tagged test compound, after contacting with the tagged test compound, it may be contacted with the second untagged test compound, or it may be contacted simultaneously with both the second untagged test compound and the tagged test compound.

[0107] The test compound and the second test compound may be the same or different (i.e., they may be the same chemical compound or different chemical compounds).

[0108] In some embodiments, the test compound and the second test compound may be a peptide or small organic molecule as described above. In other embodiments, the test compound may be a peptide or small organic molecule as described above, and the second test compound may be a protein such as an antibody, a nucleic acid, or other macromolecule. For example, the second test compound may be a polypeptide having more than 50 amino acids or an organic molecule having more than 5 kDa.

[0109] Also provided are kits for use in the methods described herein and the use of such kits in such methods. The kit comprises (i) a tag covalently linked to or linkable to a test compound, (ii) A first binding member that specifically binds to a tag, (iii) A second binding member that specifically binds to the first binding member, (iv) A nuclease associated with or attachable to the second binding member, may be included.

[0110] Suitable kit components are described above. A linkable tag is a tag that can be attached to a test compound by covalent bond. Suitable linkable tags include tags containing a first click chemistry group. Suitable click chemistry groups are known in the art and include, for example, one of an azide group or an alkyne group. The linkable tag can react with a test compound containing a second click chemistry group that reacts with the first click chemistry group, for example, the other of an azide group or an alkyne group, to covalently link the compound and the tag.

[0111] The kit may further include (v) one or more sequencing adapters. The sequencing adapter may be associated with or attachable to the transposase.

[0112] The kit may further include a suitable buffer and washing solution; a fixative and a permeabilization agent.

[0113] The kit may further include a solid support. Suitable solid supports may include capture molecules that bind to eukaryotic cells, such as lectins.

[0114] The kit may further include nucleic acid extraction and purification reagents. Suitable reagents are known in the art and include, for example, spin chromatography columns.

[0115] The kit may further include amplification reagents. Suitable reagents are known in the art and include, among others, primers, dNTPs, and heat-resistant polymerase. In some embodiments, the kit may include amplification primers for amplifying one or more target regions or loci within the genome.

[0116] The kit may include instructions for use in the methods described herein.

[0117] Other aspects and embodiments of the present invention provide the above-described aspects and embodiments with the term "comprising" replaced by the term "consisting of", and the above-described aspects and embodiments with the term "comprising" replaced by the term "consisting essentially of".

[0118] It is understood that this application discloses all combinations of any of the above aspects and the embodiments described above with each other, unless the context requires otherwise. Similarly, this application discloses all combinations of the preferred and / or optional features alone or with any other aspects, unless the context requires otherwise.

[0119] Variations, further embodiments, and variations of the above embodiments will be apparent to those skilled in the art upon reading the present disclosure, and are themselves included within the scope of the present invention.

[0120] All documents and sequence database entries referred to herein are hereby incorporated by reference in their entirety for all purposes.

[0121] As used herein, "and / or" is understood as a specific disclosure that includes or does not include each of the other of two recited features or components. For example, "A and / or B" is understood as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually recited herein.

[0122] Experiment In this application, a generalized approach is disclosed for establishing interaction maps of small molecules that bind to genomic DNA or chromatin proteins in situ. The approach is illustrated using three distinct classes of DNA- or protein-interacting molecules. The bromodomain inhibitor JQ1 is used to validate the inventors' method at high signal-to-noise ratios in small samples of cells. Next, the genomic target sites within cells are mapped for the first time for three widely used chemicals, including two benchmark molecules that bind to guanine quadruplex DNA and the first-choice anti-cancer drug doxorubicin. Finally, the effect of HDACi on doxorubicin binding to genomic DNA is investigated.

[0123] Materials and Methods General Chemicals and reagents were purchased from Sigma-Aldrich, MedChemExpress, and Fluorochem. All organic solvents were distilled by standard purification methods before use or purchased in anhydrous form from Sigma-Aldrich. Unless otherwise specified, all reactions were carried out under argon in glassware dried in a desiccator. NMR spectra were recorded on a Bruker 400 MHz Advance III HD spectrometer or a 500 MHz DCH Cryoprobe spectrometer, operating at 400 MHz and 500 MHz for 1H NMR and 125 MHz for 13C NMR in DMSO-d6, respectively. NMR data were reported as follows: chemical shifts in parts per million (ppm) referenced to the solvent residual peak, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet; m = multiplet, br = broad), and coupling constant values in Hz. LC-MS was performed on an Amazon ESI-MS (Bruker) connected to a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific). High-resolution mass spectra (HRMS) were obtained using a Waters Vion IMS QTOF spectrometer. Flash column chromatography was carried out using CombiFlash Rf (Teledyne ISCO) with a C18 puriFlash column (Interchim). Pyridostatin (PDS) and PhenDC3 were prepared according to previously reported procedures.

[0124] Chemical synthesis 1. Synthesis of JQ1-bio

Chem.

[0125] 2. Synthesis of PDS-bio

Chemical Structure

[0126] 3. Synthesis of PhenDC3-bio

Chemical formula

[0127] PhenDC3-in (7, 5.4 mg, 6.5 μmol) and biotin-PEG3-azide (6 mg, 13 μmol) were suspended in 75 μL of DMSO. Tris(2-carboxyethyl)phosphine (TCEP, 0.33 mmol, freshly prepared 100 mM solution in water) was added, followed by copper(II) sulfate pentahydrate (0.33 mmol, freshly prepared to 10 mM in water). Tris(benzyltriazolylmethyl)amine (TBTA, 0.33 mmol, stock solution, 40 mM in a 1:1 water / tert-butanol mixture) was added to the solution, and the mixture was stirred at room temperature under argon for 3 h. The mixture was purified using flash column chromatography (C18 column, gradient elution over 30 min at a flow rate of 18 mL per minute: water (0.1% (v / v) TFA)~MeCN (0.1% (v / v) TFA)). The solvent was removed by lyophilization to obtain the product as a yellow powder (PhenDC3-bio, 2.9 mg, 41%). HRMS (ESI-QTof) m / z: [M2+] + C 61 H 72 N 14 O8S 2+ Calculated value: 580.26836; Found: 580.26768, 387.18184.

[0128] 4. Synthesis of Dox-bio1

Chemical Structure

[0129] 5. Synthesis of Dox-bio2

Chemical Structure

[0130] Cell culture and treatment with compounds Mycoplasma-free human chronic myelogenous leukemia K562 cells (RRID: CVCL_0004) derived from a 53-year-old female were purchased from ATCC and cultured in RPMI 1640 (Gibco, 21875034) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, A3840401). Human osteosarcoma epithelial U-2 OS cells (RRID: CVCL_0042) derived from a moderately differentiated sarcoma of the tibia of a 15-year-old female's osteosarcoma were obtained from ATCC and cultured in DMEM (Gibco, 41966029) supplemented with 10% FBS. Both cell lines were grown according to the ENCODE cell culture protocol, tested regularly for mycoplasma contamination, and their identity was confirmed by short tandem repeat (STR) typing. HDAC inhibitor (HDACi) was prepared as a stock solution at 10 mM in DMSO. Cells were treated with HDACi dissolved at a final concentration of 1 μM or an equal concentration of vehicle (0.1% DMSO) for 72 hours.

[0131] CUT&Tag Transposome assembly using recombinant pA-Tn5 and DNA adapters has been described in detail elsewhere (W. W. I. Hui, et al. Scientific Reports, 11 (2021) 1930). BRD4 CUT&Tag was performed as previously described (H.S. Kaya-Okur, et al. Nat. Protoc., 15 (2020) e23641). Briefly, cells were incubated with magnetic beads (Bangs Labs, BP531) coated with activated concanavalin A. Cells bound to the beads were permeabilized and incubated with anti-BRD4 (E2A7X) rabbit antibody (Cell Signalling Technology, 13440), and then incubated with rabbit anti-mouse IgG antibody (Antibodies-Online, ABIN101961, RRID:AB_10775589). Then, diluted pA-Tn5 adapter complex was added, and then tagmentation reaction was performed. The extracted DNA fragments were used for library preparation and Illumina sequencing.

[0132] Chemmap Cell preparation. U-2 OS cells were detached using Accutase and immediately quenched using complete culture medium. U-2 OS and K562 floating cells were collected by centrifugation, fixed in 0.1% formaldehyde (Thermo Scientific, 28906) in PBS for 2 minutes at room temperature, and then quenched with glycine to a final concentration of 75 mM. The fixed cells were collected by centrifugation at 600×g for 4 minutes, and then resuspended in nuclease-free cold wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, and 0.5 mM spermidine (Sigma, S0266)) supplemented with cOmplete Protease Inhibitor, EDTA-free (Sigma, 11873580001). (Note: For the Chemmap of G4 ligand, NaCl in all buffers was replaced with equimolar KCl to maintain G4 stability. To obtain optimal results, the cell concentration needs to be adjusted based on the target abundance and the relative affinity of the probe. Cells were used at 1500 cells / μL (JQ1-bio) and 6000 cells / μL (G4 ligand and biotinylated doxorubicin).

[0133] Probe-primary antibody complex assembly. The compound stock solution in DMSO was diluted to 10 μM in antibody buffer (2 mM EDTA in wash buffer, 0.1% BSA (Sigma, A8577), 0.05% digitonin (EMD Millipore, 300410)). For five samples, 20 μL of probe solution (10 μM) and 16.7 μL of anti-biotin (D5A7) rabbit mAb (Cell Signaling Technology, 5597, concentration is approximately 10 μM) were added to 200 μL of antibody buffer and incubated on ice for 1 hour to perform complex formation at a high concentration (probe excess 1.2:1 to avoid non-specific antibody binding). Next, 300 μL of antibody buffer was added to the probe-primary antibody complex solution to bring the final small molecule concentration to 0.4 μM.

[0134] Bead capture. For five samples, 50 μL (JQ1-bio) or 75 μL (G4 ligand and biotinylated doxorubicin) of concanavalin A beads (Bangs Labs, BP531) were washed twice in 1 mL of binding buffer (nuclease-free water containing 20 mM HEPES, pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) and resuspended in 75 μL of binding buffer. 100 μL of cell suspension was incubated with 10 μL of pre-washed concanavalin A beads at 25 °C and 600 rpm for 10 minutes. Cells bound to the beads were gently washed twice with wash buffer and then resuspended in 100 μL of probe-primary antibody solution and incubated overnight at 4 °C and 600 rpm.

[0135] Secondary antibody-Tn5 transpososome complex assembly. For five samples, 2.5 μL of secondary antibody (Antibodies-Online, ABIN101961, RRID:AB_10775589) and 5 μL of pA-Tn5 were added to 200 μL of Dig-300 buffer (nuclease-free water supplemented with Complete Protease Inhibitor, EDTA-free, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin) and incubated on ice for 1 hour. 300 μL of antibody buffer was added to the secondary antibody-Tn5 complex solution (ratio of secondary antibody-Tn5 complex 2:1).

[0136] Tagmentation. Cells were washed three times with 500 μL of Dig-wash buffer (0.05% digitonin in wash buffer), resuspended in 100 μL of secondary antibody-Tn5 transpososome solution and incubated at 25 °C and 600 rpm for 1 hour. Then, the cells were washed three times in 500 μL of Dig-300 buffer and then incubated in 300 μL of tagmentation buffer (10 mM MgCl2 in Dig-300 buffer) at 37 °C and 600 rpm for 1 hour.

[0137] DNA extraction. After tagging, the cells were washed twice with 500 μL of TAPS wash buffer (nuclease-free water, 10 mM TAPS (Alfa Aesar, J63268.AE), 0.2 mM EDTA). 150 μL of extraction buffer (10 mM Tris-HCl, pH 8.0, 0.5 mg / mL proteinase K (Thermo Scientific, EO0491), 0.5% SDS) was added, vortexed, and incubated at 55 °C and 800 rpm for 1 hour. Next, 100 μL of phenol-chloroform-isoamyl alcohol (Invitrogen, 15593049) was added and mixed. The mixture was transferred to a MaXtract High Density Tube (QIAGEN, 129046) and centrifuged at room temperature at 16000×g for 3 minutes. 150 μL of chloroform was added to the upper aqueous phase, mixed by inverting the phase lock tube, and centrifuged at room temperature at 16000×g for 3 minutes. The upper aqueous layer was transferred to a 1.5 mL DNA Lo-bind tube (Eppendorf, 022431021). 6 μL of 5 M NaCl and 375 μL of cold ethanol were added, mixed, and incubated overnight at -20 °C. The sample was centrifuged at 4 °C at 21130×g for 30 minutes. The supernatant was carefully discarded, the DNA pellet was rinsed with 1 mL of cold 100% ethanol, and then centrifuged at 4 °C at 21130×g for 2 minutes. The wash solution was discarded, the residual liquid was blotted with a paper towel, and the pellet was air-dried. Finally, the pellet was resuspended by vortexing in 25 μL of elution buffer 1 (nuclease-free water, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 1 / 400 RNAse A (Thermo Scientific, EN0531)) and incubated at 37 °C and 800 rpm for 10 minutes.

[0138] Library Preparation. To a 0.2 mL PCR tube, add 25 μL of NEBNext HiFi 2× PCR Master Mix (NEB, M0541), 2 μL of 10 μM Ad1 (J.D. Buenrostro, et al., Nature, 523 (2015) 486-490), 2 μL of 10 μM Ad2 (J.D. Buenrostro, et al) and 21 μL of tagged DNA, and subject to PCR (5 minutes at 72°C, 30 seconds at 98°C, then 10 cycles of 10 seconds at 98°C and 10 seconds at 63°C, and 1 cycle of 1 minute at 72°C). The library was purified using 1.3× ratio (65 μL) of Ampure XP beads (Beckman Coulter, A63882). After incubating for 10 minutes at room temperature, the DNA bound to the beads was washed twice with 80% ethanol, and the library was eluted with 25 μL of 10 mM Tris-HCl for 5 minutes at room temperature.

[0139] Library Sequencing The size and concentration of the library were measured using TapeStation HSD1000 ScreenTape (Agilent, 5067-5584). The library was equilibrated and pooled for size selection using Ampure XP beads. 0.4× ratio of Ampure XP beads was added to the pooled library, left at room temperature for 15 minutes, and then the supernatant was transferred to a new tube. Then, 1.3× ratio of Ampure XP beads was added to the supernatant and incubated at room temperature for 15 minutes. The beads were washed twice with 80% ethanol, and the library was eluted with 40 μL of 10 mM Tris-HCl. The library was sequenced on a NextSeq 500 sequencer (Illumina) using the High Output kit (Illumina, FC-404-2005) with a 36 bp × 2 paired-end format.

[0140] FRET Melting Assay The 400 nM FAM-TAMRA dual-labeled oligonucleotide (Biomers) was annealed in assay buffer (60 mM potassium cacodylate, pH = 7.4) at 95 °C for 5 minutes and then gradually cooled to 20 °C. A series of probe concentrations were prepared in 8-well strip tubes: 150 μL of 6 μM ligand in assay buffer was prepared as the initial concentration. Subsequent serial dilutions were performed by adding 100 μL of the probe solution to 50 μL of assay buffer, thereby obtaining 12 concentrations including the control (1% DMSO). 25 μL per solution was transferred to a 96-well plate, and then 25 μL of the annealed oligonucleotide solution was added to each well. The plate was then sealed with an adhesive transparent cover and gently shaken at room temperature for 10 minutes. The measured values of the recovered FAM signal were recorded using a Bio-Rad CFX96 Touch Real-Time PCR detection system with a temperature gradient of 25 °C to 95 °C at 0.5 °C per minute. The melting temperature was determined by the maximum value of the first derivative of the relative fluorescence unit (RFU) value with respect to time, and ΔT m was calculated by baseline correction of the melting temperature subtracting the control group. The 1-site binding model of GraphPad Prism 7 was used to perform FRET T m curve fitting. The average value was calculated from two replicate experiments.

[0141] Cell imaging Approximately 40,000 U-2 OS cells diluted in 1 ml of DMEM medium supplemented with 10% FBS were plated in a 12-well plate. After incubating for 18 hours, the live cells were treated with doxorubicin (1 μM), Dox-bio1 (1 μM), Dox-bio2 (1 μM), and 0.1% DMSO (control) in fresh medium for 6 hours. Then, the cells were washed twice with PBS and gently fixed with 1% formaldehyde for 2 minutes. Next, the cells were incubated with Hoechst 33342 at 37 °C for 10 minutes to label the cell nuclei. Imaging was captured with an EVOS M5000. A blue light filter cube with wavelengths of Ex 357 / 44 and Em 447 / 60 was used for nuclear visualization, and a red light filter cube with wavelengths of Ex 531 / 40 and Em 593 / 40 was used for visualization of doxorubicin and its derivatives.

[0142] Bioinformatics data processing 1. Data demultiplexing and duplicate removal. The Illumina sequencing paired-end output files were demultiplexed using demuxIllumina version 3.0.9 with the flags; -c -d -i -e -t 1 -r 0.01 -R -l 9. The resulting fq.gz files were subjected to sequencing quality control using FastQC v0.11.8, and their summaries were visualized by MultiQC v1.11. Bases with quality scores below 20 were removed from both reads using cutadapt (cutadapt -q20). The Fastq files were aligned to a combination of the hg38 and E. coli genomes using bwa 0.7.17 - r1188, and only reads within the regions included in the hg38 white list were continued through the processing pipeline. Duplicates were removed using Picard version 2.20.3 (Picard MarkDuplicates), and seacr version 1.3 was used to call peaks at the top 1% and 5% of the AUC using both lenient and stringent criteria without using input controls. The duplicate-removed bam files were sorted and indexed.

[0143] 2. Peak calling. The deduplicated bam files were converted to bedpe files (bedtools bamtobed -bedpe), and then only the fragments smaller than 1000 bp in size were retained (awk '{if($1==$4&&$6-$2<1000)print $0}'). Next, the coverage of fragments less than 1000 bp across the human genome was computationally calculated using bedtools genomecov and reported in bedgraph format. Then, SEACR was used to identify regions of local enrichment. A SEACR stringent search was performed to select the top 5% of peaks based on the total signal within the peaks (SEACR_1.3 $bdg_1000 0.05 non-stringent). Peaks with a minimum coverage of 8 reads were retained for further analysis.

[0144] 3. Consensus regions and reference comparison. Different thresholds were applied to obtain peak files with the minimum total signal at 5, 8, and 10 peak coordinates. For each threshold, the overlap between 5 technical replicates was calculated using the intervene tools (venn, upset, and pairwise), and multiIntersectBed was used with the -wa wb flags of bedtools v.2.30.0 to create a series of bed files containing at least 1 (integration of all technical replicates) to 5 (common to all technical replicates). The same pipeline can be followed for the intersection between biological replicates. This peak classification enables quantification and ranking of peaks according to the normalized (cpm) signal intensity and peak reproducibility.

[0145] Assessment of G4 enrichment quality using Chemmap-qPCR G4 enrichment by Chemmap-qPCR was assessed by quantifying the relative enrichment of G4 DNA regions significantly enriched beyond the background region. For Chemmap-qPCR, Chemmap 10× PCR samples were used. DNA concentration and size distribution were confirmed using an Agilent HS D1000 Tapestation and normalized to the same amount for qPCR.

[0146] PCR reaction conditions: A master mix was prepared for each reaction. Chemmap samples were diluted 10-fold. 5 μl of 2× Premix SYBRgreen qPCR mixture and in addition 2.5 μl of primer mix were added to each well, and then 2.5 μl of the diluted Chemmap sample was added. A plate seal was applied using a roller, and after rotating the plate at high speed (1000 rpm for 1 minute), qPCR was performed. qPCR was carried out for 40 cycles to 45 cycles. RPA3 and MAZ were used as positive controls, and ESR1 and TMCC1 were used as negative controls. The primers used are included below.

[0147]

Table 1

[0148] Statistical analysis Data are presented as mean ± s.d. The sample size (n) in the figure legends indicates the number of replicates in each experiment and is presented in the corresponding figure legends. The peak of the heatmap or the size (N) of the gene indicates the number of peaks or genes included. Statistical analysis in the relevant figures was performed by unpaired Student's t-test. P-values are shown in each figure.

[0149] Results Chemmap is based on the introduction of affinity tags to small molecules. When the small molecule approaches its binding site in the nucleus, antibodies recognizing the tags of the small molecule-antibody pre-complex can be recruited to mobilize a secondary antibody pre-loaded with a synthetic sequencing adapter and transposase Tn5, resulting in in situ insertion of the sequencing adapter proximal to where the small molecule is bound (Figure 1A).

[0150] Mobilization of Tn5 to proteins bound to chromatin in lysate chromatin and permeabilized cells by this method was exemplified by TAM-ChIP (Active Motif, M. Tedesco, et al., Nat. Biotechnol., 40 (2022) 235-244.) and CUT&Tag (9). Once the DNA is extracted, the adapter fragments in the vicinity of the small molecule binding site can be selectively amplified by sequencing and then mapped by alignment to the genome. To validate the Chemmap approach and compare the signal quality with established methods, biotinylated molecule JQ1, which had been previously mapped by Chem-Seq, was synthesized (Figure 1B) (3, 5).

[0151] JQ1 is an inhibitor of the BET family of bromodomain proteins that has been well characterized and has high binding affinity (10). In parallel, mapping of the genome-wide binding sites of its major target, BRD4, was also performed using a specific antibody with CUT&Tag (9). Mapping experiments using JQ1-bio were carried out on 150,000 human leukemia K562 cells, with two biological replicates and five technical replicates to assess the robustness and reproducibility of our approach. In each experiment, approximately 10,000 JQ1 binding sites were observed, with excellent reproducibility across both technical and biological replicates (r S(see >0.9, FIGS. 1C, 2A, and 2B). Next, a set of high-confidence binding sites was compared from the findings of JQ1 Chemmap and BRD4 CUT&Tag, where 93% of the JQ1 peaks overlap with BRD4 binding sites (see the method). BRD4 binding sites that did not overlap with the high-confidence JQ1 peak sites were investigated, and after a slight optimization of the peak calling parameters based on the difference in the binding affinity of the two probes, BRD4 binding sites with a higher coverage with JQ1 (84%) were obtained (FIG. 2C). Similarly, differential binding analysis revealed a strong overlap between the JQ1 binding sites and the BRD4 binding sites (1213 / 45667 discriminative sites, 2.7%) (FIG. 2D). Furthermore, principal component analysis (PCA) confirmed that the JQ1 data and the BRD4 data were clustered together and clearly separated from the biotin control (FIG. 2E). Overall, this clearly summarizes the binding profile of JQ1 to its protein target by Chemmap and suggests that the observed differences are mainly due to the suboptimal peak calling thresholds.

[0152] To assess the signal quality of Chemmap, the inventors' findings were compared with publicly available JQ1 Click-Chem-seq data in K562 cells, revealing that Chemmap substantially improves the signal-to-noise ratio (FIG. 1E)(5). To quantitatively compare the methods, the average read counts of Chemmap and Click-Chem-seq near the highest-confidence BRD4 binding sites (7772 peaks present in all replicate experiments) obtained by BRD4 CUT&Tag were plotted. Despite using two orders of magnitude fewer cells, Chemmap was found to result in approximately 150-fold signal accumulation compared to Click-Chem-seq (maximum average read counts of 8.17 cpm and 0.05 cpm, respectively) (FIG. 1F).

[0153] Since this method has been verified using known protein inhibitors, we then attempted to map the binding sites of two widely used guanine quadruplex (G4)-targeting molecules, PDS and PhenDC3, which were previously impossible (Fig. 3a) (11, 12). G4 is a four-stranded structure that can form G-rich DNA sequences. G4s are associated with gene regulation and are considered potential drug targets for treating cancer (13). Small molecules that bind to G4s can interfere with transcription and replication forks, thereby causing DNA damage.

[0154] G4 itself has only recently been mapped to chromatin using the specific antibody BG4 (14). Given the lack of understanding of how BG4 recognizes its binding target and potential off-targets, there is an urgent need to cross-validate G4 topography in cells using prominent alternative methods. Also, as the number of trials utilizing G4 ligands in preclinical and clinical studies is increasing, it is necessary to demonstrate the justification that these chemicals can actually bind to their targets in situ. G4 ligands interact with the entire G-tetrad surface of G4 via π-π stacking in the crystal structure and have moderate binding affinities in the μM range (15). However, despite many attempts, it has been impossible to directly map the binding sites of such small molecules in the nucleus. Success with pull-down enrichment sequencing that has provided only evidence of binding in telomeric repeats has been limited (16). Perhaps the most compelling evidence of target engagement at G4 loci has arisen by indirect methods such as mapping the sites of strand breaks induced as a downstream consequence of small molecule binding events (17). A major challenge in mapping DNA-small molecule interactions is that the ligand dissociates from the DNA target during the washing step, which leads to low recovery rates and poor signal-to-noise ratios.

[0155] Previous attempts by the inventors to map G4 ligands using conventional Chem-seq were unsuccessful, most likely because the affinity of small molecules was relatively low (16). Considering the substantially improved sensitivity obtained using JQ1 Chemmap, it was inferred that the transposon-based method in permeabilized cells (Figure 1a) simply requires the DNA-small molecule interaction to persist for a long enough time to allow the catalytic insertion of adapters to mark and select the binding sites. Ligands PDS-biotin and PhenDC3-azide were synthesized, evaluated in biophysical assays as previously reported, and conjugated by copper-assisted click reaction with PhenDC3-biotin (16, 18). To minimize binding perturbation and steric hindrance between antibody binding and DNA fragmentation, flexible long PEG4 linkers were inserted into both probes (Figure 3A). Fluorescence resonance energy transfer (FRET) melting assays were used to verify the binding of tagged G4 ligands to different G4 oligomers in vitro (Figure 4A). Both probes were used in the inventors' established Chemmap protocol, but 600,000 K562 cells per experiment were used to compensate for the relatively weak binding affinity of G4 ligands. Additionally, to mimic intracellular ionic conditions and maintain the endogenous G4 landscape during the experiment, a buffer using potassium salts instead of sodium was used. For both G4 ligands, high-quality maps were obtained, and for both G4 ligands, approximately 20,000 high-confidence peaks were revealed over two biological replicates and five technical replicates (Figure 3B). In contrast, principal component analysis confirmed a clear separation from the biotin control experiment (Figure 4C). By comparing the high-confidence binding sites with publicly available G4-seq data (OQS) (19) containing all sites with the potential to form G4 structures in human genomic DNA, a large overlap was found for both PDS (∼89%) and PhenDC3 (∼88%) (Figures 3C and 4B).Next, the small molecule binding sites were compared to the published maps of endogenous G4s observed in the BG4 CUT&Tag experiments. By validating our approach, strong overlaps of high-confidence binding sites as well as signal correlations were observed compared to antibody and G4 ligand maps (r s > 0.7), and even higher correlations were observed for PDS and PhenDC3 (r s > 0.9) (Figures 3D, 3E, and 4B). These results are consistent with previous in vitro binding experiments which found that PDS, PhenDC3, and BG4 are relatively promiscuous G4 binders with moderate G4 structure specificity (20). Nevertheless, the small molecules capture different spaces of the binding sites that are not captured by BG4 (5690 consensus loci), which could be due to different binding preferences of the probes or accessibility of the G4 structures (Figure 3E). From this data, due to the orthogonality of the antibody and small molecule probes, a strong validation of the mapped endogenous G4 landscape is provided. Furthermore, similar trends were observed in the mapping of both G4 ligands in U-2 OS cells, thus confirming the robustness of the Chemmap approach (Figure 4F). Overall, high-resolution mapping of G4 ligands should directly confirm drug-target engagement in chromatin and also serve as a useful tool to guide the development of improved G4 ligands and therapeutics.

[0156] To evaluate the general applicability of this method for detecting DNA-small molecule interactions, we tested doxorubicin, a clinically approved drug that is thought to act by targeting DNA, but for which direct mapping to genomic DNA in cells has not been done. Doxorubicin belongs to the anthracycline class of antitumor antibiotics, which can interact with DNA by an intercalation mechanism. The major anticancer effects of anthracyclines are DNA intercalation, inhibition of topoisomerase II, and formation of free radicals, which cause structural changes in DNA, DNA damage, and cytotoxicity (1). Approximately 1 million cancer patients are treated with doxorubicin or its variants each year. However, despite numerous studies over the past 50 years on doxorubicin and its clinical use, its mode of action remains poorly understood (21).

[0157] To use the Chemmap method, we first had to design appropriately tagged doxorubicin. Following previous reports, we tested two conjugation points, 14-OH and 3’-NH2, and obtained biotinylated derivatives Dox-bio1 and Dox-bio2 (Figure 5A). Next, we investigated intracellular uptake in U2OS cells using a fluorescence microscope that utilized the intrinsic fluorescence of doxorubicin (Figure 5B). In particular, doxorubicin and Dox-bio1 accumulated mainly in the nucleus, while Dox-bio2 was mainly located in the cytoplasm. Similarly, the importance of 3’-NH2 for DNA binding events was emphasized because in several attempts at Chemmap using Dox-bio2 in permeabilized K562 cells, we were unable to recover significant amounts of DNA. In contrast, for Chemmap using Dox-bio1 at different probe concentrations, substantial amounts of target DNA fragments were recovered from permeabilized K562 cells. In particular, when using 200 nM Dox-bio1, up to 30-fold more material was recovered compared to other probes tested under the same conditions (Figure 6B).

[0158] Depending on the probe concentration, a highly reliable Dox-bio1 binding site of approximately 14k was observed. Interestingly, the Dox-bio1 peaks mapped by ATAC-seq were mainly (95%) located in open chromatin regions (Figure 5D). Considering the use of Dox-bio1 saturation conditions and the fact that heterochromatin histone marks and protein binding can be easily mapped by the CUT&Tag type method (9), the possibility that this binding property is due to technical biases of the Chemmap method is low. From the inventors' findings, on the contrary, it is suggested that chromatin accessibility is a prerequisite for doxorubicin-DNA binding in cells (Figure 5D) (22).

[0159] Next, Chemmap was applied to explore the mechanistic evidence that epigenetic drugs (epi-drugs) can be synergized with chemotherapy by profiling the dynamics of doxorubicin binding. Histone deacetylases (HDACs) are important chromatin silencing modifiers, generally dysregulated in cancer, and thus attractive therapeutic targets for cancer treatment. Preclinical and clinical trials have demonstrated that HDAC inhibitors (HDACi) can effectively sensitize cancer responses to doxorubicin treatment, causing cell apoptosis and cell death (23, 24). Tucidinostat (tidamide) was selected. This is a selective HDAC inhibitor for class I HDAC1, HDAC2, HDAC3, and class IIb HDAC10, clinically approved for peripheral T cell lymphoma (PTCL) and adult T cell leukemia-lymphoma (ATLL) (25, 26), and combination treatment with doxorubicin in preclinical and clinical trials is ongoing ((27), NCT04231448).

[0160] After 72 hours of treatment in K562 cells, a similar number of total high-confidence peaks were recovered. Notably, differential binding analysis demonstrated that treatment with tucidinostat enhanced the binding of doxorubicin and generated an overwhelming level of new binding sites (Figures 7A and 7D). Additionally, pretreatment with HDACi enlarged the size of the original binding loci. To investigate the genomic distribution of the new binding sites, tucidinostat generated new binding sites outside the initially open chromatin regions (42% in tucidinostat compared to 95% in the vehicle group), indicating significant chromatin remodeling progression (Figure 4B). Overall, these results highlight that HDACi sensitizes cancer cells by expanding and newly establishing binding sites, enhancing the accessibility of doxorubicin to chromatin. Thus, Chemmap is a mechanism-based approach for establishing the rationale for combinatorial epigenetic therapy drugs that rejuvenate the field once again.

[0161] To confirm that Chemmap reflects small molecule binding sites in living cells, K562 cells were treated with unmodified PDS (4 μM, 3 hours), followed by PDS Chemmap (Figure 9). With increasing concentrations and treatment times of the competitor, a decrease in the recovered DNA material and a substantial decrease in the number of peaks were observed (approximately 6000 sites (60%) were lost compared to untreated) (Figure 10).

[0162] To confirm that Chemmap can be performed using different nucleases, the binding of biotinylated doxorubicin was mapped genome-wide using both pA-Tn5 and pA-MNase. Doxorubicin mapped using pA-Tn5 and doxorubicin mapped using MNase were found to show similar peak distributions (Figure 11). This demonstrates that nucleases such as MNase can be used to map binding sites by Chemmap.

[0163] Pyrrole-imidazole polyamides are molecules that bind in a sequence-specific manner to the minor groove of DNA. By adjusting the composition of the PIPs, it is possible to modify the target DNA sequences that are recognized. Three biotinylated PIPs (Figure 12) that bind to dsDNA with the following sequence specificities were used: WGCWGCW (PIP1-bio), WGCWGCW (PIP2-bio), and WWCWWWGW (PIP3-bio) (where W represents A or T). After the Chemmap protocol and data analysis, PIP1-bio and PIP2-bio showed enrichment of binding at genomic loci having the DNA sequence WGCWGCW (Figure 13).

[0164] Chemmap has been demonstrated to be a robust, convenient, and unambiguous method for mapping the interaction sites of small molecules in the genomic DNA and chromatin proteins of the cell nucleus. This method has provided insights into probe molecules and clinically used drugs that were previously impossible, and has also demonstrated the general utility of the method by mapping the interaction sites of chromatin protein inhibitors, two molecules that selectively interact with guanine quadruplex structures, and finally, doxorubicin, a known DNA-interacting compound that has been used as an anticancer drug for many years. Importantly, Chemmap helps to clarify the binding specificity of small molecules, cross-validate the fidelity of drug targets (e.g., G4 structures), and understand the mechanistic rationale for combination treatments for diseases.

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Claims

1. A method for mapping the location of one or more binding sites of a test compound within a nucleic acid, (i) A step of contacting the nucleic acid with a tagged test compound comprising a test compound linked to a tag by covalent bond, wherein the tagged test compound binds to the nucleic acid or a protein associated with the nucleic acid at one or more locations within the nucleic acid; (ii) A step of bringing the tagged test compound into contact with a first binding member that specifically binds to the tag, wherein as a result, the first binding member binds to the tagged test compound. (iii) A step of contacting the nucleic acid with a second binding member that specifically binds to the first binding member and is accompanied by an activatable nuclease, wherein the second binding member binds to the first binding member bound to the tagged test compound at one or more binding sites, (iv) A step of activating the nuclease, wherein as a result, the nucleic acid is cleaved at one or more binding sites by the nuclease, and fragments are generated. (v) A step of determining the sequence of the generated fragments, Methods that include...

2. The method according to claim 1, wherein the sequence of the nucleic acid fragment indicates the location of one or more binding sites of the test compound within the nucleic acid.

3. The method according to claim 1 or 2, wherein the test compound binds to the nucleic acid at one or more locations within the nucleic acid.

4. The method according to claim 1 or 2, wherein the test compound binds to one or more sites within the nucleic acid to a protein associated with the nucleic acid.

5. The method according to claim 1 or 2, wherein the test compound is covalently bound to the nucleic acid or a protein associated with the nucleic acid.

6. The method according to claim 1 or 2, wherein the test compound binds to the nucleic acid or a protein associated with the nucleic acid by a non-covalent bond.

7. The method according to claim 1 or 2, wherein the test compound is an organic small molecule with a density of less than 5 kDa.

8. The method according to claim 1 or 2, wherein the tag is biotin.

9. The method according to claim 1 or 2, wherein the nuclease is fused with an immunoglobulin-binding moiety as a fusion protein, and the fusion protein is non-covalently bonded to the second binding member through the immunoglobulin-binding moiety.

10. The method according to claim 1 or 2, wherein the activatable nuclease is a micrococcal nuclease.

11. The method according to claim 1 or 2, wherein the activatable nuclease is a transposase.

12. The method according to claim 11, wherein the transposase is Tn5.

13. The method according to claim 1 or 2, wherein steps (i) and (ii) are performed simultaneously.

14. The method according to claim 13, comprising contacting the nucleic acid with a complex composed of the tagged test compound and the first binding member.

15. The method according to claim 1 or 2, wherein steps (i) and (ii) are carried out sequentially.

16. The method according to claim 1 or 2, wherein the nucleic acid is contained in a eukaryotic nucleus or an extract thereof.

17. The method according to claim 1 or 2, wherein the nucleic acid is contained within a cell or cell extract.

18. The method according to claim 17, wherein the cell is a prokaryotic cell.

19. The method according to claim 17, wherein the cell is a eukaryotic cell.

20. The method according to claim 17, wherein step (i) comprises culturing viable cells in the presence of the tagged test compound.

21. The method according to claim 20, further comprising permeabilizing the cells before step (ii).

22. The method according to claim 1 or 2, wherein the nucleic acid is RNA.

23. The method according to claim 22, wherein the RNA is a cellular transcriptome or a fraction thereof.

24. The method according to claim 1 or 2, wherein the nucleic acid is DNA.

25. The method according to claim 24, wherein the DNA is a cell genome or a fragment thereof.

26. The method according to claim 1 or 2, wherein the first binding member is an antibody.

27. The method according to claim 1 or 2, wherein the second binding member is an antibody.

28. The method according to claim 1 or 2, wherein the arrangement of the generated fragments is determined by sequencing the fragments.

29. The method according to claim 1 or 2, comprising generating a set of sequence reads of the nucleic acid fragment.

30. The method according to claim 29, comprising mapping sequence reads within the population to one or more locations in a reference genome.

31. The method according to claim 1 or 2, wherein the arrangement of the generated fragments is determined by amplifying the fragments.

32. The method according to claim 31, wherein the amplification of the fragment is performed using a set of primers specific to the nucleic acid sequence containing the binding site of the test compound.

33. The method according to claim 1 or 2, comprising mapping the locations of one or more binding sites of a test compound in a first nucleic acid and a second nucleic acid, and identifying the locations of one or more binding sites that are present in the first nucleic acid but not in the second nucleic acid, or present in the second nucleic acid but not in the first nucleic acid.

34. The method according to claim 33, wherein the first nucleic acid is present in cells or cell extracts subjected to treatment, and the second nucleic acid is present in cells or cell extracts not subjected to treatment.

35. The method according to claim 34, wherein the treatment is selected from exposure to one or more compounds, exposure to or irradiation of light, or exposure to cell culture conditions.

36. The method according to claim 1 or 2, wherein the arrangement of the generated fragments is determined by amplifying the fragments.

37. The method according to claim 32, wherein the amplification of the fragment is performed using a set of primers specific to the nucleic acid sequence containing the binding site of the test compound.

38. The nucleic acid mentioned above, (i) A group of tagged test compounds, wherein each tagged test compound in the group includes a test compound covalently linked to the tag, and binds to the nucleic acid or a protein associated with the nucleic acid at one or more sites within the nucleic acid, (ii) A group of primary binding members, wherein each primary binding member within the group of primary binding members specifically binds to a different tagged test compound within the group of tagged test compounds, and (iii) A group of secondary binding members accompanied by an activatable nuclease, wherein each secondary binding member in the group specifically binds to a different primary binding member bound to a tagged test compound at its binding site. The method according to claim 1 or 2, wherein the object is brought into contact with the object.

39. The method according to claim 1 or 2, wherein step (i) further comprises contacting the nucleic acid with a second untagged test compound, the untagged second test compound optionally binding to the nucleic acid or a protein associated with the nucleic acid at one or more locations within the nucleic acid.

40. The method according to claim 39, comprising determining the effect of the presence of the untagged second test compound on the sequence of fragments generated by the tagged test compound.

41. A kit for mapping the location of one or more binding sites of a test compound within a nucleic acid, A tag covalently linked to or capable of linking with the test compound, A first binding member that specifically binds to the aforementioned tag, A second binding member that specifically binds to the first binding member, A nuclease attached to or capable of being attached to the second binding member, A kit that includes this.

42. A kit according to claim 41, used in the method described in claim 1 or 2.

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