Epigenetic profiling methods

Abstract

A method for analyzing DNA includes cleaving genomic DNA into DNA fragments, selectively functionalizing any unmethylated CpG sites present in the DNA with a linker containing a hydrolyzable moiety, and attaching a label to the linker to form labeled DNA fragments. The method further includes separating the labeled DNA fragments from any unlabeled DNA fragments, hydrolyzing the hydrolyzable moiety of the linker of the separated labeled DNA fragments to release the DNA fragments from the label, and sequencing the released DNA fragments.

Description

[Technical field]

[0001] The present invention relates to a method for epigenetic profiling, in particular a method for epigenetic profiling comprising selectively functionalizing unmethylated CpG sites of DNA with a linker comprising a hydrolyzable moiety. [Background technology]

[0002] Epigenetics is the study of genetic modifications to the genome that do not involve alterations in the DNA sequence. Such modifications include DNA methylation, histone modifications, and non-coding RNA-associated gene silencing. Epigenetic modifications play a crucial role in regulating gene expression in cells, most notably in eukaryotes.

[0003] DNA methylation occurs by the addition of a methyl (CH3) group to DNA. The most extensively characterized DNA methylation process is the addition of a methyl group to the 5-carbon of the cytosine ring, resulting in 5-methylcytosine (5mC). In somatic cells, 5-methylcytosine is primarily restricted to CpG (cytosine-phosphate-guanine) sites (also referred to as "CG" sites). When CpG sites in the promoter region of a gene are methylated, the expression of the gene is silenced. The addition of methyl groups to DNA is carried out by enzymes called DNA methyltransferases.

[0004] Alterations in the organization of epigenetic modifications have been linked to an increasing number of diseases. In recent years, there has been considerable research interest in investigating the role of epigenetic modifications in a variety of diseases, including cancer and heart disease, as well as in mechanisms of aging and human development. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Wojciechowski et al., Proc Natl Acad Sci USA, 2013 January 2; 110(1): 105-110 [Non-Patent Document 2] Darii et al., Molecular Biology 41, pp. 110-117 (2007) [Non-Patent Document 3] Kumar et al., Biochemistry (1992), 31 (36), 8648-8653 [Non-Patent Document 4] Hlz et al., Nucleic Acids Res. 26, pp. 1076-1083 (1998) [Non-Patent Document 5] Lukinavicius et al., Nucleic Acids Research, 40, 22 (2012) pp. 11594-11602 [Non-Patent Document 6] Sambrook J, Russell DW, eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Summary of the Invention [Problem to be solved by the invention]

[0006] There are several methods available to interrogate the epigenome, however, some of the current approaches suffer from uneven coverage, lack of specificity, harsh chemical treatments of DNA, and / or high cost.

[0007] The present invention was devised with these problems in mind. [Means for solving the problem]

[0008] According to a first aspect of the present invention there is provided a method for analysing DNA, for example for epigenetic profiling, the method comprising: (a) cleaving genomic DNA into DNA fragments; (b) selectively functionalizing any unmethylated CpG sites present in the DNA with a linker that contains a hydrolyzable moiety; and (c) attaching a label to the linker forming labeled DNA fragments by; - separating the labeled DNA fragments from any unlabeled DNA fragments; - hydrolyzing the hydrolyzable moieties of the linkers of the separated, labeled DNA fragments to release the DNA fragments from the labels; and - sequencing the released DNA fragments Includes.

[0009] It will be understood that the above steps (a), (b), and (c) can be performed in any order. For example, the label can be attached to the linker before the DNA is functionalized with the linker. The DNA can be functionalized with a linker (which may or may not already have a label attached) before or after cleaving the DNA. Thus, it will be understood that step (b) can be performed on genomic DNA or on DNA fragments.

[0010] In some embodiments, step (c) occurs before step (b). In some embodiments, step (a) occurs after step (b) or after step (c). In some embodiments, the method is performed in the order described, i.e., step (a), followed by step (b), followed by step (c).

[0011] In some embodiments, the step of forming the labeled DNA fragments is carried out by performing steps (a), (b), and (c) in the order described. Thus, in some embodiments, the method of the present invention comprises: - cleaving the genomic DNA into DNA fragments; - selectively functionalizing any unmethylated CpG sites present in the DNA fragments with a linker that includes a hydrolyzable moiety, thereby forming functionalized DNA fragments; - attaching a label to the linker of the functionalized DNA fragment, thereby forming a labeled DNA fragment; - separating the labeled DNA fragments from any unlabeled DNA fragments; - hydrolyzing the hydrolyzable moieties of the linkers of the separated, labeled DNA fragments to release the DNA fragments from the labels; and - sequencing the released DNA fragments Includes.

[0012] The genomic DNA may be present in a sample. The sample may be obtained from an animal, such as a human. The sample may further comprise a suitable diluent or buffer.

[0013] Cleavage of genomic DNA can be performed enzymatically, for example, using a restriction enzyme. Alternatively, genomic DNA can be cleaved into fragments using mechanical techniques, for example, sonication or shearing. In some embodiments, cleavage is performed using a restriction enzyme that generates sticky ends (i.e., overhangs or extensions of unpaired nucleotides at the ends of the resulting DNA fragments). Alternatively, a restriction enzyme that results in DNA fragments with blunt ends (both strands of the resulting DNA fragments terminate in a base-paired state) can be used. Any restriction enzyme that is insensitive to CpG methylation can be used. An example of a suitable restriction enzyme that produces sticky ends and is insensitive to CpG methylation is SaqAI, which cuts DNA at the T^TAA recognition site.

[0014] Optionally, the method further comprises isolating genomic DNA before cleavage. Genomic DNA can be isolated from cultured cells. Protocols for isolating genomic DNA from cells will be known by those skilled in the art. Genomic DNA isolation can be carried out using a commercially available kit (e.g., sold by Qiagen) according to the manufacturer's instructions.

[0015] In some embodiments, the hydrolyzable moiety comprises a Schiff base. In some embodiments, the Schiff base is an N-substituted hydrozone or an O-substituted oxime.

[0016] In some embodiments, the hydrolyzable moiety comprises a disulfide (SS) bond.

[0017] The linker may have the following general formula: [ka] [wherein FG represents a functional group containing a reactive center; Z represents a non-reactive group selected from one of an aliphatic or aromatic linkage; ABC represents a hydrolyzable moiety (e.g., a Schiff base moiety); Y represents a non-reactive group selected from an aliphatic or aromatic linkage; and U represents an unsaturated bond selected from one of an alkene, an alkyne, an aryl group, a carbonyl group, or a group containing one or two S=O bonds.

[0018] In some embodiments, the hydrolyzable moiety comprises one of the following structures: [ka] [In the formula, R x represents one of a hydrogen atom, a deuterium atom, an aromatic group, or an aliphatic group.

[0019] The step of selectively functionalizing any unmethylated CpG sites present in the DNA may comprise forming a covalent bond between the unmethylated CpG site and a reactive centre of a functional group of the linker.

[0020] In some embodiments, the covalent bond formed between the DNA and the reactive center can be, for example, a carbon-carbon bond, a carbon-nitrogen bond, a carbon-sulfur bond, or a carbon-oxygen bond. In some embodiments, the covalent bond is a carbon-carbon bond.

[0021] A covalent bond can be formed between the reactive center and position 5 of the cytosine ring of an unmethylated CpG site.

[0022] Methyltransferases (MTases) have emerged as important tools for the site-selective modification of DNA, RNA, and proteins. In nature, methyltransferase enzymes catalyze the highly specific transfer of a methyl group from an S-adenosyl-L-methionine cofactor to DNA or RNA. The introduction of a methyl group into these classes of biomolecules helps to regulate gene expression levels in cells.

[0023] In mTAG labeling, S-adenosyl-L-methionine cofactor analogues are employed, and the methyl group of the natural S-adenosyl-L-methionine cofactor is exchanged with a different moiety, such as a linker moiety. A methyltransferase enzyme can then be used to functionalize the target biomolecule with a different moiety using the modified cofactor. By manipulating the chemical structure of the naturally occurring S-adenosyl-L-methionine cofactor, it is possible to use this labeling process as a method for the covalent introduction of functional groups into biomolecules. The linker moiety can contain additional functionality, which can be used to further modify the biomolecule, for example, with labels, tags, or additional biomolecules. One of the most common applications explored using this methodology is the introduction of clickable groups into DNA for the introduction of fluorophores for mapping.

[0024] Thus, in some embodiments, the step of selectively functionalizing any unmethylated CpG sites in DNA (either genomic DNA or DNA fragments) with a linker may be performed using a DNA methyltransferase enzyme. In other words, the method of the invention may comprise mTAG labeling. The methyltransferase may be capable of selectively transferring a transferable group from an S-adenosyl-L-methionine (AdoMet) cofactor analogue to the unmethylated CpG sites of DNA. In such an embodiment, the transferable group of the S-adenosyl-L-methionine cofactor analogue constitutes the linker that functionalizes the DNA.

[0025] As known in the art, cytosine-5 methyltransferases can be engineered to direct the transfer of extended groups from AdoMet analogs to CpG sites in DNA. Thus, in some embodiments, DNA is functionalized using a methyltransferase that exhibits sensitivity to CpG methylation. In other words, the methyltransferase specifically methylates CpG sites. The methyltransferase can be a cytosine-5 methyltransferase. Suitable enzymes include M.HhaI, M.SssI, M.MpeI, M.TaqI, and variants thereof. M.MpeI can be obtained using the method described by Wojciechowski et al., Proc Natl Acad Sci USA, 2013-01-02; 110(1): 105-110. The preparation of M.SssI is described by Darii et al., Molecular Biology 41, 110-117 (2007). The purification of M.HhaI is described by Kumar et al., Biochemistry (1992), 31 (36), 8648-8653. The preparation of M.TaqI is described by Hlz et al., Nucleic Acids Res. 26, 1076-1083 (1998).

[0026] In some embodiments, DNA is functionalized using the CpG-specific cytosine-5 methyltransferase M.MpeI. In some embodiments, the methyltransferase is a double mutant (Q136A / N374A) of M.MpeI. These mutations facilitate the enzyme's use of AdoMet analogs, such as those described herein. Those skilled in the art will be able to engineer additional cytosine-5 methyltransferases for site-specific labeling of DNA using standard molecular biology techniques and the teachings of Lukinavicius et al., Nucleic Acids Research, 40, 22 (2012) 11594-11602.

[0027] In some embodiments, the S-adenosyl-L-methionine (AdoMet) analog has the general formula: [ka] [wherein R represents a transferable group (i.e., a linker); FG represents functional group; Z represents a non-reactive group selected from one of an aliphatic or aromatic linkage; ABC represents a hydrolyzable moiety; Y represents a non-reactive group selected from one of an aliphatic or aromatic linkage; U represents an unsaturated bond selected from one of an alkene, an alkyne, an aryl group, a carbonyl group, or a group containing one or two S=O bonds; and and k represents an integer of 1 or 2.

[0028] FG may be selected from one of an azide, alkyne, isothiocyanate, or isocyanate moiety, hi some embodiments, FG is an azide moiety.

[0029] AdoMet analogs can be associated with a counterion, which is a carbonate anion (CO 2- ), hydrogen carbonate (HCO3 -), tetrafluoroborate anion (BF4 - ), hexafluorophosphate anion (PF6 - ), acetate (OAc - ), trifluoroacetate anion, formate anion, halides (e.g., F - , Cl - , Br - , I - ), or a sulfonate anion.

[0030] Y may represent a non-reactive aliphatic or aromatic linkage containing 1-15 atoms, e.g., 2-10 or 3-5 atoms, in the backbone of the linker. In some embodiments, Y represents a non-reactive aliphatic or aromatic linkage containing 1-15 CH2 moieties, e.g., 2-10 or 3-5 CH2 moieties.

[0031] In some embodiments, Y is an alkyl linkage.

[0032] In some embodiments, Z comprises a polyether chain, optionally a polyethylene glycol chain comprising up to five monomers of ethylene glycol. Additionally or alternatively, Z may comprise an aromatic group, such as a CH(C=0)NH group.

[0033] The methods of the invention using linker molecules can be described as bioorthogonal.

[0034] The unsaturated moiety U at the β position to the trivalent sulfonium center is believed to enhance the reactivity of the linker towards alkylation of polynucleotide biomolecules using methyltransferase enzymes.

[0035] In some embodiments, U may represent an alkyne. Thus, in some embodiments, the AdoMet analog has the following general formula: [ka] has.

[0036] In alternative embodiments, U may represent an alkene. In embodiments, the AdoMet analogue has the following general formula: [ka] may have:

[0037] In some embodiments, the AdoMet analog has the general formula: [ka] wherein the hydrolyzable moiety is a Schiff base moiety that includes a C=NXCQ moiety; p represents an integer from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; or an integer from 2 to 10; or an integer from 3 to 5; for example, p can be 4; Q represents one oxygen atom or two hydrogen atoms independently bonded to a carbon center; U represents an unsaturated bond selected from the group consisting of an alkene, an alkyne, an aryl group, a carbon atom constituting a carbonyl group, and a sulfur atom constituting one or two S=O bonds; X represents either an oxygen atom or a nitrogen atom; Z represents a non-reactive group selected from one of an aliphatic or aromatic linkage; FG represents a functional group, which may be selected from, for example, one of an azide, alkyne, isothiocyanate, or isocyanate moiety; and and k represents an integer of 1 or 2.

[0038] In some embodiments, the AdoMet analog has the general formula: [ka] wherein the hydrolyzable moiety is a Schiff base moiety containing a -C=NNC=O bond; p represents an integer from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; or an integer from 2 to 10; or an integer from 3 to 5; for example, p can be 4; q represents an integer from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; or an integer from 2 to 10; or an integer from 3 to 5, for example q may represent 2 or 3; k represents an integer of 1 or 2; and FG represents a functional group, which may be selected from, for example, one of an azide, alkyne, isothiocyanate, or isocyanate moiety.

[0039] In some embodiments, the AdoMet analog has the general formula: [ka] wherein the hydrolyzable moiety is a Schiff base moiety containing a -C=NO- bond; p represents an integer from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; or an integer from 2 to 10; or an integer from 3 to 5; for example, p can be 4; wherein q represents an integer from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; or an integer from 2 to 10; or an integer from 1 to 5, for example q can be 2 or 3; k represents an integer of 1 or 2; and FG represents a functional group, which may be selected from, for example, one of an azide, alkyne, isothiocyanate, or isocyanate moiety.

[0040] In some embodiments, the AdoMet analog has the general formula: [ka] wherein k is 1 or 2, p is 4, and q is 2 or 3.

[0041] In some embodiments, the AdoMet analog has the general formula: [ka] wherein k is 1 or 2, p is 4, and q is 2 or 3.

[0042] In any of the above embodiments, k can be 1 or 2. In some embodiments, k is 2.

[0043] Attaching the label to the linker may include forming a covalent bond between the label and the reactive center of the functional group (FG) of the linker. In some embodiments, the covalent bond formed between the linker and the label may be a carbon-nitrogen bond.

[0044] It should be understood that the functional group (FG) can react with the label via a chemical reaction (e.g., click chemistry) or a chemoenzymatic reaction (e.g., in an enzyme-mediated reaction). Any suitable functional group that is bioorthogonal to the hydrolyzable moiety ABC can be selected as the functional group for the reaction. For example, suitable reactions involving a functional group and a label include click chemistry, cycloaddition, Staudinger reaction, epoxide ring-opening reaction, nucleophilic substitution, and / or nucleophilic addition.

[0045] In some embodiments, the functional group may be selected from one of a halide (e.g., an F, Cl, Br, or I atom), an unsaturated bond (e.g., an alkene, an alkyne), an azide, an activated ester, an activated carbonate, a carbamate, an epoxide, an isothiocyanate, or an isocyanate moiety.

[0046] In some embodiments, the functional group includes an azide or alkyne moiety, for example, for reacting with an alkyne or azide, respectively, on the label.

[0047] The label may include a second functional group that can react with the functional group of the linker molecule to form a covalent bond. Any suitable group that is bioorthogonal to the hydrolyzable moiety ABC may be selected as the second functional group for the reaction. In some embodiments, the second functional group is an alkyne or an azide.

[0048] The label may comprise any suitable molecule that allows labeled DNA fragments to be separated from unlabeled DNA fragments, for example, the label may comprise a ligand (such as a protein or antibody) that can be selectively captured by a capture agent (such as a protein receptor or further antibody).

[0049] Those skilled in the art will be aware of many possible types of label / ligand-capture agent interactions that can be utilized in the context of the present invention. These can be broadly classified according to the nature of the interaction. In some embodiments, the interaction between the label (or the ligand forming part of the label) and the capture agent is a covalent bioorthogonal coupling, such as azide-alkyne, amine-carboxylic acid, sulfhydryl-maleimide, alkoxyamine-aldehyde, or azide-amine coupling. For example, the label may contain a functional group that can interact with a functional group of the capture agent to form one of the covalent couplings mentioned above.

[0050] In some embodiments, the interaction between the label (or ligand) and the capture agent is non-covalent, eg, biotin-streptavidin, digoxenin-antidigoxenin, desthiobiotin-streptavidin.

[0051] In some embodiments, the label comprises or consists of a protein tag. Suitable protein tags include CLIP tags, SNAP tags, or maltose binding protein (which can be captured using amylose agarose).

[0052] In some embodiments, the label comprises biotin. The use of biotin is advantageous because it allows for the selective capture of labeled DNA using streptavidin or avidin as a capture agent.

[0053] In some embodiments, the label comprises a ligand conjugated to a moiety that comprises a second functional group, for example, the label may comprise biotin conjugated to an alkyne-containing chemical moiety to form a covalent bond with the azide functional group of the linker.

[0054] The click reaction between azides and alkynes is often catalyzed by a copper(I) catalyst in a process known as CuAAC (copper-catalyzed azide-alkyne cycloaddition). An alternative reaction that avoids the use of copper(I), which is toxic to biological systems, is the strain-promoted azide-alkyne cycloaddition (SPAAC) between azides and strained alkyne groups. Suitable strained alkyne groups include cyclooctyne, MOFO (monofluorinated cyclooctyne), DIFO (difluorinated cyclooctyne), DBCO (dibenzocyclooctyne, also abbreviated DIBO), and BARAC (biarylazacyclooctynone).

[0055] Thus, in some embodiments, the second functional group of the label comprises a strained alkyne group, such as DBCO. The label may further comprise a ligand conjugated to the strained alkyne group, such as biotin.

[0056] An example of a suitable biotin-DBCO conjugate has the following structure: [ka] Includes:

[0057] Separating the labeled DNA fragment from any unlabeled DNA fragments can include using a capture agent that selectively binds to the label of the labeled DNA fragment. The capture agent does not bind to the unlabeled DNA fragment. Since only the DNA fragments that contain unmethylated CpG are labeled, the method of the present invention allows the isolation of the unmethylated DNA sequence present in genomic DNA.

[0058] It will therefore be understood that the capture agent will be selected by one of skill in the art according to the type of label used, for example, if the label includes a protein, an antibody specific for that protein may be selected as the capture agent.

[0059] In some embodiments in which the label comprises biotin, the capture agent may comprise avidin or streptavidin.

[0060] The capture agent can be immobilized, for example, on a solid support, such as a bead, a column, or an array.

[0061] In some embodiments, the capture agent comprises or is streptavidin immobilized on magnetic beads (i.e., magnetic beads with streptavidin covalently coupled to the surface). In such embodiments, DNA fragments labeled with a label containing biotin can be separated from unlabeled DNA by mixing the labeled DNA fragments with streptavidin-coated beads. The streptavidin capture agent binds to the biotin label, thereby binding the labeled DNA fragments to the beads. The captured DNA fragments are then separated from the unlabeled DNA fragments using a magnet. Those skilled in the art will be aware of other ligand-capture agent pairs and methods suitable for separating labeled DNA.

[0062] After separation of the labeled DNA fragments, the hydrolyzable portion of the linker is hydrolyzed to remove the label and release the DNA fragments. Hydrolysis can be carried out by treating the separated and labeled DNA fragments with a hydrolysis agent. It will be understood that the hydrolysis agent will be selected by the skilled artisan according to the nature of the hydrolysis moiety. In some embodiments, hydrolysis is carried out using a hydrolysis agent, such as an acid or hydroxylamine, and / or by heating. For example, hydrolysis can be carried out by treatment with hydroxylamine in an ammonium acetate buffer solution.

[0063] In some embodiments, the method further comprises ligating the released DNA fragments together before sequencing.Ligating DNA fragments together to generate longer sequences may be advantageous when DNA is to be sequenced using a certain sequencing platform, for example, nanopore sequencing.However, it will be understood that various sequencing platforms that do not require ligation steps can be used.

[0064] Ligation of the released DNA fragments can be carried out enzymatically using DNA ligase, such as T4 DNA ligase. Ligation protocols are considered to be well known by those skilled in the art and are described by Sambrook J, Russell DW, eds. (2012) Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Alternatively, ligation can be carried out using the protocol described herein. The ligation step of the method results in a sequence of DNA that is composed of many fragments. It will be understood that DNA fragments are ligated randomly, so that in the sequence produced, the fragments are in a different order from the order in which they appear in the original genomic DNA.

[0065] Optionally, the DNA sequence is amplified prior to sequencing. Amplification can be carried out after ligation. Several methods are known for amplifying DNA in vitro, including PCR (polymerase chain reaction), rolling circle amplification, and strand displacement.

[0066] In some embodiments, the DNA sequences are amplified by PCR, which not only removes the portions of the linker molecules that remain attached to the DNA fragments after hydrolysis, but also increases the amount of DNA available for sequencing.

[0067] As known in the art, PCR involves subjecting a reaction mixture to repeated heating and cooling cycles, including denaturation at a relatively high temperature (e.g., around 95° C.), primer annealing at a relatively low temperature (e.g., 50-65° C.), and chain extension at a moderate temperature (e.g., around 70-80° C.). The reaction mixture contains the DNA sequence to be amplified (e.g., ligated DNA sequences), a pair of primers (single-stranded oligonucleotides having sequences complementary to a region of the target DNA or a PCR adapter sequence), deoxyribonucleoside triphosphates (dNTPs), and a DNA polymerase enzyme (e.g., Taq polymerase) in a suitable buffer. PCR protocols are well known to those of skill in the art, and commercial PCR kits are widely available.

[0068] In some embodiments, amplification is performed before hydrolysis of the hydrolyzable portion of the linker to release DNA fragments from the label.In some embodiments, the labeled DNA sequence is amplified while being bound to the capture agent immobilized on a solid support.Surprisingly, the inventors have found that DNA polymerase can process DNA with biotin label even when the biotin label is not located at the end of DNA but toward the center of the DNA strand.

[0069] In some embodiments, before amplification and after any ligation, the method further comprises adding PCR adapters to the ends of the released DNA fragments or ligated DNA sequences. As known in the art, PCR adapters are DNA oligonucleotides that contain sequences to which PCR primers can bind.

[0070] Prior to the addition of PCR adaptors, DNA fragments or ligated DNA sequences can be modified. For example, the ends of the fragments or sequences can be blunt-ended and optionally dA-tailed. The addition of a single adenine advantageously improves the efficiency of ligation to the adaptor sequence compared to ligation of blunt ends. Sequencing adaptors can be added to the modified ends of the DNA sequences by ligation, for example using T4 DNA ligase. Such techniques are believed to be well known to those skilled in the art and are described herein.

[0071] In some embodiments, the method further comprises adding a sequencing adapter to the end of the released DNA fragment or the ligated DNA sequence, optionally after amplification. As known in the art, a sequencing adapter is a DNA oligonucleotide of known sequence that is attached to the end of a DNA sequence prior to sequencing. The adapter sequence may include one or more of the following: a sequencing primer site (i.e., a binding site for a sequencing primer); a platform-specific sequence (e.g., a sequence that allows binding to a flow cell); a sample index (a short sequence that allows multiplexing, typically 6-10 bases); and a molecular identifier (a unique code for each molecule in the library). Adapter sequences are commercially available. Alternatively, adapter sequences may be custom designed for a particular application.

[0072] Sequencing can be carried out using any suitable technique, for example, next-generation sequencing (NGS), also known as high-throughput sequencing.NGS technology includes Illumina (Solexa) sequencing, Roche 454 sequencing, massively parallel signature sequencing (MPSS), polony sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, and microfluidic systems.

[0073] In some embodiments, sequencing is performed using nanopore DNA sequencing, a technique in which a sequence is determined by measuring the change in charge as a DNA strand passes through a nanopore.

[0074] Thus, the method of the present invention provides affinity-based enrichment of fragmented DNA using methyltransferase-directed DNA modification that targets only unmethylated CpG sites of the genome. Subsequent labeling and capture leads to the isolation of the unmethylated fraction of the genome. This fraction can then be religated for efficient sequencing. This method allows for faster and more cost-effective epigenetic profiling compared to direct sequencing of the entire native genome. Furthermore, by functionalizing the DNA fragments with linkers that contain hydrolyzable moieties, the label used to isolate the unmethylated fraction can be efficiently removed for downstream processing.

[0075] It will be understood that any of the embodiments described herein may be combined with any other embodiment, unless stated otherwise.

[0076] Embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings. [Brief description of the drawings]

[0077] [Figure 1] 1 is a reaction scheme for the formation of S-adenosyl-L-methionine cofactor analogs. [Diagram 2] FIG. 1 provides an overview of a method according to an embodiment of the present invention. [Diagram 3] FIG. 2 illustrates a molecular structure produced by a method according to an embodiment of the present invention. [Figure 4] FIG. 1 is a plot showing the efficiency of capture of biotinylated DNA fragments onto streptavidin-coated beads, where the DNA fragments contain 0, 1, 2, 3, or 4 unmethylated CpG sites. [Diagram 5] FIG. 1 is a plot showing the efficiency of capture and release of biotinylated DNA fragments using streptavidin-coated beads, where the DNA fragments were generated by shearing human genomic DNA. [Figure 6] 1 is a graph showing the lengths of DNA sequences created by ligating and amplifying the released DNA fragments. [Figure 7] A plot of aligned read length versus sequenced read length. [Figure 8] Alignment of sequencing reads of gene promoter regions obtained by the method of the present invention (SUURF IDs 1, 2, and 3) with sequencing reads obtained by MeDIP. [Figure 9] FIG. 1 shows the results of Illumina sequencing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] Referring to FIG. 1, a reaction scheme for the formation of S-adenosyl-L-methionine cofactor (AdoMet) analogs is shown. EXAMPLES

[0079] Synthesis of S-adenosyl-l-methionine cofactor analogues Synthesis of precursor 1 Synthesis of 8-hydroxyoct-6-ynoic acid 7 A solution of 6-heptynoic acid (2 g, 15.87 mmol) was made in dry THF (42 ml) under argon, to which HMPA (34.9 mmol, 6.13 ml) was added and the solution was cooled to -78°C. To this was added nBuLi (1.6 M in hexanes, 34.9 mmol, 21.8 ml) dropwise while maintaining the temperature below -60°C. The solution was then warmed to -40°C and stirred for 1 h. After 1 h, paraformaldehyde (1.47 g, 47.6 mmol) was added through a powder funnel under argon flow. The reaction mixture was then warmed to 45°C for 4 h. After the reaction, the mixture was quenched with 1 M HCl to pH 4-5 and extracted with EtOAc. The solvent was then dried and the EtOAc was removed by rotary evaporation to give the crude product. Purification was completed using flash column chromatography (silica gel, Hex:EtOAc, 6:4): Yield=68%, Rf=0.27 (Hex:EtOAc, 6:4); 1H NMR (300 MHz, DMSO-d6) δ 12.03 (s, 1 H), 5.03 (s, 1H), 4.02 (d, J = 2.6 Hz, 2H), 2.29 - 2.14 (m, 4H), 1.63 - 1.50 (m, 2H), 1.50 - 1.39 (m, 2H); MS: m / z [MH] = 155.46.

[0080] Synthesis of tert-butyl 2-(8-hydroxyoct-6-ynoyl)hydrazine-1-carboxylate 8 8-Hydroxyoct-6-ynoic acid 7 (1.35 g, 8.65 mmol) and tert-butyl carbazate (1.4 g, 10.38 mmol) were dissolved in 2:1 THF:HO (13.5:6.75 ml). To this was added EDC.HCl (1.87 g, 9.52 mmol) slowly over 15 min. The mixture was allowed to stir for 3 h and then extracted with EtOAc. The organic layer was washed with 0.1M HCl, water, and brine, then the organic layer was collected, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to afford the product as a white solid: yield=63%; 1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.66 (s, 1H), 5.04 (t, J = 5.9 Hz, 1H), 4.02 (dt, J = 5.9, 2.2 Hz, 2H), 2.19 (tt, J = 7.1, 2.2 Hz, 2H), 2.06 (t, J = 7.2 Hz, 2H), 1.58 (p, J = 7.3 Hz, 2H), 1.50 - 1.32 (m, 12H); 13C NMR (101 MHz, DMSO) δ 172.01, 84.36, 80.94, 79.42, 49.59, 33.06, 28.53, 28.08, 24.70, 18.24; MS: m / z [M+Na] = 294.15.

[0081] Synthesis of tert-butyl 2-(8-bromooct-6-ynoyl)hydrazine-1-carboxylate 1 A solution of tert-butyl 2-(8-hydroxyoct-6-ynoyl)hydrazine-1-carboxylate 8 (300 mg, 1.11 mmol) was made in dry DCM (3.33 ml) and cooled on ice. Triphenylphosphine (437 mg, 1.67 mmol) was added and dissolved, and once dissolved, tetrabromomethane (552 mg, 1.67 mmol) was added slowly. The reaction was then allowed to reach room temperature and stirred for 1 hour. After the reaction, the solvent was removed under reduced pressure and the crude mixture was purified by flash column chromatography (silica gel Hex:EtOAc, 7:3): Yield=55%; Rf=0.15 (Hex:EtOAc 7:3); 1H NMR (300 MHz, DMSO-d6) δ 9.48 (s, 1H), 8.67 (s, 1H), 4.21 (t, J = 2.3Hz, 2H), 2.27 (tt, J = 6.9, 3.4Hz, 2H), 2.06 (t, J = 7.4Hz, 2H), 1.65 - 1.31 (m, 13H); 13C NMR (101 MHz, DMSO) δ 171.4, 155.2, 87.7, 78.9, 76.3, 54.9, 39.5, 32.5, 28.0, 27.3, 24.1, 17.9, 17.2; MS: m / z [M+Na] = 355 / 357.08.

[0082] Synthesis of precursor 4 Synthesis of 7-bromo-hept-1-yne 9 A solution of 6-heptyn-1-ol (5 g, 44.6 mmol) was made in dry DCM (60 ml) and cooled on ice. To this was added triphenylphosphine (17.6 g, 67 mmol) and once completely dissolved tetrabromomethane (22.2 g, 67 mmol) was added slowly. The reaction mixture was allowed to reach room temperature and stirred for 1 hour. After completion, the solvent was removed under reduced pressure. Hexane was added to the crude to form a white suspension. The hexane fraction was filtered, collected and then the solvent was removed. An oily residue remained, which was purified by flash column chromatography with hexane: Yield=91%, Rf=0.45(hexane);%); □max(neat) / cm-1 540(C-Br);1H NMR (300 MHz, DMSO-d6) δ 3.53 (t, J = 6.7 Hz, 2H), 2.75 (t, J = 2.7 Hz, 1 H), 2.23 - 2.10 (m, 2H), 1.89 - 1.74 (m, 2H), 1.50 - 1.43 (m, 4H).

[0083] Synthesis of 8-bromooct-2-yn-1-ol 10 A solution of 7-bromohept-1-yne 9 (20.56 mmol, 3600 mg) was prepared in dry THF (12.3 ml) under argon and cooled to -78°C. To this was added a solution of nBuLi in hexanes (1.6 M, 13 ml) dropwise while maintaining the temperature below -60°C. The reaction mixture was then warmed to 0°C in an ice bath at which point paraformaldehyde (1718 mg, 55.5 mmol) was added under a flow of argon and stirred for 30 minutes. The mixture was then warmed to room temperature and allowed to stir, maintaining the temperature below 30°C until the exothermic reaction ceased. The mixture was then heated to 45°C for 2 hours. Upon completion, the reaction was extracted with ether and sat. NH4Cl. The organic layer was collected and the solvent was removed under reduced pressure to yield the crude product as an oil. Upon drying, purification was completed by flash column chromatography (silica gel, hexane:ethyl acetate, 9:1). The product was then collected as a colorless oil: Yield=55%, Rf=0.15 (Hex:EtOAc 9:1), 1H NMR (300 MHz, DMSO-d6) δ 5.04 (t, J = 5.7 Hz, 1H), 4.03 (dt, J = 5.5, 2.1 Hz, 2H), 3.54 (t, J = 6.7 Hz, 2H), 2.20 (m, 2H), 1.88 - 1.75 (m, 2H), 1.52 - 1.40 (m, 4H).

[0084] Synthesis of tert-butyl ((8-hydroxyoct-6-yn-1-yl)oxy)carbamate 11 To a solution of N-Boc hydroxylamine (890 mg, 6.55 mmol) in DMF (4.3 ml) was added 8-bromooct-2-yn-1-ol 10 (1200 mg, 5.85 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (1000 mg, 6.55 mmol). The solution was stirred at 50° C. for 20 h. Upon completion, the reaction was extracted with DCM and 15% citric acid solution. The organic phase was dried, collected and the solvent was removed under reduced pressure. A colorless oil was collected as the crude product. This was further purified by flash column chromatography (silica gel, hexane:ethyl acetate, 8:2). The product was collected as a colorless oil: Yield=73%, Rf=0.27; 1H NMR (300 MHz, DMSO-d6) δ 9.91 (s, 1H), 5.03 (t, J = 5.9 Hz, 1H), 4.02 (dt, J = 5.9, 2.2 Hz, 2H), 3.66 (t, J = 6.2 Hz, 2H), 2.17 (tt, J = 6.7, 1.7 Hz, 2H), 1.40 (m, 15H); MS: m / z [M+H] = 258.2.

[0085] Synthesis of tert-butyl ((8-bromooct-6-yn-1-yl)oxy)carbamate 4 A solution of tert-butyl ((8-hydroxyoct-6-yn-1-yl)oxy)carbamate 11 (1 g, 3.89 mmol) was made in dry DCM (5.2 ml) and cooled on ice. To this was added triphenylphosphine (1.53 g, 67 mmol). Once completely dissolved, tetrabromomethane (1.94 g, 67 mmol) was added slowly. The reaction mixture was allowed to reach room temperature and stirred for 1 hour. After completion, the solvent was removed under reduced pressure. Purification was completed using flash column chromatography (silica gel, hexane:ethyl acetate, 8:2): yield=67%, Rf 0.52 (Hex:EtOAc, 8:2); λmax(neat) / cm-1 1712 (C=O), 607 (C-Br); 1H NMR (300 MHz, DMSO-d6) δ 9.90 (s, 1 H), 4.21 (t, J = 2.4 Hz, 2H), 3.66 (t, J = 6.2 Hz, 2H), 2.25 (tt, J = 6.9, 2.4 Hz, 2H), 1.40 (m, 15H); 13C NMR (101 MHz, DMSO) δ 156.04, 87.85, 79.37, 76.22, 75.05, 39.52, 28.05, 27.64, 27.04, 24.76, 18.06, 17.25; MS: m / z [M+Na] = 342.35 / 344.35, [M-tBuOH] = 246.38 / 248.38.

[0086] General Coupling Procedure Precursors 1, 4 were reacted with S-adenosyl-L-homocysteine ​​under acidic conditions to give reversible and reprogrammable Boc-protected AdoMet derivatives.

[0087] A solution of S-adenosyl-l-homocysteine ​​(15 mg, 0.04 mmol) was made in a 1:1 mixture of formic acid and acetic acid (300 μl). Then, precursor 1 or 4 (tert-butyl 2-(8-bromooct-6-ynoyl)hydrazone-1-carboxylate or tert-butyl ((8-bromooct-6-yn-1-yl)oxy)carbamate) (1.2 mmol, 30 equiv.) was added dropwise on ice. The reaction mixture was warmed to 35° C. and allowed to stir overnight. After stirring overnight, the reaction mixture was extracted with diethyl ether and the aqueous layer was collected and dried by lyophilization: MS: m / z [M+H]=638 (2), [M+H]=624 (5).

[0088] Cofactor deprotection The AdoMet analogs were deprotected under acidic conditions to expose the hydrazone or alkoxyamine moieties. The crude products were dissolved in TFA (400 μl) and allowed to stir at room temperature for 2 h. After the reaction, the acid was removed under a flow of argon.

[0089] Cofactor purification Any excess precursor was removed by purification.

[0090] Both diastereomers of the deprotected cofactor could be separated by HPLC, a separation not possible at a later stage.

[0091] The crude reaction mixture was then dissolved in water (2 ml). Purification of the AdoMet analogues was carried out by preparative reversed-phase HPLC (ACE 5 C-18 25 x 2.12 cm) eluting with a 20 mM ammonium acetate pH 5.5 water (A) / MeOH (B) gradient at a flow rate of 10 ml / min. Gradient system: 3-30% B in 30 min, 30-97% B over 30 min, 5 min hold at 97% B, program stop. Retention times: hydrazide iso.1 = 17.51 ​​min, iso.2 = 18.73 min, hydroxylamine iso.1 = 25.47 min, iso.2 = 28.24 min: MS: m / z [M+H] = 538 (2), [M+H] = 524 (5).

[0092] The deprotected AdoMet derivatives decompose slowly via multiple pathways, especially after freeze-drying, giving additional peaks at higher retention times.

[0093] Aldehyde Coupling To mitigate decomposition, the AdoMet derivative was reacted with commercially available benzaldehyde immediately after purification by HPLC to minimize side reactions due to the nucleophilic nature of the hydrazone and alkoxyamine moieties.

[0094] To the collected HPLC fractions, Ald-PEG3-N3 (1.2 equiv.) was added and rotated at room temperature for 30 min. The fractions were then dried by lyophilization. Upon drying, the solid was dissolved in 100 μl of 0.1% acetic acid and stored at -20°C. The concentration was determined by UV absorption analysis with ε260=15.400 dm-3 mol-1 cm-1: MS: m / z [M+H]=867 (3), [M+H]=856 (6).

[0095] The resulting AdoMet analogs contain a reactive terminal azide that can be readily conjugated with a wide range of functional groups, while condensation of aldehydes with hydrazones or alkoxyamines introduces dynamic functionality that can be reversibly functionalized.

[0096] A slight excess of aldehyde (1.2 equiv.) was employed to ensure complete functionalization of the deprotected intermediate.

[0097] No degradation of the freeze-dried AdoMet analogues was observed.

[0098] 2, the method according to the invention is used for epigenetic profiling of genomic DNA (10), for example human genomic DNA. In a first step (A), the genomic DNA is digested into DNA fragments using a restriction enzyme, for example SaqAI. The DNA fragments generated by enzymatic digestion include fragments that are not methylated at CpG sites (12) and fragments that are methylated at CpG sites (14).

[0099] Step (B) involves methyltransferase-directed unmethylated CpG functionalization. In this step, unmethylated CpG sites present in DNA fragments (12) are functionalized using a methyltransferase enzyme (such as M.MpeI) and the S-adenosyl-L-methionine (AdoMet) analog AdoHCY-8-HY (shown in FIG. 3A). The methyltransferase transfers a transferable group, i.e., a linker (16), from a cofactor to the 5-position of the cytosine of the unmethylated CpG site, thereby generating a functionalized DNA fragment (18).

[0100] As shown in FIG. 3A, the linker that is transferred from the cofactor to the DNA fragment contains a hydrolyzable C═N moiety (Schiff base) and a terminal azide (N3) group.

[0101] In step (C), the functionalized DNA fragment (18) is reacted with diazobiotin-DBCO to form the labeled DNA fragment (20). A click reaction between the terminal azide of the linker and the alkyne of the DBCO moiety forms a covalent bond between the linker and the biotin label, resulting in the structure shown in Figure 3B.

[0102] In step (D), the labeled DNA fragments (20) are captured using streptavidin-coated beads (22). The beads (22) are then washed to remove any non-specifically bound (i.e., unlabeled) DNA. The captured DNA fragments (24) are then released by hydrolyzing the hydrolyzable moieties (step (E)), giving the structure shown in Figure 3C.

[0103] The released fragments are then religated together in a random fashion using DNA ligase (step (F)). This creates a long sequence of DNA (26) composed of many ligated DNA fragments that contain CpG sites that were not methylated in the original genomic DNA sequence. Optionally, the ligated DNA is amplified by PCR to remove the linkers (step (G)). Finally, the amplified DNA (28) is sequenced. EXAMPLES

[0104] Capture and release of control DNA fragments containing different numbers of capture sites methodology PCR amplification of CpG site-containing fragments DNA fragments (approximately 150 bp) containing 0, 1, 2, 3, or 4 CpG sites were generated by PCR amplification of fragments of the lambda genome (NEB) using Q5® High-Fidelity DNA Polymerase (NEB) according to the manufacturer's instructions. The following amplification program was used: 98°C for 30 s, 30 cycles of 98°C for 10 s, 61°C for 30 s, 72°C for 20 s, and a final extension at 72°C for 2 min. After amplification, DNA was purified using 2x AMPure XP beads and eluted in 100 μl EB (10 mM Tris-HCl, pH 8.5). DNA concentration was quantified using a Qubit™ 4 Fluorometer (Thermo Fisher) using the dsDNA BR Assay Kit and size measured using a High Sensitivity D5000 ScreenTape (Agilent) on a TapeStation 2200.

[0105] CpG capture analysis 750 ng of PCR fragments containing CpG sites (0, 1, 2, 3, or 4) were mTAG labeled in a 35 μl reaction containing 10× cutsmart buffer (3.5 μl) (NEB), 500 μM AdoHcy-8-Hy cofactor (1.17 μl), M.MpeI enzyme (double mutant (Q136A / N374A), 2.5 μl) (1.7 mg / ml), and water. Samples were incubated at 37° C. for 1 hour. After this, 1 μl of proteinase K (800 units / ml) (NEB) was added and samples were incubated at 50° C. for 1 hour. Next, 1 μl of diazobiotin-DBCO (Jena bioscience) was added and samples were incubated at 37° C. for 1 hour at 1000 rpm. Samples were then purified using 2x AMPure XP beads (washed twice with 500 μl of 80% ethanol) and eluted in Tris Buffer A (10 mM Tris, 1 mM NaCl, pH 7.5). DNA concentrations were quantified using a Qubit™ 4 Fluorometer and dsDNA HS Assay Kit (Thermo Fisher).

[0106] 5 μl of Dynabeads™ MyOne™ Streptavidin C1 beads (Thermo Fisher) for each sample were washed twice with an equal volume of Tris Buffer A. The wash was removed and 250 ng of biotin-labeled PCR DNA fragment in 5 μL Tris Buffer A was added to 5 μl of washed streptavidin beads and incubated at RT, 1000 rpm for 20 min. Samples were placed on a magnet and the supernatant was removed and saved. The beads were then washed twice with 5 μl Tris Buffer A and the washes saved. The percentage of captured DNA was calculated using a Qubit™ 4 Fluorometer (Thermo Fisher) with the dsDNA HS Assay Kit.

[0107] result The results of this experiment are shown in Figure 4. It demonstrates the ability to capture control fragments of DNA containing various numbers of CpG sites. DNA that does not possess a capture site (0CG) was not captured, while DNA that contains one (1CG), two (2CG), three (3CG), or four (4CG) capture sites was efficiently captured, respectively. This demonstrates the specificity and efficiency of the method. EXAMPLES

[0108] Epigenetic research using human DNA methodology Genomic DNA extraction Human genomic DNA was extracted from cultured GM12878 human cells (Coriell: GM12878). Cell cultures were performed using Epstein-Barr virus (EBV) transformed B lymphocyte cultures from the GM12878 cell line, grown in RPMI-1640 medium supplemented with 2 mM L-glutamine, 15% FBS, and incubated at 37°C. Genomic DNA was extracted using the QIAGEN Genomic-tip 500 / G kit (Qiagen) according to the manufacturer's instructions.

[0109] Digestion of genomic DNA Human genomic DNA (NA12878) was digested using Anza™ 64 SaqAI (Thermo Fisher) in an 80 μL reaction containing 4 μg DNA, 8 μL buffer, 4 μl SaqAI enzyme, and water. The reaction was then incubated at 37° C. for 1 hour. Fragmented DNA was cleaned up using a QIAquick PCR Purification Kit (Qiagen) and eluted in 30 μl EB (10 mM Tris-HCl, pH 8.5). If additional DNA was required, the reaction was repeated. DNA concentration was quantified using a Qubit™ 4 Fluorometer and dsDNA HS Assay Kit (Thermo Fisher) and sized using a High Sensitivity D5000 ScreenTape (Agilent) on a TapeStation 2200.

[0110] mTAG-directed functionalization of human DNA and enrichment of unmethylated CpG-containing fragments mTAG labeling and biotin tagging of human DNA fragments were performed in triplicate. Each reaction contained 1.5 μg SaqAI digested human DNA, 7 μl 10× cutsmart buffer, 2.33 μl AdoHcy-8-Hy cofactor (500 μM), 5 μL M.MpeI enzyme (double mutant (Q136A / N374A)) (1.7 mg / ml), and water to a final volume of 70 μl. Samples were incubated at 37° C. for 1 hour. To these reactions, 2 μl Proteinase K (800 units / ml) (NEB) was added and incubated at 50° C. for 1 hour. Then, 2 μl Diazobiotin-DBCO (Jena bioscience) was added and the reactions were incubated at 37° C., 1000 rpm for an additional hour. Each sample was purified using 2x AMPure XP beads (washed twice with 1000 μl 80% ethanol) and eluted in 30 μl Tris Buffer A (10 mM Tris, 1 mM NaCl, pH 7.5). DNA concentration was quantified using a Qubit™ 4 Fluorometer and dsDNA HS Assay Kit (Thermo Fisher).

[0111] 20 μl of Dynabeads™ MyOne™ Streptavidin C1 beads (Thermo Fisher) for each sample were washed twice with an equal volume of Tris buffer A. 1 μg of labeled and biotin-tagged DNA in 30 μl Tris buffer A from each sample was added to 20 μl of washed streptavidin beads and incubated at RT, 1000 rpm for 20 min. Samples were then placed on a magnet, the supernatant removed, and the beads washed twice in 20 μl Tris buffer A. To release the captured DNA from the beads, 80 μl of release buffer (11.2 mM ammonium acetate (pH 6.5), 1 M NaCl) was added along with 20 μl of 0.85 M hydroxylamine solution (170 mM final) and incubated at 50° C., 1000 rpm for 1 h. The released DNA was then purified using 2x AMPure XP beads and eluted in 14 μl EB (10 mM Tris-HCl, pH 8.5). DNA concentration was quantified using a Qubit™ 4 Fluorometer (Thermo Fisher) with a dsDNA HS Assay Kit and sized using a High Sensitivity D5000 ScreenTape (Agilent) on a TapeStation 2200.

[0112] Ligation and PCR amplification of released fragments After quantification and sizing of released human DNA fragments, approximately 170-190ng of DNA remained in each 10.5μl sample. To this, an equal volume (10.5μl) of Anza™ T4 DNA Ligase Master Mix (Thermo Fisher) was added and the samples were incubated for 1h at RT. Each sample was then purified using 2x AMPure XP beads and eluted in 13μl EB (10mM Tris-HCl, pH 8.5). DNA concentration was quantified using a Qubit™ 4 Fluorometer (Thermo Fisher) with a dsDNA HS Assay Kit and sized using a Genomic DNA ScreenTape (Agilent) on a TapeStation 2200.

[0113] After this, the remaining 10 μl of each sample was end-repaired and dA-tailed using the NEBNext® Ultra™ II End Repair / dA-Tailing Module (NEB). The DNA was then cleaned up using 2×AMPure XP beads and eluted in 15 μl EB (10 mM Tris-HCl (pH 8.5)). To the 15 μl of end-repaired and dA-tailed DNA, 10 μl PCA (Oxford Nanopore Technologies (ONT)) and 25 μl Blunt / TA Ligase Master Mix were added and the samples were incubated at 25° C. for 1 hour. The DNA was then purified using 2×AMPure XP beads and eluted in 12 μl EB. The DNA concentration was quantified using a Qubit™ 4 Fluorometer (Thermo Fisher) with the dsDNA HS Assay Kit.

[0114] Next, 3x 50 μl PCR reactions were prepared containing 4.5 μl (10 ng) PCA-ligated DNA from the previous step, 1.5 μl dNTPs (10 mM), 2 μl PRM (Oxford Nanopore Technologies (ONT)), 10 μl LongAmp® Taq reaction buffer (NEB), 2 μl LongAmp® Taq DNA Polymerase (NEB), and 30 μl water. The following amplification program was used: 94°C for 2 min, 21 cycles of 94°C for 30 sec, 62°C for 15 sec, 65°C for 15 min, and a final extension at 65°C for 15 min. DNA was purified using 2x AMPure XP beads, DNA concentration was quantified using a Qubit™ 4 Fluorometer (Thermo Fisher) with a dsDNA BR Assay Kit, and size measured using a Genomic DNA ScreenTape (Agilent) on a TapeStation 2200.

[0115] Library preparation and MinION sequencing 1 μg of religated and PCR amplified DNA in 50 μl nuclease-free water was end-repaired and dA-tailed using the NEBNext® Ultra™ II End Repair / dA-Tailing Module (NEB). Samples were then purified using 2×AMPure XP beads and eluted in nuclease-free water. Sequencing adapters (AMX) were then ligated using NEBNext Quick T4 DNA Ligase (NEB) according to the manufacturer's instructions (1D genomic DNA by ligation (SQK-LSK109) (Oxford Nanopore Technologies (ONT)).

[0116] Each library was loaded onto an R9.4.1 flow cell (FLO-MIN106D) according to the manufacturer's instructions (ONT) and sequenced for 48 hours using standard parameters specified for the library preparation protocol. Base calling was performed using Guppy (2.0.10) with parameters based on the library preparation method.

[0117] Read Alignment Map the sequenced reads to minimap2 with the "-ax map-ont -K 500M" option. 1 were used to map against the human genome reference (hg19).

[0118] result Initial digestion of human genomic DNA with SaqAI enzyme was found to produce DNA fragments approximately 150 bp in length. After mTAG functionalization of the DNA fragments using AdoHcy-8-Hy cofactor and labeling with diazobiotin-DBCO, the labeled DNA fragments were captured on streptavidin-coated beads. Consistent capture of approximately 24% of DNA was observed across all three samples. In addition, highly efficient recovery (approximately 95%) of the captured DNA from the streptavidin beads was achieved in a single step for all samples (Figure 5).

[0119] The released DNA fragments from each sample were then randomly glued together to form long fragments of DNA. Figure 6 shows the successful religation and PCR amplification of the captured and released DNA fragments in all three replicates of the experiment.

[0120] The released and religated human DNA from each sample was then sequenced using a MinION nanopore sequencing instrument. Because each sequencing read consists of many short fragments of DNA randomly ligated / glued together, a publicly available algorithm was used to align each individual fragment in the read to the genomic location from which it originated.

[0121] Evidence supporting the correct alignment of individual DNA fragments within each read can be seen in Figure 7. This figure shows the "sequenced read length" compared to the "aligned read length", which is the length of each sequencing read (each read consists of multiple small fragments glued together). If the short fragments within each sequencing read were aligned to their correct location on the genome, one would expect the "aligned read length" to be shorter than the "sequenced read length", which is exactly what is shown in Figure 7. This demonstrates that the alignment algorithm can align the short fragments within each sequencing read.

[0122] To ensure that the correct sites were captured, the acquired sequencing data was compared with MeDIP sequencing data. MedIP is a method for capturing methylated sites in the genome, in contrast to the method of the present invention, which captures unmethylated sites. Therefore, the method of the present invention should not capture the same positions in the genome as MeDIP.

[0123] Figure 8 shows a comparison of the sequencing reads obtained using the present method (SUURF ID1, ID2, and ID3) with the sequencing reads obtained using MeDIP. The region shown in the box is a gene promoter sequence that is known to be unmethylated. It can be seen that in this region, there is an increase in sequencing reads from the SUURF sample (capture of unmethylated DNA) and a decrease in sequencing reads from the MedIP sample (capture of methylated DNA). This demonstrates that the method of the present invention can be used to successfully capture and sequence unmethylated regions of the genome. EXAMPLES

[0124] Capture and release of human head and neck cancer for Illumina sequencing Head and neck squamous cell carcinoma (HNSCC) (VU40T) DNA was fragmented to 150 bp in the following reaction: 50 μl DNA (5.6 μg), 6.5 μl 10× Fragmentase Reaction Buffer v2, 3 μl NEBNext dsDNA Fragmentase enzyme (M0348), 3.5 μl 200 mM MgCl2, 2 μl nuclease-free water to a final volume of 65 μl. The reaction was incubated at 37° C. for 35 min. To stop the reaction, 35 μl of 400 mM EDTA was added. The DNA was then purified using 2.5× AMPure XP beads (Beckman) and size checked using a 1% agarose gel and prestained with GelRed® (Biotium) (120V for 55 min).

[0125] Next, a labeling reaction was prepared containing 19.4 μl of fragmented head and neck cancer DNA (800 ng) (150 bp), 10 μl M.MpeI enzyme (double mutant (Q136A / N374A)) (stock 1.7 mg / ml), 7 μl 10× CutSmart® Buffer (NEB), 2.33 AdoHcy-8-Hy cofactor (500 μM final), and 31.27 μl nuclease-free water to a final volume of 70 μl, and the reaction was then incubated at 37° C. for 1 hour. Next, 4 μl of Proteinase K (800 units / ml) (NEB) was added and the sample was incubated at 50° C. for an additional hour. Finally, 4 μl of Sulfo-DBCO-Biotin conjugate (15 mM stock) (Jena Bioscience) was added and it was incubated at 37° C. for 1 hour. Samples were then purified using 2.5x AMPure XP beads (Beckman) and DNA concentrations were quantified using a Qubit™ 4 Fluorometer and dsDNA HS Assay Kit (Thermo Fisher).

[0126] After this, 600 ng of fragmented and biotinylated DNA in 50 μl Tris Buffer A (10 mM Tris, 1 mM NaCl, pH 7.5) was incubated with 60 μl of washed Dynabeads™ MyOne™ Streptavidin C1 beads (Thermo Fisher) for 20 min at RT. The beads were then washed twice with 100 μl Tris Buffer A to remove any non-specifically bound DNA. The captured DNA was released from the beads using 90 μl of release buffer (11.2 mM ammonium acetate (pH 6.5), 1 M NaCl) and 10 ul of 0.85 M hydroxylamine solution (170 mM final) at 50° C., 1200 rpm for 1 h. The released DNA was then purified using 2.5x AMPure XP beads (Beckman) and DNA concentration was quantified using a Qubit™ 4 Fluorometer and dsDNA HS Assay Kit (Thermo Fisher). Samples were then prepared for Illumina sequencing using the KAPA HyperPrep Kit according to the manufacturer's instructions.

[0127] FIG. 9 shows a comparison of data from a genome browser showing the number of reads that mapped to the region containing the CpG island at the start of the MLM1 gene, for our unmethylome chemistry (top), showing the location of unmethylated CpG sites, and MeDIP data (antibody-based capture) (middle and bottom), showing complementary capture of methylated regions of the genome. EXAMPLES

[0128] Amplification of captured DNA Labeling of unmethylated genomic DNA in fixed cells using methyltransferase enzymes and AdoMet analogues 1×10 6Single cells (MCF7 or MCF10A) were seeded in a 10 cm dish and incubated for 24 hours. The cells were then fixed with 5 ml of chilled MeOH / AcOH (95:5) at -20°C for 10 minutes and washed twice with PBS. The fixed cells were then resuspended in 5 ml of the following solution in 1x CutSmart buffer: Taq-37.4μl M.TaqI(WT) (1.1mg / ml), 4μM AdoHcy-6-N3 M.MpeI-9 0.9 μl M.MpeI (double mutant (Q136A / N374A)) (6.9 mg / ml), 65 μM AdoHcy-6-N3 The cells were incubated with 100 μM sulfo-DBCO-biotin in PBS for 1 h at 37° C., washed twice with PBS, and incubated with 100 μM sulfo-DBCO-biotin in PBS overnight at RT. The cells were then washed three times with PBST. 1 ml of PBST was added and the cells were scraped. DNA was purified using a QIAGEN Genomic-tip 20 / G. The biotin-labeled DNA was then resuspended in 100 mM Tris-HCl pH 8.5 and sonicated to 150 bp.

[0129] In vitro labeling of unmethylated genomic DNA using methyltransferase enzymes and AdoMet analogues 2 μg of extracted genomic DNA from MCF7 or MCF10A cells was incubated with 0.5 μl of M.Taq (1.1 mg / ml) or M.MpeI (6.9 mg / ml) and AdoHcy-6-N3 (4 μM and 65 μM, respectively) in a total volume of 20 μL of 1× CutSmart buffer at 37° C. for 1 h. 3 μL of proteinase K was added and the samples were incubated at 50° C. for 1 h, followed by incubation with 2 mM DBCO-sulfo-biotin at 37° C. for 1 h. Biotin-labeled DNA was then purified using the GenElute Bacterial Genomic DNA Kit. DNA was eluted twice in 10 mM Tris-HCl pH 8.5.

[0130] Library construction (following the procedure from Ponnaluri, VKC et al., Genome Biol 18, 122 (2017)) 1 μg of DNA was end-repaired, dA-tailed, and ligated using the NEBNext Ultra8482 II DNA Library Prep Kit. Without further purification, the ligation product was mixed with 50 μL of streptavidin magnetic beads (Invitrogen 65001, blocked overnight at 4° C. using 0.1% chilled fish gelatin in 1×PBS) in 1 mL of B&W buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl). Biotin-labeled DNA was captured by streptavidin magnetic beads for 2 hours at 4° C. using end-over-end rotation. The beads were washed four times with B&W buffer + 0.05% Triton X-100, followed by one wash with TE + Triton X-100. The beads were resuspended in 40 μL of nuclease-free water, and 4 μL was used for library amplification using standard PCR. The presence of the biotin label was found to have no effect on the amplification process.

Claims

1. 1. A method for analyzing DNA, the method comprising: - forming labeled DNA fragments, (a) cleaving genomic DNA into DNA fragments; (b) selectively functionalizing any unmethylated CpG sites present in the DNA with a linker that comprises a hydrolyzable moiety, the hydrolyzable moiety comprising an imine moiety, an oxime moiety, or a hydrazone moiety; and (c) attaching a label to the linker forming labeled DNA fragments by; - separating the labeled DNA fragments from any unlabeled DNA fragments using a capture agent that selectively binds to the label of the labeled DNA fragments; - hydrolyzing the hydrolyzable moieties of the linkers of the separated, labeled DNA fragments to release the DNA fragments from the labels; and - sequencing the released DNA fragments Including, the step of selectively functionalizing any unmethylated CpG sites in the DNA with the linker is carried out using a DNA methyltransferase enzyme capable of selectively transferring a transferable group from an S-adenosyl-L-methionine analogue to the unmethylated CpG sites of the DNA, the transferable group constituting the linker; The S-adenosyl-L-methionine analog has the following general formula: 【Chemistry 1】 [In the formula, R represents a transferable group constituting the linker; FG represents a functional group; Z represents a non-reactive group selected from one of an aliphatic or aromatic linkage; ABC represents said hydrolyzable moiety; Y represents a non-reactive group selected from one of an aliphatic or aromatic linkage; U represents an unsaturated bond selected from one of an alkene, an alkyne, an aryl group, a carbon atom constituting a carbonyl group, and a sulfur atom constituting one or two S=O bonds; and k represents an integer of 1 or 2. The method comprising:

2. The method of claim 1, wherein step (c) is performed before step (b).

3. 3. The method of claim 1 or claim 2, wherein step (a) is performed after step (b) or after step (c).

4. 4. The method of claim 1, wherein the DNA methyltransferase is a cytosine-5 methyltransferase.

5. The method of claim 4, wherein the cytosine-5 methyltransferase is M.MpeI, and optionally the DNA methyltransferase is a double mutant (Q136A / N374A) of M.MpeI.

6. 6. The method of claim 1, wherein the hydrolyzable moiety comprises a Schiff base.

7. 7. The method of claim 1, wherein Z comprises a polyether chain.

8. The method of claim 7, wherein the polyether chain is a polyethylene glycol chain containing up to five monomers of ethylene glycol.

9. 9. The method of claim 1, wherein FG is selected from one of an azide, an alkyne, an isothiocyanate, or an isocyanate moiety.

10. The S-adenosyl-L-methionine analog has the following general formula: 【Chemistry 2】 10. The method according to claim 1 , comprising the steps of:

11. The S-adenosyl-L-methionine analog has the following general formula: 【Chemistry 3】 10. The method according to claim 1 , comprising the steps of:

12. The linker molecule has the general formula: 【Chemistry 4】 wherein the hydrolyzable moiety is a Schiff base moiety that includes a C=NXCQ moiety; p represents an integer from 1 to 15; Q represents an oxygen atom or one of two hydrogen atoms independently bonded to the carbon center; X represents either an oxygen atom or a nitrogen atom; Z represents a non-reactive group selected from one of an aliphatic or aromatic linkage; U represents an unsaturated bond selected from the group consisting of an alkene, an alkyne, an aryl group, a carbon atom constituting a carbonyl group, and a sulfur atom constituting one or two S=O bonds; k represents an integer of 1 or 2; and FG represents a functional group], 12. The method according to any one of claims 1 to 11.

13. The linker molecule has the general formula: 【Chemistry 5】 wherein the hydrolyzable moiety is -C=NNC=O; p represents an integer from 1 to 15; q represents an integer from 1 to 15; k represents an integer of 1 or 2; and FG represents a second functional group], 13. The method of any one of claims 1 to 10 or 12.

14. The linker molecule has the general formula: 【Chemistry 6】 wherein the hydrolyzable moiety is a -C=NO- bond; p represents an integer from 1 to 15; q represents an integer from 1 to 15; k represents an integer of 1 or 2; and FG represents a second functional group], 13. The method of any one of claims 1 to 10 or 12.

15. 15. The method of claim 13 or claim 14, wherein FG is an azide moiety, p is 4, and q is 2 or 3.

16. The method of claim 14 or claim 15, wherein k is 2.

17. 17. The method of claim 1, wherein the step of attaching a label to the linker comprises forming a covalent bond between a reactive center of a functional group of the linker and the label.

18. 18. The method of any one of claims 1 to 17, wherein the label comprises a ligand conjugated to a moiety that includes a second functional group capable of reacting with a functional group of the linker molecule to form a covalent bond.

19. 20. The method of claim 18, wherein the label comprises biotin conjugated to an alkyne-containing moiety.

20. 20. The method of any one of claims 1 to 19, wherein separating the labeled DNA fragments from any unlabeled DNA fragments comprises using an immobilized capture agent that selectively binds to the label.

21. The method of claim 20, wherein the capture agent comprises avidin or streptavidin.

22. 22. The method of any one of claims 1 to 21, further comprising the step of ligating the released DNA fragments together prior to sequencing.

23. 23. The method of any one of claims 1 to 22, wherein the DNA is sequenced using nanopore sequencing.

24. 24. The method of any one of claims 1 to 23, further comprising amplifying the DNA prior to sequencing.

25. 25. The method of any one of claims 1 to 24, wherein the cleavage of the genomic DNA is carried out using a restriction enzyme.

Images