Dialysis-based method for affinity mapping of chromatin interactions

Abstract

Alternative chromatin mapping methods, other than ChIP, typically map a chromatin associated biomolecule by successive binding of a specific antibody in situ in permeabilized nuclei, and then tethering chromatin-cleaving or -modifying enzymes. These methods are however limited by their requirement for a nuclear envelope to hold the chromatin together during wash steps and buffer exchanges. This results in large input material requirements due to sample losses, limitations in what types of samples can be processed, increased variability due to handling steps, and an inability to study chromatin outside of its compacted form in the nucleus. A new method for preparing chromatin from cells for downstream genomic chromatin mapping is disclosed. The method uses dilution, precipitation, and dialysis to replace wash steps and buffer exchanges, allowing the entire protocol to take place in a single tube with no removal of material prior to DNA extraction.

Description

DIALYSIS-BASED METHOD FOR AFFINITY MAPPING OF CHROMATIN INTERACTIONS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 606,883 filed on December 6, 2023, the disclosure of which is hereby incorporated by reference in its entirety herein.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under contract GM074728 awarded by the National Institutes of Health. The Government has certain rights in the invention.FIELD

[0003] The present disclosure relates generally to methods for preparing chromatin from cells for downstream genomic chromatin mapping.BACKGROUND

[0004] Cellular DNA is packaged into a highly heterogeneous fiber known as chromatin, which is composed of a variety of molecules whose precise locations and interactions are critical for establishing and maintaining proper cell states. Chromatin Immunoprecipitation followed by next-generation sequencing (ChlP-seq) is widely used to map the genomic location of chromatin elements, such as histone post-translational modifications (PTMs) and chromatin associated proteins (ChAPs; e.g., transcription factors (TFs) or chromatin binding proteins (CBPs)) (Collas 2010, Nakato and Shirahige 2017). In this approach, specific antibodies (or analogous affinity reagents) are used to enrich chromatin fragments containing specific PTMs or ChAPs. The associated DNA is then isolated and quantified using next-generation sequencing (NGS), providing a genome-wide view of the target under study. However, ChlP-seq remains challenging with small samples and is fraught with signal-to-noise issues and artifacts, although recent modifications of the basic strategy have greatly increased resolution (Rossi MJ, Lai WKM & Pugh BF. 2018 Nat Commun 9, 28423- 6; He Q, Johnston J & Zeitlinger J. 2015. Nat Biotechnol 33, 395-401 ; Skene PJ & Henikoff S A. 2015. eLife 4, e09225; Kasinathan S et al. 2014. Nature Methods 11 , 203-9) and efficiency (Ai S et al. 2019. Grosselin K et al. 2019. Nat Genet 51 , 1060-1066; Nat Cell Biol 21 , 1164-1172; Schmidl et al. 2015. Nat Methods 12, 963-965).

[0005] An alternative chromatin profiling strategy that is becoming increasingly popular is enzyme tethering in situ, whereby the chromatin protein or modification of interest is targeted by an antibody or fusion protein, and the underlying DNA is marked or released. A succession of enzyme-tethering methods have been introduced over the past two decades,including DamID (DNA adenine methylase identification) (van Steensel B, Delrow J, Henikoff S. 2001. Nat Genet. 27:304-308; van Steensel B, Henikoff S. 2000. Nat Biotech not. 18:424- 428), ChEC (Chromatin Endogenous Cleavage), and ChIC (Chromatin ImmunoCleavage) (Schmid M, Durussel T, Laemmli UK. 2004. Mol Cell. 16(1 ):147-57), Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (Skene PJ, Henikoff S. 2017. Elife. e21856; Skene PJ, Henikoff JG, Henikoff S. 2018. Nat Protoc. 13(5):1006-1019), Cleavage Under Targets and Tagmentation (CUT&Tag) (Kaya-Okur et al. 2019. Nat Common.10(1 ):1930; Kaya-Okur et al. 2020. Nat Protoc. 15(10):3264-3283), and directed methylation with long-read sequencing (DiMeLo-seq) (Altemose et al. 2022. Nat Methods.19(6) :711-723; Maslan et al. bioRxiv 2022.07.03.498618; WO 2022 / 256469).

[0006] These techniques however suffer some drawbacks which limits their utility under conditions where samples are limiting. Some of the assays are carried out by incubating permeabilized cells with enzymes, and then halting the reactions after a specified amount of time. In permeabilized cells, the nuclear envelope retains chromatin and interacting biomolecules within the nucleus during wash steps. However, reliance on a nuclear envelope precludes the use of those assays in anucleated cells such as bacterial cells, and cells undergoing cell division (i.e., mitotic cells) whose nuclear envelopes have been disassembled. Moreover, due to spatial constraint in the nucleus, chromatin is compacted through extensive higher-order organization where short-range chromatin interactions regulate the expression of specific loci and long-range interactions allow the communication between regions kilobases away in the genome. Limiting the analysis of chromatin interactions to the confines of the nucleus impairs the ability to distinguish between short and long-range chromatin interactions. This reduces the interpretability of data generated from these experiments, as signal may be due to direct contacts with the underlying DNA or through long-range interactions with the site in question (Datta V, Hannenhalli S, Siddharthan R. 2019. PLoS Comput Biol. 15(3): e1006921). Additionally, existing protocols require numerous wash and centrifugation steps, which can introduce substantial variability in analysis based on the duration and efficiency of each wash step. Furthermore, the degree of handling can produce unwanted negative impacts on chromatin quality which can hamper downstream analysis. For example, increased handling can lead to DNA shearing, which limits the lengths of DNAs that can be readout and hence the “mappability” of DNA within repetitive regions. Wash steps can also lead to the possible extraction of proteins that are weakly bound to chromatin, weakening overall target protein signal across the genome. Therefore, there is a need in the art for improved methods for sample preparation for chromatin mapping with lower starting material requirements, minimal handling steps, and which do not rely on the presence of a nuclear membrane.SUMMARY OF THE INVENTION

[0007] One embodiment of the present disclosure provides a single vessel method of determining the genomic location of at least one biomolecule-genomic DNA interaction, said method comprising the steps of: (a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence; (b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest; (c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest; (d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of modifying genomic DNA; (e) precipitating excess or unbound enzyme by adding a dialysis membrane to the vessel, wherein the dialysis membrane contains a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme; (f) activating the enzyme of (d) under conditions that allow modification of genomic DNA; (g) isolating and preparing the genomic DNA for sequencing; and (h) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

[0008] According to some embodiments of the present disclosure, the single vessel method comprises the steps of: (a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence; (b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest; (c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest; (d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of modifying genomic DNA; (e) transferring the contents of steps (b)-(d) into a dialysis membrane; (f) incubating the dialysis membrane of (e) in the vessel, said vessel further comprising a buffer comprising a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme; (g) activating the enzyme of (d)under conditions that allow modification of the genomic DNA; (h) isolating and preparing the genomic DNA for sequencing; and (i) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

[0009] According to some embodiments of the present disclosure, the single vessel method comprises the steps of: (a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence; (b) permeabilizing the cells in (a) under conditions that expose the genomic DNAbound by the biomolecule of interest; (c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest; (d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of modifying genomic DNA; (e) precipitating excess or unbound enzyme using a dialysis membrane comprising a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme; (f) activating the enzyme of (d) under conditions that allow modification of genomic DNA; and (g) isolating biomolecule-bound chromatin.

[0010] The present disclosure also provides a method for determining the genomic location of at least one biomolecule-genomic DNA interaction, said method comprising the steps of: (a) incubating in a first vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence; (b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest; (c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest; (d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of modifying genomic DNA; (e) transferring the contents of steps (b)-(d) into a dialysis membrane; (f) incubating the dialysis membrane of (e) in a second vessel, said vessel comprising a dialysis buffer capable of diluting out unbound first moiety and / or second moiety conjugated to the enzyme; (g) activating the enzyme of (d) under conditions that allow modification of the genomic DNA; (h) isolating and preparing the genomic DNA for sequencing, wherein said preparing does not require removal of the DNA from the dialysis membrane or amplification of the DNA; and (i) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

[0011] In some embodiments, the enzyme capable of modifying genomic DNA is a DNA methyltransferase, wherein the DNA methyltransferase is selected from the group consisting of DNA adenine methyltransferase (Dam) or a biologically active fragment thereof, EcoGII methyltransferase or a biologically active fragment thereof, Hia5 or a biologically active fragment thereof, M.CviPI or a biologically active fragment thereof, M.CviQIX or a biologically active fragment thereof, and M.Sssl or a biologically active fragment thereof.

[0012] In another embodiment, the DNA methyltransferase is Hia5 or a biologically active fragment thereof.

[0013] In some embodiments, the second binding moiety comprises an antibody, nanobody, single-domain antibody (dAB), camelid nanobody (sdAb), single-chain variable fragment (scFv), or a Fab.

[0014] In a further embodiment, the second binding moiety comprises an anti-IgG nanobody, wherein the anti-IgG nanobody is fused to a Hia5 DNA methyltransferase or biologically active fragment thereof, and a maltose binding protein (MBP) tag.

[0015] In other embodiments, the interaction is in a cell and the incubating of step (a) comprises incubating a plurality of cells.

[0016] In some embodiments, the permeabilization of step (b) comprises contacting the cells with a nuclear membrane permeabilization agent and optionally a chromatin decondensation agent.

[0017] In some embodiments, the nuclear membrane permeabilization agent comprises one or more detergents selected from the group consisting of Triton X-100, Tween 20, saponin, digitonin, or a combination thereof.

[0018] In other embodiments, the chromatin de-condensation agent is selected from the group consisting of heparin, polyglutamic acid (PGA), maltotriose per-O-sulfate (MTS), and methyl cellobiose sulfate (mCBS).

[0019] In one embodiment, the chromatin de-condensation agent is heparin. In a further embodiment, the heparin is deactivated by the addition of heparinase.

[0020] In another embodiment, the dialysis membrane is selected from a group consisting of a dialysis button, a dialysis pouch, a sealed dialysis membrane, a membrane envelope, and a dialysis tubing.

[0021] In further embodiments, the dialysis membrane is an ultra-high molecular weight cutoff (UHMWCO) dialysis button comprising a resin capable of precipitating unbound enzyme.

[0022] In yet another embodiment, the dialysis button comprises a sealable ultra-high molecular weight cutoff (UHMWCO) dialysis button capable of holding the contents of steps (b)-(d).

[0023] In one embodiment, the ultra-high molecular weight cutoff is between 300 kDa and 1000 kDa.

[0024] In some embodiments, the resin comprises magnetic beads. In a further embodiment, the magnetic beads comprise anti- Maltose binding protein (MBP) antibodies.

[0025] In another embodiment, the first binding moiety is an antibody.

[0026] In yet other embodiments of the present disclosure, the cell is selected from the group consisting of a bacterial cell, a eukaryotic cell, prokaryotic cell, a plant cell, an archaeal cell and a virus.

[0027] In one embodiment, the cell is a mammalian cell.

[0028] In another embodiment, the cell is a human cell.

[0029] The present disclosure also provides an aforementioned method, wherein the biomolecule of interest is selected from the group consisting of a protein, a chromatinbinding small molecule, a RNA, and a RNA-DNA hybrid.

[0030] In some embodiments, the biomolecule is a RNA selected from the group consisting of ncRNA, tRNA, rRNA, snRNA, snoRNA, miRNA, mRNA, and TERC.

[0031] In still other embodiments, the biomolecule is a protein selected from the group consisting of a nuclear lamina protein, a nucleolar protein, a transcription factor, a histone or histone or histone variant, centromere protein A, an intracellular scFV, a chromatinmodifying enzyme, an RNA polymerase, a DNA polymerase, a DNA helicase, a DNA repair protein, a Cas9 protein, a dCas9 protein, a zinc finger protein, a TALE protein, a CTCF protein, a cohesin protein, a synaptonemal complex protein, a telomere -binding protein, a centromere -binding protein, an outer kinetochore protein, a splicing protein and a chromatin remodeling protein. In another embodiment, the plurality of cells is induced to express the protein of interest. In yet another embodiment, the protein of interest is a recombinant protein and is expressed from an expression vector.

[0032] In some embodiments, of the present disclosure, an aforementioned method is provided wherein 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more biomolecule-genomic DNA interactions are determined.

[0033] In some embodiments the modifying genomic DNA of step (f) or (g) comprises modifying one or more nucleotides at one or more locations selected from the group consisting of (a) within 1 -50 nucleotides of the genomic DNA binding site of the biomolecule, (b) topologically near the genomic DNA binding site of the biomolecule, and (c) both (a) and (b).

[0034] In still other embodiments, the incubating of step (f) comprises incubating in the presence of bovine serum albumin (BSA) and low salt conditions.

[0035] In yet other embodiments, the isolating and preparing the genomic DNA for sequencing of step (g) comprises high molecular weight DNA extraction.

[0036] In another embodiment, the sequencing of step (g) comprises long read or short sequencing.

[0037] The present disclosure further provides a single vessel method for determining the genomic location of at least one biomolecule-genomic DNA interaction, said method comprising the steps of: (a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence; (b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest; (c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest; (d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of cutting genomic DNA; (e) precipitating excess or unbound enzyme by adding a dialysis membrane to the vessel, wherein the dialysis membrane contains a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme; (f) activating the enzyme of (d) under conditions that allow cutting of genomic DNA; (g) isolating and preparing the genomic DNA for sequencing, wherein said isolating comprises diffusing fragments of genomic DNA bound by the enzyme into the dialysis membrane and binding genomic DNA complex with the enzyme to the resin, and wherein said preparing comprises amplification of the DNA; and (h) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

[0038] Provided herein also is a single vessel method for determining the genomic location of at least one biomolecule-genomic DNA interaction, said method comprising the steps of: (a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence; (b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest; (c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest; (d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of cutting genomic DNA; (e) transferring the contents of steps (b)-(d) into a dialysis membrane; (f) incubating the dialysis membrane of (e) in the vessel, said vessel further comprising a buffer comprising a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme, (g) activating the enzyme of (d) under conditions that allow cutting of the genomic DNA; (h) isolating and preparing the genomic DNA for sequencing, wherein said isolating comprises diffusing fragments of genomic DNA bound by the enzyme into the dialysis membrane and binding genomic DNA in complex with theenzyme to the resin, and wherein said preparing comprises amplification of the DNA; and (i) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

[0039] The disclosure additionally provides a method for determining the genomic location of at least one biomolecule-genomic DNA interaction, said method comprising the steps of: (a) incubating in a first vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence; (b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest; (c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest; (d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of cutting genomic DNA; (e) transferring the contents of steps (b)-(d) into a dialysis membrane; (f) incubating the dialysis membrane of (e) in a second vessel, said vessel comprising a dialysis buffer capable of diluting out unbound first moiety and / or second moiety conjugated to the enzyme; (g) activating the enzyme of (d)under conditions that allow cutting of the genomic DNA; (h) isolating and preparing the genomic DNA for sequencing, wherein said preparing comprises amplification of the DNA; and (i) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figures 1A-1C provides schematics showing the reliance of previously disclosed chromatin profiling methods, like CUT&RUN or CUT&TAG, on a nuclear envelope. Figure 1 A is a simplified schematic depicting compacted chromatin in the nucleus and long-range cis- and trans- interactions with chromatin-bound proteins. Figure 1 B is a schematic showing the utility of a permeabilized nuclear envelope in allowing access to reagents e.g., antibody tethered enzymes into the nucleus, retaining bulk chromatin during wash steps for removal of unbound reagents or inhibitory intermediates, and allowing diffusion of smaller fragments of cleaved DNA out of the nucleus and into the supernatant where it is extracted and sequenced after making a DNA library. Figure 1C is a close-up schematic depicting sample processing in the permeabilized nucleus by DNA cutting enzymes (MNase for CUT&RUN or TN5 for CUT&TAG), which are tethered by an antibody to a protein of interest followed by cleavage of the DNA around the protein binding site after enzyme activation (middle panel), liberating DNA fragments for sequencing (right panel).

[0041] Figure 2 is a drawing showing an exemplary one-pot workflow for sample preparation in accordance with the present teachings, which optionally includes one-pot CUT&RUN or CUT&TAG. Step 1 shown in the workflow is cell harvesting to obtain a cell pellet. Step 2 shown in the workflow is cell permeabilization, and optionally, chromatin decompaction. Step 3 shown in the workflow is target binding of the biomolecule of interest with a primary antibody reagent then a secondary antibody or fragment thereof that tethers TN5 or MNase enzymes to antibody-bound sites. Step 4 shown in the workflow is precipitation of unbound or excess enzyme using a resin sealed in a dialysis membrane in lieu of a wash step. Step 5 shown in the workflow is activation of the bound enzyme for example by the addition of Mg2+or Ca2+for TN5 or MNase respectively. After DNA cutting, a liberated nucleosome in complex with an antibody and the tethered MNase would be small enough to diffuse into the dialysis membrane rather than be retained (assuming the nucleosome -180 kDa + Ab 150 kDa + 2x Nb-MNase -30 kDa = -400 kDa), similar to the way it diffuses out of the permeabilized nucleus in conventional CUT&RUN or CUT&TAG. Accordingly, following the five steps, target DNA fragments diffuse across the dialysis membrane and bind to the resin. DNA is then isolated from the resin, amplified, and sequenced.

[0042] Figure 3 is a drawing of an exemplary one-pot workflow for sample preparation in accordance with the present teachings, which optionally includes one-pot CUT&RUN or CUT&TAG. Step 1 shown in the workflow is cell harvesting to obtain a cell pellet. Step 2 shown in the workflow is cell permeabilization, and optionally, chromatin decompaction. Step 3 shown in the workflow is target binding of the biomolecule of interest with a primary antibody reagent then a secondary antibody or fragment thereof that tethers TN5 or MNase enzymes to antibody-bound sites, and transfer of the contents into a dialysis pouch. Step 4 shown in the workflow is precipitation of unbound or excess enzyme by adding the dialysis membrane and its contents into an activation buffer containing a resin capable of precipitating the unbound or excess enzyme. Step 5 shown in the workflow is activation of the bound enzyme for example by the addition of Mg2+or Ca2+for TN5 or MNase respectively. Following the five steps, target DNA fragments diffuse across the dialysis membrane and bind to the resin. DNA is then isolated from the resin, amplified and sequenced.

[0043] Figure 4 is a drawing showing an exemplary workflow for sample preparation, which does not require the use of an affinity resin, in accordance with the present teachings which optionally includes one-pot CUT&RUN or CUT&TAG. Step 1 shown in the workflow is cell harvesting to obtain a cell pellet. Step 2 shown in the workflow is cell permeabilization, and optionally, chromatin decompaction. Step 3 shown in the workflow is target binding ofthe biomolecule of interest with a primary antibody reagent then a secondary antibody or fragment thereof that tethers TN5 or MNase enzymes to antibody-bound sites, and transfer of the contents into a dialysis pouch. Step 4 shown in the workflow is the dilution of unbound or excess enzyme by adding the dialysis membrane and its contents into a vessel containing a buffer capable of diluting out the unbound or excess enzyme. Step 5 shown in the workflow is activation of the bound enzyme for example by the addition of Mg2+or Ca2+for TN5 or MNase respectively. Following the five steps, target DNA fragments diffuse into the supernatant, DNA is then isolated from the supernatant, amplified and sequenced.

[0044] Figures 5A-5B is a schematic comparing the present Maltose-binding protein / Nanobody / N6-adenine methyltransferase (Hia5) fusion protein (MBP-Nb-Hia5) (Figure 5A) and a previously disclosed protein A-N6-adenine methyltransferase (Hia5) fusion protein (pA-Hia5) (WO 2022 / 256469) (Figure 5B). pA is divalent and can lead to aggregation, while MBP-Nb-Hia5 is monovalent.

[0045] Figures 6A-6B shows drawings of exemplary designs of dialysis pouches in accordance with certain embodiments.

[0046] Figures 7A-7B is a schematic of the presently described chromatin preparation methods and their combination with previously disclosed DiMeLo-Seq. Figure 7A is a schematic showing processing of permeabilized cells containing condensed (left panel) or decondensed chromatin (right panel), where a dialysis pouch of the present disclosure plays the role of the nuclear envelope, allowing buffer exchanges and size selective diffusion of chromatin fragments. Figure 7B is a close-up schematic of DiMeLo-seq where a primary antibody is used to tether Hia5 to the antibody bound proteins where it methylates adenines proximal to the target protein after enzyme activation, in cells with condensed chromatin (left panel) or relaxed chromatin (right panel).

[0047] Figure 8 provides plots of percentage methylation (mA / A) across all low-coverage DiMeLo reads overlapping the top 3000 strongest CTCF ChlP-seq peaks from the GM12878 cell line. The left panel is the plot achieved using the standard DiMeLo-seq protocol with an anti-CTCF antibody and with a no-antibody control. The right panel is the plot achieved with the One-Pot DiMeLo-seq (without chromatin decompaction, like in Figure 7B) with the same anti-CTCF antibody or no antibody.

[0048] Figure 9 provides a graph of samples containing different input cell numbers were carried through mock One-Pot or standard DiMeLo-seq protocols, followed by DNA extraction and quantification.DETAILED DESCRIPTION

[0049] The present disclosure provides methods and compositions to address the aforementioned unmet needs. For example, provided herein in one embodiment is a single vessel (i.e., a “one-pot”) method for preparing samples for genome wide mapping of protein- DNA interactions. As described herein, the workflows may include DiMeLo-seq or other methods, such as CUT&RUN or CUT&TAG. The presently disclosed methods in some embodiments eliminate wash steps or the need for containment of chromatin within a nuclear envelope by replacing wash steps with a series of carefully designed dilution and / or precipitation steps that enable addition of components that would otherwise interfere with downstream workflow steps above a certain concentration. Excess components that require removal are in some embodiments removed using an ultra-high molecular weight cutoff (UHMWCO) dialysis vessel or membrane such as a dialysis button designed to selectively remove reaction components without interaction with chromatin (Figures 2, 3). This is done by including resin with selective affinity for the component to be removed, such as the active enzyme construct being used (for example, an MNase fusion protein in the case of CUT&RUN (see WO 2019 / 060907), a Hia5 fusion protein in the case of DiMeLo-seq, (see, e.g., WO 2022 / 256469, which is incorporated by reference herein), and other enzymes or fusions as described herein).DEFINITIONS

[0050] The phrases “single vessel,” and “one-pot” with respect to the methods provided herein are used interchangeably to refer to a process or method in which a starting material optionally undergoes two or more chemical transformations in a single reaction vessel, wherein all the reactants are added simultaneously at the beginning of the first reaction or sequentially during the course of the reaction, and in which no separation and / or purification of the intermediate products is required before the final product is produced.

[0051] As described herein, the combination of multiple steps in a one-pot method would offer the possibility to drastically shorten the overall time and workload. However, the design of such a one-pot method bears the challenge to ensure the compatibility of all reagents and intermediates, and the present disclosure addresses these and other concerns.

[0052] The term “intermediate product” or “intermediate material,” as used herein, refers to whatever components are present following the chemical or enzymatic treatment or transformation of a suitable starting material and prior to formation of the final product.

[0053] The term “starting material” or “starting product,” as used herein, refers to any mixture or composition, containing a material that is suitable for the preparation of the final product. In one embodiment, the starting material refers to cells. In some embodiments, the final product refers to genomic DNA.

[0054] As used herein, the term “vessel,” generally refers to any container in which a reaction can occur in accordance with the present teachings. In some embodiments, a vessel means any receptacle capable of holding a cell suspension. In some embodiments, a vessel can be an Eppendorf tube, a PCR tube, and other containers of the sort in common practice in modern molecular biology laboratories. In some embodiments, a vessel can be a well in microtitre plate. For example, a plurality of reaction vessels can reside on the same support. In another embodiment, a vessel can be a dialysis membrane or pouch described herein. The vessel may be of any shape suitable for mixing or incubating biological materials. It will be recognized that a variety of reaction vessels are available in the art and can be used in the context of the present teachings.

[0055] As used herein, the phrase, “expose the genomic DNA,” refers to the dissolution of the lipids in cell membranes and permeabilization of nuclear membranes so as to provide macromolecules that are part of the reagents described herein such as antibodies and fusion proteins access to nuclear contents where they modify the genomic DNA and / or the associated biomolecules contained therein.

[0056] As used herein, the term “biomolecule,” refers to any molecule exhibiting biological activity. Exemplary biomolecules include peptides, polypeptides, proteins, nucleic acids e.g., a RNA, and a RNA-DNA hybrid, carbohydrates, or sugars, enzymes, hormones, growth factors, chromatin-binding small molecules, cell surface receptors, antigens, cytokines, neurotransmitters, soluble extracellular receptors, antibodies, soluble matrix proteins, antigens, allergens, cofactors and the like.

[0057] As used herein, the term, “biomolecule of interest,” “analyte,” or “target,” refers to any molecule or aggregate of molecules of interest for detection in a biological sample. Nonlimiting examples of a biomolecule of interest e.g., a RNA of interest include, without limitation, Non-coding RNA (ncRNA), long noncoding RNA (IncRNA), ribosomal RNA (rRNA), small nuclear RNAs (snRNA), messenger RNA (mRNA), miscellaneous RNA (miscRNA), and Telomerase RNA (TERC). The RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Small RNAs mainly include 5.8S ribosomal RNA (5.8S rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single-stranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

[0058] Non-limiting examples of a biomolecule of interest e.g., a protein or protein of interest include, without limitation, a nuclear lamina protein (e.g., LMNB1 and LMNA), anucleolar protein (e.g., NPM1 and NCL), a transcription factor (e.g., NPAT and SOX9), a histone or histone variant (e.g., centromere protein A (CENPA) and H3K9ac), centromere protein A, a modification- specific internal antibody (mintbody) (e.g., H3K9ac mintbody and H4K20mel mintbody), an intracellular scFV, a nanobody, a chromatin-modifying enzyme (e.g., PRDM9 and HDAC2), an RNA polymerase subunit or modifier (e.g., RPB1 and CDK9), a DNA polymerase subunit or modifier (e.g., POLB and POLA2), a DNA helicase (e.g., MCM2 and RECQ1), a DNA repair protein (e.g., RAD51 and FANCD2), a Cas9 protein, a dCas9 protein, a zinc finger protein (e.g., PRDM9 and ZNF212), an engineered TALE protein, a CTCF protein, a cohesin protein (e.g., RAD21 and SMC1 A), a synaptonemal complex protein (e.g., SYCP1 and SYCP2), a telomere-binding protein (e.g., TRF1 and TRF2), a centromere-binding protein (e.g., CENPC and CENPT), and an outer kinetochore protein (e.g., SPC24 and SPC25). Additional proteins are described herein.

[0059] A “biological sample” is any sample obtained or obtainable from a biological source and / or including material from a biological source. A “biological sample” includes, but is not limited to bacteria and other microorganisms, plants, animals, human biological materials such as blood, lymph, cerebrospinal fluid, sputum, semen, urine, feces, hair follicles, lachrymal fluid, epithelial scrapings, a cell sample, a tissue sample, an organ sample, a biopsy sample, cultured cells, as well as extracts and / or homogenates of cells, tissues or organs, etc.

[0060] The term "chromatin" as used herein refers to a complex of DNA and protein, both in vitro and in vivo. This includes all proteins that are directly contacting DNA, and also proteins that are part of a protein or ribonucleoprotein complex that may be associated with DNA. A chromatin protein may or may not directly contact DNA. Chromatin also includes proteins that are transiently associated with DNA, with DNA-protein, or with DNA- ribonucleoprotein complexes, i.e., only during part of the cell cycle. "Chromatin protein" includes, but is not limited to histones, transcriptional factors, centromere proteins, heterochromatin proteins, euchromatin proteins, condensins, cohesins, origin recognition complexes, histone kinases, dephosphorylases, acetyltransferases, deacetylases, methyltransferases, demethylases, and other enzymes that covalently modify histone, DNA repair proteins, proteins involved in DNA replication, proteins involved in transcription, proteins part of dosage compensation complexes and X- chromosome inactivation, proteins that are part of chromatin remodeling complexes, telomeric proteins, and the like.

[0061] The term "polypeptide" refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. A "fragment" refers to a portion of a polypeptide having typically at least 10 contiguous amino acids, more typically at least 20,still more typically at least 50 contiguous amino acids of the protein. A "derivative" is a polypeptide which is identical or shares a defined percent identity with the wild-type protein or nucleotide modification enzyme. The derivative can have conservative amino acid substitutions, as compared with another sequence. Derivatives further include, for example, glycosylations, acetylations, phosphorylations, and the like. Further included within the definition of "polypeptide" are, for example, polypeptides containing one or more analogs of an amino acid (e.g., unnatural amino acids, and the like), polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-naturally occurring. Ordinarily, such polypeptides will be at least about 50% identical to the native protein or nucleotide modification enzyme acid sequence, typically in excess of about 90%, and more typically at least about 95% identical. The polypeptide can also be substantially identical as long as the fragment, derivative or analog displays similar functional activity and specificity as the wild-type protein or nucleotide modification enzyme.

[0062] The term "isolated" refers to a nucleic acid or polypeptide that has been removed from its natural cellular environment. An isolated nucleic acid is typically at least partially purified from other cellular nucleic acids, polypeptides and other constituents.

[0063] The term "affinity resin" means a solid phase or substrate with ligands bound to its surfaces, wherein the functional groups of the ligands are capable of binding target compounds via a "lock / key" mechanism, such as antibody / antigen; enzyme / receptor; biotin / avidin; Protein A / antibody etc. Affinity resins are known to bind target compounds with high specificity, and said binding is normally based on more than one kind of interaction. Well known affinity resins are e.g. Protein A coupled to particles, such as Protein A Sepharose™ or Prosep A™. In one embodiment, the affinity resin comprises a solid support coupled to an anti-MBP antibody. In a further embodiment, the solid support is selected from the group consisting of: a magnetic bead, a glass slide, a chip, a gelatin, and agarose.

[0064] As used herein, the term, “dialysis membrane,” refers to a thin film or layer of a selectively semi-permeable material that selectively allows the passage of some molecules or ions under certain conditions and inhibits the passage of others. The membrane may be of a low-binding material to minimize adsorptive losses, and should be durable, cleanable and chemically compatible with the buffers to be used. A number of suitable membranes are commercially available, and are made from polymeric materials including e.g., polyvinylidene fluoride (PVDF), polyacrylonitrile, a polyacrylonitrile copolymer, polysulfone, sulfonated polysulfone, polyethersulfone, cellulose acetate, cellulose triacetate, polymethylmethacrylate or a mixture thereof. In one embodiment, the membrane material is a cellulose ester membrane with a MWCO of 1000kDa.

[0065] As used herein, the expression, "Molecular weight cut-off" or "MWCO" refers to the lowest molecular weight solute (in daltons) in which 90% of the solute is retained by the membrane, or the molecular weight of the molecule (e.g. globular protein) that is 90% retained by the membrane. The expression "ultra-high molecular weight cutoff" or " IIHMWCO " as used herein refers to dialysis membranes having a MWCO between 300 and 1000 kDa.

[0066] Solute retention can vary due to molecular shape, structure, solute concentration, presence of other solutes and ionic conditions. Hence, the person skilled in the art will appreciate that MWCO ratings are based on globular molecules, while more linear molecules, which may have a small diameter in two of three dimensions, may be able to pass through the pores more freely despite having molecular weights that exceed the stated MWCO. To ensure proper retention of the biomolecule of interest, the person skilled in the art can select a membrane having the required properties by performing some routine experimentation. A good general rule is to select a membrane with a MWCO that is 3 to 6 times lower than the molecular weight of the biomolecule of interest to be retained.

[0067] As used herein, the term "contacting" refers to the process of bringing into contact at least two distinct species such that they can react or bind together. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0068] In one embodiment, “contacting” will generally comprise any suitable means for delivering, or exposing, a sample to one of or a plurality of the detection reagents described herein. In some embodiments, the term "contacting" refers to adding the detection reagents (e.g., suspended in a solution) directly to the sample. In some embodiments, the term "contacting" can further comprise mixing the sample with the detection reagents by any means known in the art (e.g., vortexing, pipetting, and / or agitating). In some embodiments, the term "contacting" can further comprise incubating the sample together with the detection reagents for a sufficient amount of time, e.g., to allow binding of the probe reagents to the target analytes. The contact time can be of any length, depending on the binding affinities and / or concentrations of the probe reagents and / or the analytes, concentrations of the detection reagents, and / or incubation condition (e.g., temperature). For example, the contact time can be reduced if the sample and detection reagents are incubated at a higher temperature. In some embodiments, the contact time between the sample and the detection reagents can be at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at leastabout 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours or longer. One of skill in the art can adjust the contact time accordingly.

[0069] In another embodiment, “contacting,” involves bringing a biomolecule of interest or target into contact with a genomic DNA sequence. Such contact can result in the biomolecule of interest directly interacting with or directly binding to the genomic DNA or it could result in an indirect interaction (e.g., through a mediating protein that binds directly to the DNA). In another embodiment, physical contact is not necessary - a biomolecule of interest just needs to be in close enough proximity to the DNA to permit the desired activity to occur. An example includes contacting the genomic DNA sequence with a protein of interest in sufficient proximity to allow for a DNA modifying enzyme that is targeted to the protein of interest to have the desired effect on the DNA in its vicinity such as DNA cleavage or methylation.

[0070] Nucleotide modifying enzymes (e.g., “enzyme capable of modifying genomic DNA”), fragments, derivatives and analogs thereof useful in the present invention are those which can modify one or more nucleotides in a nucleic acid sequence, such as an RNA, DNA, or the like, under conditions found in vitro or in situ or in a live cell and in a manner which is detectable. The enzyme, in some embodiments, will optionally modify the nucleotides in a manner which is not toxic to the cell. In other words, the cell or organism must be able to continue to proliferate and differentiate in a normal manner. For the modification to be detectable, an enzyme is selected which modifies the nucleotide in a manner which is not typical of a modification commonly found in the cell being assayed. For instance, in eukaryotic cells it is typical to select as the modification enzyme, for example, DNA adenine methyl transferase because methylation of adenine is not common in eukaryotic cells. Additional nucleotide modification enzymes useful in the present invention include, for example, but are not limited to, adenine methyltransferases, cytosine methyltransferases, thymidine hydroxylases, hydroxymethyluracil b-glucosyl transferases, adenosine deaminases, and the like. In other embodiments, the enzyme capable of modifying genomic DNA includes ten-eleven translocation (TET) dioxygenase (e.g., enzymes that generate 5-hydroxymethylcytosine, 5-carboxylcytosine, 5-formylcytosine). In still another embodiment, the use of a methyltransferase with modified SAM is provided to deposit a different mark rather than a methyl group. Optionally this would still involve using a (potentially modified or unmodified) methyltransferase but it would not be a methyl group being deposited. In still other embodiments, the enzyme is miniSOG or SOPP2 which, when excited with blue light, generate highly reactive singlet oxygen molecules, which oxidize guanines in their vicinity.METHODS

[0071] The present disclosure provides methods for determining the genomic location of at least one biomolecule-genomic DNA interaction. As will be appreciated by those in the art, and as described further herein, the present disclosure also contemplates that biomolecule-DNA interactions can also be mapped (i.e., interactions of a biomolecule on or with nucleic acid that may not conventionally understood to be “genomic,” such as plasmid DNA and others described herein). For example, the methods also contemplate chloroplast DNA, plasmid DNA, artificial chromosome DNA and artificially-introduced DNA, and pathogen DNA. Thus, the methods also contemplate, in certain embodiments, first isolating or otherwise preparing mitochondria, chloroplasts or synthetic chromatin as “inputs” to a particular workflow (e.g., rather than chromatin derived from cells).

[0072] In one embodiment, the method is a single vessel method comprising one or more or all of the following steps, (a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence; (b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest; (c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest; (d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of modifying genomic DNA; (e) precipitating excess or unbound enzyme by adding a dialysis membrane to the vessel, wherein the dialysis membrane contains a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme; (f) incubating the first binding moiety and second binding moiety of (d) under conditions that allow modification of genomic DNA; (g) isolating and preparing the genomic DNA for sequencing; and (h) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

[0073] In some embodiments, the population of cells used for the methods may be composed of any number of cells, e.g., about 500 to about 106or more cells, about 500 to about 100,000 cells, about 500 to about 50,000 cells, about 500 to about 10,000 cells, about 50 to 1000 cells, about 1 to 500 cells, about 1 to 100 cells, about 1 to 50 cells, or a single cell. In some embodiments, the cell sample includes less than about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1 ,000,000 cells. In some embodiments, the cell sample includes more than about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000,25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1 ,000,000 cells.

[0074] In some embodiments, the cells can be permeabilized to facilitate transfer of analytes out of the sample, and / or to facilitate transfer of species (such as binding moieties) into the sample. If a sample is not permeabilized sufficiently, the amount of analyte captured from the sample may be too low to enable adequate analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationship of the analytes within the tissue sample can be lost. Hence, a balance between permeabilizing the tissue sample enough to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution in the sample is desirable.

[0075] In general, cells can be permeabilized by exposing them to one or more permeabilizing agents. Suitable permeabilizing reagent (e.g., detergents) are those that permeabilize cells and retain surface epitope integrity of the proteins being detected. Detergents which are usually used, are roughly classified as cationic, anionic, amphoteric, and nonionic. The nonionic detergents are exemplified by digitonin, polyoxyethylene alkylether (Brij series), polyoxyethylene sorbitan (Tween series), p-dodecylmaltoside, p- octylglucoside, p-nonylglucoside, p-heptylglucoside, p-octylthioglucoside, sucrose monodecanoate, sucrose mono-dodecanoate, octyltetraoxyethylene, octylpentaoxyethylene, and dodecyloctaoxyethylene. The anionic detergents are, for example, taurodeoxycholic acid and the like. The amphoteric ionic detergents are exemplified by N,N-dimethyldecylamine- N-oxide, N,N-dimethyldodecylamine-N-oxide, N,N-dimethyldodecylammonio propanesulfonate, octyl (hydroxyethyl)sulfoxide, octanoyl-N-methylglucamide, nonanoyl-N- methylglucamide, decanoyl-N-methylglucamide, and (3-[(3-cholamidepropyl) dimethylammonio]-1 -propanesulfonate (CHAPS).

[0076] These detergents can be used as single species or as a combination of more than one species. The working concentration of these detergents is preferably adjusted depending on the kinds of target proteins, however, the concentration of usually used is preferably 1 to 50 times of critical micelle concentration (CMC) of the detergent, more preferably 3 to 10 times thereof. In certain embodiments, the detergent does not substantially interfere with the activity of the modifying enzyme as described herein.

[0077] In one embodiment, the detergent is Digitonin. In another embodiment, the digitonin is added at a final concentration of 0.01 -0.02% (w / v).

[0078] In some embodiments, nuclei are permeabilized by incubating the cells for 5 minutes on ice in Buffer 1 , wherein Buffer 1 comprises 0.02% digitonin, 20 mM HEPES pH7.5, 150 mM Sodium Glutamate, 0.5 mM spermidine, 0.05% BSA (w / v), and 1X Roche Complete tablet EDTA-free protease inhibitor. In one embodiment, the permeabilized cells are contacted with a first binding moiety capable of specifically binding to the biomolecule of interest.

[0079] In some embodiments, the methods optionally comprise contacting the permeabilized cells with a chromatin de-condensation agent.

[0080] As used herein, the term “de-condensing” or “de-compacting” means one or more processes for decreasing and / or reversing the condensation of chromatin including one or more chromosomes, one or more portions of chromosomes, one or more genomes, or one or more portions of genomes. As used herein, the term “condensed chromatin” means the more tightly packaged DNA / protein complex that occurs to varying extents during various stages of mitosis & meiosis, for example. Condensed chromatin, in some embodiments, can refer to interphase chromatin inside permeabilized nuclei. This chromatin, though not as compact as mitotic chromatin, is still highly compacted by being constrained to the volume of the nucleus. The condensation of chromatin is characterized by a reduction of volume due to a spatial organization into densely packed higher-order structures. The consequences of this compaction into condensed chromosomes at mitosis are reduced free volumes and less exposed surface of the chromatin substructures on which molecules could interact. In the presently disclosed methods, the presence of a nuclear envelope is no longer necessary as the wash steps have been eliminated. As such, chromatin may be removed from the nucleus and completely de-condensed prior to subsequent method steps, eliminating higher- order structure that confound chromatin mapping efforts. Chromatin de-condensation provides access to DNA and binding of nuclear proteins.

[0081] In some embodiments, chromatin de-condensation is performed by adding de- condensation reagents to the sample prior to downstream target marking. In some embodiments, the chromatin de-condensation agent can be selected from the group consisting of heparin and other polyanions, dithiothreitol, glutathione, polyglutamic acid (PGA), maltotriose per-O-sulfate (MTS), and methyl cellobiose sulfate (mCBS). Additional reagents for disruption of nuclear lamina are contemplated and include, for example, bile salts (including deoxycholate), lysolecithin, nonionic detergents, sarkosyl, cyclin dependent kinases, phosphatase inhibitors, sucrose, NaF+NaVO4, protein kinase C and urea.

[0082] In some embodiments, chromatin is extracted from nuclei by addition of polyanions, which promote nuclear swelling and rupture. In one embodiment, polyanions interact with the positively charged tails of histones and promote chromatin de-condensation. In another embodiment, targeted degradation or modification of lamin proteins which providestructural support to the nuclear envelope could be performed followed by chromatin decondensation with polyanions. In a further embodiment, the method described herein further comprises the removal of de-condensation agents to prevent with interference with downstream steps. In one embodiment, the de-condensation agents are removed by targeted sequestration with the dialysis membrane described herein. In another embodiment, the de-condensation agents are removed by targeted degradation using enzymes. In one embodiment, the chromatin de-condensation agent is heparin. In a further embodiment, the heparin is deactivated by the addition of the heparin degrading enzyme, heparinase.

[0083] In some embodiments, the method described herein further comprises contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest.

[0084] As used herein, the term "binding moiety" refers to a moiety capable of specifically binding to a target molecule or target epitope. Suitable binding moieties in the context of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof, antibody-like proteins, ligands and receptors, and the like.

[0085] The term "specifically binds" refers to, with respect to an antigen, the preferential association of an antibody or other ligand, in whole or part, with a specific polypeptide, such as a specific protein bound to chromatin DNA, for example a transcription factor. A specific binding agent binds substantially only to a defined target, such as a specific chromatin associated factor. It is recognized that a minor degree of non-specific interaction may occur between a molecule, such as a specific binding agent, and a non-target polypeptide.Nevertheless, specific binding can be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they can do so with low affinity. Specific binding typically results in greater than 2-fold, such as greater than 5 -fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to a target polypeptide, such as compared to a non-target polypeptide. A variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0086] The term "antibody" is used herein in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological activity. Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). An "antibody fragment" comprises a portion of a full-length antibody, e.g. antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997;10: 949-57); camelid nanobody; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 23, 1 126-1 136; W02005040219, and published U.S. Patent Applications 20050238646 and 20020161201 .

[0087] In some embodiments, the first binding moiety is an antibody.

[0088] In a further embodiment, the method described herein further comprises contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety.

[0089] In some embodiments, the second binding moiety is selected from the group consisting of an antibody, a nanobody, a single-domain antibody (dAB), a camelid nanobody (sdAb), a single-chain variable fragment (scFv), a Fab, Fv, scFv, dsFv, Fab', or (Fab')2.

[0090] In some embodiments, second binding moiety is conjugated to an inducibly active enzyme capable of modifying genomic DNA once activated. In some embodiments, the enzyme is conjugated to a monovalent second binding moiety. Monovalency is key for avoiding aggregation or chromatin compaction. Monovalent antibody fragments suitable for conjugating to the enzymes described herein can be chosen from the group consisting of scFv, Fab, F(ab'), single-domain antibodies (sdAbs), disulfide-stabilized Fv (dsFv), or a camelid nanobody (sdAb).

[0091] In some embodiments, the monovalent second binding moiety that is conjugated to the DNA modifying enzyme is a camelid nanobody.

[0092] In other embodiments, the monovalent second binding moiety that is conjugated to the DNA modifying enzyme is a Fab or a scFv.

[0093] In some embodiments, the second binding moiety is an anti-IgG nanobody.

[0094] In some embodiments, the second binding moiety binds to the target directly. In other embodiments, the second binding moiety binds to an adapter molecule such as an antibody, that binds to the target. In one embodiment, the antibodies may be recognized through the aforementioned monomeric proteins. In a different embodiment, the second binding moiety comprises an engineered protein domain that is specific for the antibody of interest.

[0095] The phrase, "modifying genomic DNA," as used herein, refers to the alteration of DNA in a detectable manner. Exemplary modifications include DNA nicking or cleavage or introduction or removal of chemical moieties from the DNA. DNA modifying enzymes that do not result in DNA cleavage include, but are not limited to, DNA methyltransferases. DNA modifying enzymes resulting in DNA cleavage include, but are not limited to, Micrococcal Nuclease (MNase). In yet another embodiment, the DNA modifying enzymes are enzymes without or with minimal intrinsic DNA sequence specificity or those that cut and / or insert DNA sequences.

[0096] In an embodiment, DNA base editors are used, which convert the identity of one base to another, which can be distinguished based on its mutation compared to reference sequences.

[0097] In some embodiments, the DNA methyltransferase is selected from the group consisting of a DNA adenine methyltransferase (Dam), EcoGII methyltransferase, Hia5, M.CviPI, M.Sssl, M.CviQIX, and biologically active fragments thereof.

[0098] In an embodiment, the DNA methyltransferase is Hia5 or a biologically active fragment thereof.

[0099] The disclosure contemplates the use of fusion binding moieties with different fusion partners that may be arranged in any suitable order, depending on the purpose of the fusion protein. For example, in some embodiments, the first binding moiety is fused directly to (i) a second moiety or (ii) an enzyme capable of modifying genomic DNA. In other embodiments, the fusion proteins could comprise different affinity tags that allows it to bind the precipitation resin for example SpyTag. For example, proteins tagged with SpyTag and a SpyCatcher- Hia5 fusion to bind it directly, and covalently, is contemplated in one embodiment. In another exemplary related embodiment, fusing Hia5 to a nanobody directly targeting the protein of interest or an affinity tag on it is contemplated herein.

[0100] In some embodiments, the second binding moiety is a fusion protein termed MBP- Nb-Hia5, wherein the MBP-Nb-Hia5 comprises an anti-IgG nanobody, fused to a Hia5 DNAmethyltransferase or biologically active fragment thereof, and a maltose binding protein (MBP) tag.

[0101] In one embodiment, a buffer comprising glutamate instead of chloride as a counter-ion enhances the enzymatic activity of the enzyme disclosed herein. This is because glutamate is the predominant counterion in the bacterial cytoplasm, and the disclosed enzyme is a bacterial enzyme. In some embodiments, the enzyme disclosed herein is diluted using Buffer 2, wherein Buffer 2 comprises, 20 mM HEPES pH 7.5, 150 mM Sodium Glutamate, 0.5 mM spermidine, 0.1% Tween-20, 0.05% BSA (w / v), and 1X Roche Complete tablet EDTA-free protease inhibitor.

[0102] In some embodiments, the method described herein can further comprise removing any unbound reagents before modifying the genomic DNA in a temporally- sequential manner. The term "unbound reagents" as used herein refers to reagents that are in excess or that have not bound to or interacted with target analytes. The unbound reagents can be removed from the sample by any methods known in the art. For example, it is common to include intermediate washing steps between addition of reagents to remove excess / unbound reagents. Other methods include buffer exchanges, precipitation, or dilution as described herein.

[0103] In some embodiments, precipitation, dialysis and dilution are suitable for the removal of unbound reagents for the methods described herein. Advantageously, use of precipitation, dialysis and dilution instead of washing or buffer exchanges allows the presently disclosed methods to be carried out in a single tube without removal of material prior to DNA extraction.

[0104] In one embodiment, the methods provided herein comprise precipitating excess or unbound enzyme by adding a dialysis membrane to the vessel. The term “dialysis membrane” as used herein includes but is not limited to a dialysis button, a dialysis pouch, a sealed dialysis membrane, a membrane envelope, and a dialysis tubing. The size of the dialysis membrane may be appropriately set according to the size of the dialysis vessel. By “dialysis vessel” is meant the vessel in which the enzyme precipitation is occurring.

[0105] In a further embodiment, the dialysis membrane contains a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme.

[0106] The methods provided herein include steps of modifying, for example genomic DNA (e.g., “under conditions that allow modification of genomic DNA”). In one embodiment, the conditions promote or otherwise allow activation of an enzyme such as those described herein.

[0107] As will be appreciated by those in the art, the methods provided herein may include the separation of excess or unbound components from components that are desired for further downstream processing. As described herein, this is accomplished by optionally including membranes or other dialysis steps. In this way, cells may be placed into a membrane (e.g., button or pouch) and the placed in, for example, a cell permeabilization buffer and binding moieties such as those described herein.

[0108] In still other embodiments, the methods described herein allow for the “modification” of, for example genomic DNA. As described herein, modifying may include “cutting” genomic DNA.

[0109] In some embodiments, the method disclosed herein further comprises activating the DNA modifying enzyme. In one embodiment, enzyme activation is carried out using a buffer containing an activating agent. In some embodiments, the activating agent is S- Adenosylmethionine (SAM). In one embodiment, the activation buffer (e.g., Buffer 3) comprises 15 mM Tris pH 8, 60 mM Potassium Glutamate, 0.5 mM spermidine, 1 mM EDTA, 0.5 mM EGTA, and 1 mM DTT.

[0110] In some embodiments, the method disclosed herein further comprises reduction or removal of salt from the final activation buffer. In some embodiments, the salt reduction can be accomplished by dialysis or dilution as described herein.

[0111] In some embodiments, the modifying genomic DNA of steps (f) comprises modifying one or more nucleotides at one or more locations selected from the group consisting of (a) within 1 -50 nucleotides of the genomic DNA binding site of the biomolecule, (b) topologically near the genomic DNA binding site of the biomolecule, and (c) both (a) and (b).

[0112] In one embodiment of the present disclosure, multiple biomolecule-genomic DNA interactions are determined. In some embodiments, multiple molecules of the same biomolecule species are contemplated. In still other embodiments, multiple (different) biomolecules (e.g., biomolecules that are different species and that may or may not bind or otherwise interact with the same genomic DNA location) are contemplated.

[0113] In some embodiments, the methods as described herein can be combined with several downstream high-throughput sequencing readouts. In one embodiment, samples are processed and read out using nanopore sequencing methods such as those developed by Oxford Nanopore Technologies. In another embodiment, the methods are combined with sequencing-by-synthesis approaches in long-read (e.g. those developed by Pacific Biosciences Inc.) or short-read form (e.g. those developed by Illumina Inc.).

[0114] Both long-read sequencing approaches are capable of detecting DNA signals that do not affect the core sequence of the DNA (e.g. via DNA methyltransferase) directly without subsequent steps. Short-read sequencing can be used when the core sequence of the DNA is changed around target sites, or when the readout is coverage rather than direct modifications.EXAMPLES

[0115] Example 1

[0116] Current chromatin profiling approaches using tethered enzymes require permeabilized nuclei which gives access to reagents to label chromatin associated biomolecules and modify chromatin in situ, while simultaneously holding chromatin together during wash steps required to remove unbound antibodies and enzymes, non-specifically bound enzyme, or inhibitory reagents. However, the requirement for the presence of a nuclear envelope (Figure 1) is limiting as it necessitates large amounts of sample material to generate sufficient amounts of input DNA, increased variability due to handling steps, and an inability to study chromatin outside of its compacted form in the nucleus. Thus, new methods for sample processing are warranted that will lower sample requirements, minimize handling steps, and allow chromatin preparation in the absence of a nuclear envelope, while still providing reliable performance in downstream chromatin profiling techniques.

[0117] To address these outstanding needs, a new method for processing cells and chromatin for downstream genomic chromatin mapping that uses dilution, precipitation, and dialysis to replace wash steps and buffer exchanges. This is carried out using an ultra-high molecular weight cutoff (UHMWCO) dialysis button designed to selectively remove reaction components without interaction with chromatin (Figures 6A & B). Moreover, given that the nuclear envelope is no longer needed to carry out wash steps and buffer exchanges, chromatin may be removed from the nucleus and completely relaxed with a chromatin decondensation agent prior to reaction steps, eliminating 3D interactions that confound chromatin mapping efforts (Figure 7). Further, to avoid chromatin aggregation, the inducibly active chromatin modifying enzyme is tethered by a monomeric and monovalent affinity reagent (Figure 5A) which optionally comprises an added tag or domain for binding to the precipitating resin.

[0118] This example demonstrates the application of the disclosed sample preparation methods with DiMeLo-seq. However, the disclosed methods lend themselves to other chromatin profiling approaches using tethered enzymes such as CUT&RUN.

[0119] General materials and methods

[0120] The following materials and methods were used in in the Examples described herein.

[0121] Table 1 - List of buffer solutions

[0122] All buffers were prepared fresh on the day of use and filtered through a 0.2 pm filter and chilled on ice.

[0123] Table 2 - Additional reagents and instruments

[0124] Exemplary protocol

[0125] For each sample, prepare a replicate with an antibody targeting the molecule of interest and a separate replicate using a non-targeting antibody as a control.

[0126] For spins performed in a swinging bucket rotor, perform 3 minute spins at 300g.

[0127] For spins carried out in a fixed-angle rotor, perform a 2 minute spin at 300g and then rotate the tube 180eand spin at 300g for 1 minute to ensure full capture of material at the bottom of the tube.

[0128] In some embodiments the sample preparation method is performed as followsA. Cell permeabilization and primary antibody binding1 . Prepare replicates of 106cells.2. Wash cells in PBS. Spin and remove supernatant.3. Resuspend cells in 50 pL buffer 1 . Incubate for 5 minutes on ice.4. Add 1 pg antibody (~ 1 uL) and incubate overnight at 4°C on a rotator, alternatively, incubate at room temperature for 1 hour.B. Preparation of the dialysis pouchPrepare a dialysis button assembly for each sample on a magnet as follows:1 . Cut 8-tube per strip without caps up to just below the top to create rings2. Cut out individual caps from 8-tube cap strip (cut tightly to allow it to fit in tube)3. Place empty cap on top of a high-power magnet4. Add 50 uL beads pre-washed with buffer 3 (should rapidly settle to bottom of cap)5. Cover cap with high molecular weight cut off dialysis membrane and gently fold membrane over the edges6. Seal membrane in the cap by gently closing the cap into the ring7. Check for any subtle tears that might allow for leakage of the beadsC. MBP-Nb-Hia5 binding and optionally, chromatin decompaction1 . Combine 2.5 pL MBP-Nb-Hia5 with 22.5 pL Buffer 2, mix this 25 pL into the 50 pL from step A and incubate 1 hr at 4°C on rotator (2.5 pg, 27 pmol, 360 nM vs 133 nM Ab after dilution)2. Add heparin with sodium phosphate with buffer 2 (60pL of 10Omg / mL heparin + 60pL 10OmM Na2PC>4 per 1 mL of Buffer 2) for every condition calling for it at this step3. Add 25 pL of Buffer 2 to each sample with or without heparinase4. Add 2 pL of 1 :1 :1 heparinase 1 :2:3 mix and 1 pL 100mM CaCI2 to the relevant samples at this step to quench heparin5. Incubate at room temperature for 1 hour with end-over-end mixingPurification of MBP-Nb-Hia5 as described and used herein:-Expressed 2L of TB with .02% Rhamnose overnight at 18eC;-Lysed using emulsiflex C5 into 25 mM HEPES 300 mM NaCI 30 mM imidazole 10% glycerol -i-protease inhibitors and benzonase;-Bound to Ni-resin and washed with >5 column volumes (CVs) of lysis buffer then eluted with 250 mM Imidazole 3CVs;-Dialyzed eluate in to 25 mM HEPES 50 mM NaCI 10% glycerol 2 mM BME at 4°C;-Ran on Q HP ion exchange column using a linear gradient of 50-500 mM glycerol over 20 CV;-Fractions containing purified protein were concentrated and injected on a Superdex 200 10 / 300 increase size exclusion column; and-Single monodisperse peak was collected concentrated to 1 mg / mL (~11 uM) and flash froze in liquid nitrogen and stored at -80eC.D. Enzyme precipitation and activation1 . Briefly spin down samples and add 900 pL Buffer 3 (activation buffer) and loaded dialysis button and incubate at room temperature for 30 minutes (no agitation)2. Add 25 pL 32 mM SAM (freshly thawed), mix by gentle inversion and place in incubator at 37°C for 1 hr (no agitation)3. Remove dialysis button with forceps (wash forceps in between samples)E. DNA extractionIn some embodiments, a kit such as the Monarch Genomic DNA Purification Kit is used.1 . Pipette 4pL Proteinase K and 4 pL RNAse A (NEB stocks) per reaction2. Spin down, add 1 pL 10% SDS and incubate at 56eC for 5 minutes with shaking3. Add 500 pL 12M GuSCN and 50pL 3M NaOAc pH 5.2 to each tube4. Prep using the NEB Monarch Genomic DNA kit• Split each sample across two spin columns (750 pL each)• Elution was with 50 pL prewarmed to 60eC in EB each• Quantify yield with qubit5. Prepare sequencing library according to rapid barcoding kit6. Pool and sequence the samples according to MinlON flow cell

[0129] Results from the above protocol are shown in Figure 8.

[0130] The profile plots show the average proportion of adenines that are methylated across all low-coverage DiMeLo reads overlapping the top 3000 strongest CTCF ChlP-seq peaks from the GM 12878 cell line. On the left are the results from the standard DiMeLo-seq protocol, both with an anti-CTCF antibody (magenta) and with a no-antibody control (orange). On the right are the results from an early One-Pot DiMeLo-seq run with the same anti-CTCF antibody (magenta) or no antibody (orange and blue). Decompaction was not performed for this experiment. Anti-MBP beads were used (magenta and orange).

[0131] Comparing the magenta profiles on the left and right shows that the on-target signal appears stronger for One-Pot DiMeLo-seq. Comparing the orange profiles shows that the anti-MBP beads are similarly effective at removing excess Hia5 as the wash steps are in the standard protocol. The blue profile shows what happens if anti-MBP beads are not used: all the unbound, free-floating Hia5 in the tube methylates the accessible DNA around CTCF sites at high levels in a non-specific way (i.e. this is the pure background methylation that gets removed by the beads). These results show that immunoprecipitation can effectively replace washes and that One-Pot DiMeLo-seq may provide even better on-target signal than the standard protocol does.Example 2

[0132] To test the One-Pot method against a standard protocol, samples containing different input cell numbers were carried through mock One-Pot or standard DiMeLo-seq protocols, followed by DNA extraction and quantification and the results were plotted on the graph in Figure 9. Specifically, Digitonin-permeabilized GM24385 lymphoblastoid cells were subjected to high molecular weight DNA extraction of the indicated amount of cells eluted into 50pL elution buffer before quantification by nanodrop spectrophotometry.

[0133] The DNA extraction method is as follows:

[0134] Samples containing different cell input numbers of GM24385 lymphoblastoid cells were subject to DNA extraction. The nuclei from the different cell input numbers were isolated in 200 pL of activation buffer and were lysed by adding 10% SDS to the buffer for a final concentration of 0.1% in each tube.

[0135] 200 pg Proteinase K and 100 pg RNase A were added to each tube and digestion was carried out at 65°C for 5 minutes.

[0136] Two 3 mm glass beads were added to each tube and DNA was precipitated on beads by adding Domiphen Bromide, Guanadine thiocyanate, and PEG 800 to final concentration of 0.2% Domiphen Bromide, 2 M Guanadine thiocyanate, and 10% PEG 8000.

[0137] Each tube was rotated on their side for 5 minutes at 10 rpm to entangle DNA in the beads. The precipitated DNA was condensed on the beads using isopropanol with a final isopropanol concentration of 30% and each tube was rotated at 10 rpm for 5 minutes at room temperature.

[0138] The supernatant was aspirated and 1 mL DNA wash solution (20 mM Bis-Tris pH 7.0, 2 M LiCI, 10% PEG 8000) was added to each tube. Each tube was gently inverted 4-6 times. The supernatant was aspirated, and a wash step was repeated two more times.

[0139] Beads were added to an empty bead capture tube and lightly spun to dry the beads.

[0140] The beads were transferred to a fresh tube and eluted with 50 pL Elution buffer (10 mM Tris pH 9.0, 1 mM EDTA, 500mM 6-aminocaproic acid), was quantified using nanodrop spectrophotometry and the results were plotted on the graph in Figure 9.

[0141] Figure 9 demonstrates empirically that the One-Pot method yields more DNA at the end of the protocol compared to the standard protocol, which involves multiple wash steps.

Claims

What is claimed is:1 . A single vessel method to determine the genomic location of at least one biomolecule-genomic DNA interaction, said method comprising the steps of:(a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence;(b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest;(c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest;(d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of modifying genomic DNA;(e) precipitating excess or unbound enzyme by adding a dialysis membrane to the vessel, wherein the dialysis membrane contains a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme;(f) activating the enzyme of (d) under conditions that allow modification of genomic DNA;(g) isolating and preparing the genomic DNA for sequencing; and(h) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

2. A single vessel method to determine the genomic location of at least one biomolecule-genomic DNA interaction, said method comprising the steps of:(a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence;(b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest;(c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest;(d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of modifying genomic DNA;(e) transferring the contents of steps (b)-(d) into a dialysis membrane;(f) incubating the dialysis membrane of (e) in the vessel, said vessel further comprising a buffer comprising a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme;(g) activating the enzyme of (d)under conditions that allow modification of the genomic DNA;(h) isolating and preparing the genomic DNA for sequencing; and(i) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

3. A method to determine the genomic location of at least one biomolecule- genomic DNA interaction, said method comprising the steps of:(a) incubating in a first vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence;(b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest;(c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest;(d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of modifying genomic DNA;(e) transferring the contents of steps (b)-(d) into a dialysis membrane;(f) incubating the dialysis membrane of (e) in a second vessel, said vessel comprising a dialysis buffer capable of diluting out unbound first moiety and / or second moiety conjugated to the enzyme;(g) activating the enzyme of (d) under conditions that allow modification of the genomic DNA;(h) isolating and preparing the genomic DNA for sequencing, wherein said preparing does not require removal of the DNA from the dialysis membrane or amplification of the DNA; and(i) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

4. A single vessel method for preparing biomolecule-bound chromatin from cells, said method comprising the steps of:(a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence;(b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest;(c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest;(d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of modifying genomic DNA;(e) precipitating excess or unbound enzyme using a dialysis membrane comprising a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme;(f) activating the enzyme of (d) under conditions that allow modification of genomic DNA; and(g) isolating biomolecule-bound chromatin.

5. The method of any one of claims 1-4, wherein the enzyme capable of modifying genomic DNA is a DNA methyltransferase, wherein the DNA methyltransferase is selected from the group consisting of DNA adenine methyltransferase (Dam) or a biologically active fragment thereof, EcoGII methyltransferase or a biologically active fragment thereof, Hia5 or a biologically active fragment thereof, M.CviPI or a biologically active fragment thereof, M.CviQIX or a biologically active fragment thereof, and M.Sssl or a biologically active fragment thereof.

6. The method of claim 5, wherein the DNA methyltransferase is Hia5 or a biologically active fragment thereof.

7. The method of any one of claims 1-6, wherein the second binding moiety comprises an antibody, nanobody, single-domain antibody (dAB), camelid nanobody (sdAb), single-chain variable fragment (scFv), or a Fab.

8. The method of any claim 7, wherein the second binding moiety comprises an anti-IgG nanobody, wherein the anti-IgG nanobody is fused to a Hia5 DNA methyltransferase or biologically active fragment thereof, and a maltose binding protein (MBP) tag.

9. The method of any one of claims 1-8, wherein the interaction is in a cell and the incubating of step (a) comprises incubating a plurality of cells.

10. The method of any one of claims 1-9, wherein the permeabilization of step (b) comprises contacting the cells with a nuclear membrane permeabilization agent and optionally a chromatin de-condensation agent.11 . The method of claim 10, wherein the nuclear membrane permeabilization agent comprises one or more detergents selected from the group consisting of Triton X-100, Tween 20, saponin, digitonin, or a combination thereof.

12. The method of claim 10, wherein the chromatin de-condensation agent is selected from the group consisting of heparin, polyglutamic acid (PGA), maltotriose per-O- sulfate (MTS), and methyl cellobiose sulfate (mCBS).

13. The method of claim 12, wherein the chromatin de-condensation agent is heparin and further wherein said heparin is deactivated by the addition of heparinase.

14. The method of any one of claims 1-13, wherein the dialysis membrane is selected from a group consisting of a dialysis button, a dialysis pouch, a sealed dialysis membrane, a membrane envelope, and a dialysis tubing.

15. The method of claim 14, wherein the dialysis membrane is an ultra-high molecular weight cutoff (UHMWCO) dialysis button comprising a resin capable of precipitating unbound enzyme.

16. The method of claim 14, wherein the dialysis button comprises a sealable ultra-high molecular weight cutoff (UHMWCO) dialysis button capable of holding the contents of steps (b)-(d).

17. The method of any one of claims 1-16, wherein the resin comprises magnetic beads and wherein the magnetic beads comprise anti- Maltose binding protein (MBP) antibodies.

18. The method of claim 15 or 16, wherein the ultra-high molecular weight cutoff is between 300 kDa and 1000 kDa.

19. The method of any one of claims 1-18, wherein the first binding moiety is an antibody.

20. The method of any one of claims 1-19, wherein the genomic DNA is selected from the group consisting of nuclear DNA and mitochondrial DNA.21 . The method of any one of claims 1-19, wherein the cell is selected from the group consisting of a bacterial cell, a eukaryotic cell, prokaryotic cell, a plant cell, an archaeal cell and a virus.

22. The method of claim 21 , wherein the cell is a mammalian cell.

23. The method of claim 22, wherein the cell is a human cell.

24. The method of any one of claims 1-23, wherein the biomolecule of interest is selected from the group consisting of a protein, a chromatin-binding small molecule, a RNA, and a RNA-DNA hybrid.

25. The method of claim 24, wherein the biomolecule is a RNA selected from the group consisting of ncRNA, tRNA, rRNA, snRNA, snoRNA, miRNA, mRNA, and TERC.

26. The method of claim 24, wherein the biomolecule is a protein selected from the group consisting of a nuclear lamina protein, a nucleolar protein, a transcription factor, a histone or histone or histone variant, centromere protein A, an intracellular scFV, a chromatin-modifying enzyme, an RNA polymerase, a DNA polymerase, a DNA helicase, a DNA repair protein, a Cas9 protein, a dCas9 protein, a zinc finger protein, a TALE protein, a CTCF protein, a cohesin protein, a synaptonemal complex protein, a telomere -binding protein, a centromere -binding protein, an outer kinetochore protein, a splicing protein and a chromatin remodeling protein.

27. The method of claim 9, wherein the plurality of cells is induced to express the protein of interest.

28. The method of claim 27, wherein the protein of interest is a recombinant protein and is expressed from an expression vector.

29. The method of any one of claims 1-28, wherein 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more biomolecule-genomic DNA interactions are determined.

30. The method of any one of claims 1-29, wherein the modifying genomic DNA of step (f) or (g) comprises modifying one or more nucleotides at one or more locations selected from the group consisting of (a) within 1-50 nucleotides of the genomic DNA binding site of the biomolecule, (b) topologically near the genomic DNA binding site of the biomolecule, and (c) both (a) and (b).31 . The method of claim 1 , wherein the incubating of step (f) comprises incubating in the presence of bovine serum albumin (BSA) and low salt conditions.

32. The method of any one of claims 1 -3, wherein the isolating and preparing the genomic DNA for sequencing of step (g) comprises high molecular weight DNA extraction.

33. The method of any one of claims 1 -3, wherein the sequencing of step (g) comprises long read or short sequencing.

34. A single vessel method to determine the genomic location of at least one biomolecule-genomic DNA interaction, said method comprising the steps of:(a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence;(b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest;(c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest;(d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of cutting genomic DNA;(e) precipitating excess or unbound enzyme by adding a dialysis membrane to the vessel, wherein the dialysis membrane contains a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme;(f) activating the enzyme of (d) under conditions that allow cutting of genomic DNA;(g) isolating and preparing the genomic DNA for sequencing, wherein said isolating comprises diffusing fragments of genomic DNA bound by the enzyme into the dialysis membrane and binding genomic DNA complex with the enzyme to the resin, and wherein said preparing comprises amplification of the DNA; and(h) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

35. A single vessel method to determine the genomic location of at least one biomolecule-genomic DNA interaction, said method comprising the steps of:(a) incubating in a vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence;(b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest;(c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest;(d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of cutting genomic DNA;(e) transferring the contents of steps (b)-(d) into a dialysis membrane;(f) incubating the dialysis membrane of (e) in the vessel, said vessel further comprising a buffer comprising a resin capable of precipitating unbound first moiety and / or second moiety conjugated to the enzyme,(g) activating the enzyme of (d) under conditions that allow cutting of the genomic DNA;(h) isolating and preparing the genomic DNA for sequencing, wherein said isolating comprises diffusing fragments of genomic DNA bound by the enzyme into the dialysis membrane and binding genomic DNA in complex with the enzyme to the resin, and wherein said preparing comprises amplification of the DNA; and(i) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

36. A method to determine the genomic location of at least one biomolecule- genomic DNA interaction, said method comprising the steps of:(a) incubating in a first vessel, cells comprising a biomolecule of interest under conditions that allow the biomolecule of interest to contact a genomic DNA sequence;(b) permeabilizing the cells in (a) under conditions that expose the genomic DNA bound by the biomolecule of interest;(c) contacting the biomolecule bound to genomic DNA with a first binding moiety capable of specifically binding to the biomolecule of interest;(d) contacting the first binding moiety with a second binding moiety capable of specifically binding to the first binding moiety, wherein said second binding moiety is conjugated to an enzyme capable of cutting genomic DNA;(e) transferring the contents of steps (b)-(d) into a dialysis membrane;(f) incubating the dialysis membrane of (e) in a second vessel, said vessel comprising a dialysis buffer capable of diluting out unbound first moiety and / or second moiety conjugated to the enzyme;(g) activating the enzyme of (d) under conditions that allow cutting of the genomic DNA;(h) isolating and preparing the genomic DNA for sequencing, wherein said preparing comprises amplification of the DNA; and(i) sequencing the genomic DNA under conditions that allow determining the location of the biomolecule-DNA interaction.

Images