GB2523919A

GB2523919A - Mapping cytosine modifications

Info

Publication number: GB2523919A
Application number: GB1508094.8A
Authority: GB
Inventors: Shengxi Guan; Nan Dai; Zhenyu Zhu; Aine Quimby; Janine Borgaro; Ivan R Correa Jr
Original assignee: New England Biolabs Inc
Current assignee: New England Biolabs Inc
Priority date: 2012-11-06
Filing date: 2013-11-04
Publication date: 2015-09-09
Anticipated expiration: 2033-11-04
Also published as: GB2523919B; GB201508094D0

Abstract

Preparations are disclosed comprising Î²-glycosyltransferase; UDP-glucosamine and/or a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose; and an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI. The preparation may also contain an oxidizing agent and/or a reducing agent. Also claimed are methods for differentiating 5-(Î²-glucosyloxymethyl) cytosine from 5-(Î²-2-glucosaminyloxymethyl) cytosine in a nucleic acid comprising reacting the nucleic acid with an endonuclease capable of cleaving a nucleic acid at a glucosylated nucleotide but not at a glucosaminylated nucleotide. Also claimed are methods for placing off switches and on switches for endonuclease cleavage of a nucleic acid containing modified nucleotides comprising reacting the nucleic acid alternatively with UDP-glucosamine and UDP-glucose. Related kits are also claimed.

Description

MAPPING CYTOSINE MODIFICATIONS

BACKGROUND

5-hydroxymethylcytosine (5-hmc), 5-formylcytosine (5-fC), and 5-carboxycytosine (5-caC) were recently identified in mammalian brain and embryonic stem cells as products of the oxidation of 5-methylcytosine (5-mc) by cytosine oxygenases. The biological functions of 5-hmC, 5-fC, and 5-caC are not completely understood; however, several lines of evidence suggest that 5-hmC is involved in epigenetic regulation and DNA demethylation. Iterative oxidation of 5-h mc by cytosine oxygenase enzymes yields 5-fC and 5-caC which are hypothesized to be intermediates in the DNA demethylation process. Several challenges are associated with the identification these biologically modified nucleobases in genomic DNA samples due to their low abundance and temporal fluctuation. Mapping and quantifying 5-mC, 5-hmC, 5-fC, and 5-caC at the DNA level is, therefore, important for unraveling their role in the dynamics of gene expression and regulation.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide a variety of reagents, kits and methods for selectively altering and identifying modified nucleotides in a nucleic acid such as DNA. The modified nucleotides that can be identified include, for example, 5-mC, 5-hmC, 5-fC and 5-caC. The methods, reagents and kits of the present invention permit not merely the determination that a modified cytosine residue is present, but also permit the discrimination among or between different types of modified nucleotides, such as 5-mC and 5-hmC, or between 5-hmC and 5-fC. In this way, the invention can be used to elucidate the precise state of a modified nucleic acid, which may be a genome or genome fragment, for example.

In some embodiments, the discrimination among or between different oxidation states of a nucleotide is facilitated by alternately glucosylating or s glucosaminylating various modified nucleotides. The glucosylated or glucosaminylated forms can be distinguished based on their performance in various assays, such as by their differential sensitivity to certain restriction endonucleases. In this way, alternately reacting the nucleic acid with a UDP-GIcN (Uridine diphosphate glucosamine or UDP-Glucosamine) substrate io and a UDP-Glc (Uridine diphosphate glucose or UDP-Glucose) substrate, places off and on switches for endonuclease cleavage of a nucleic acid containing modified nucleotides. By detecting the presence of cleavage-sensitive sites, the modified nucleotides can be located and identified.

In embodiments, the term glucosylation (and any form of glucosylation such as "glucosylating" or "glucosylated") refers to the incorporation of a glucosyl moiety from UDP-Glc into the 5-hydroxy position of 5-hmC via the action of a glycosyltransferase to produce 5-gmc (5-glucosyloxymethylcytosine). Other names in common use for 5-gmC include glucosyl-5-hydroxymethyl-cytosi ne, glucosyl-5-hydroxymethylcytosine, glucosyl-oxy-5-methylcytosine, 5-glucosylhydroxymethylcytosi ne. In the present invention, the term glucosylation (and any form of glucosylation such as "glucosylating" or "glucosylated") may also refer to the incorporation of an azido modified glucosyl moiety from UDP-Azido-Glc (for example, UDP- 6-Azido-GIc or UDP-6-N3-Glc) into the 5-hydroxy position of 5-hmC via the action of a glycosyltransferase to produce a N3-5-gmc (for example, 6-azide-glucosyl-5-hydroxymethylcytosine or 6-N3gmC). In the present invention, the term glucosaminylation (and any form of glucosaminylation such as "glucosaminylating" or "glucosaminylated") refers to the incorporation of a glucosaminyl moiety from UDP-GIcN into the 5-hydroxy position of 5-hmC via the action of a glycosyltransferase to produce 5-gNmC (5-glucosaminyloxymethylcytosine). Other names for 5-gNmC include glucosaminyl-5-hydroxymethyl-cytosine, glucosaminyl-5- hydroxymethylcytosine, 5-glucosyihydroxymethylcytosine, a minoglucosyl-5- hydroxymethyl-cytosine, aminoglucosyl-5-hydroxymethylcytosine, and 5-aminoglucosyihydroxymethylcytosine. If the glycosyltransferase is an inverting glycosyltransferase such as T4 3-glucosyltransferase (BGT or 3GT or beta-GT) the product is formed with a beta glycosydic linkage (for example, 5-3-gmC, 5-3-6-N3gmC, 5-3-6-gNmC, and 5-3-2-gNmC). If the glycosyltransferase is a retaining glycosyltransferase such as T4 a-glucosyltransferase (AGI or aGI or alpha-GT) the product is formed with an alpha glycosydic linkage (for example, 5-a-gmC).

Specifically, for example, embodiments permit the differentiation of 5- 3-glucosyloxymethylcytosine (5-3-gmC) from 5-13-2-glucosaminyloxymethylcytosine (5-i3-2-gNmC) in a nucleic acid, by reacting the nucleic acid with an endonuclease capable of cleaving a nucleic acid at a glucosylated nucleotide but not at an glucosaminylated nucleotide. One suitable endonuclease is AbaSI. Other useful endonucleases include AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, and PxyI, or or one of the ZZYZ proteins or its variants described in US Patent Application Publication No. 2012/0301881, the complete disclosure of which is hereby incorporated by reference. Accordingly, one of these endonucleases, or a polypeptide at least]O% (e.g. at least 75%, at least B0%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or lOO% identical to one of those endonucleases or an active fragment thereof) can be used to selectively cleave a target nucleic acid. By controlling the conversion of glucosylation and glucosaminylation of various forms of naturally occurring modified nucleotides, the modified nucleotides can be located and identified in a manner distinguishing their original forms, such as methylcytosine, hydroxymethylcytosine, formylcytosine, a nd/or carboxycytosine.

Accordingly, in one aspect embodiments provide methods for s selectively altering modified nucleotides in a nucleic acid containing hydroxymethylated nucleotides and other modified nucleotides such as, 5-fC or 5-caC. The methods generally include reacting a first subset of the modified nucleotides (e.g. 5-hmC) in the nucleic acid with UDP-GIcN in the presence of a 3-glycosyltransferase, such as T4-3-glucosyltransferase, to convert hydroxymethylated nucleotides in the nucleic acid to glucosaminylated nucleotides.

In some embodiments, the methods include the subsequent step of reacting the nucleic acid with a reducing agent, such as NaBH4, and a UDP derivative, such as UDP-Glc or UDP-azido-glucose, to convert a second subset of nucleotides in the nucleic acid to glucosylated or azido-glucosylated nucleotides. The reducing agent promotes the reduction of a nucleotide in a higher oxidation state, such as 5-fC or 5-caC, to a hydroxymethylated nucleotide, which is then glucosylated or azido-glucosylated in a reaction typically catalyzed by a glycosyltransferase such as an a-glycosyltransferase or a -glycosyltransferase. The glycosyltransferase may or may not be the same glycosyltransferase used to catalyze the previous conversion of hydroxymethylated nucleotides to glucosaminylated nucleotides. The method can optionally differentiate pre-existing 5-hmC in a nucleic acid from newly formed as a result of the reduction of 5-fC or 5-caC, for example. When a LJDP-azido-glucose such as UDP-6-azido-glucose is used, 5-hmC in the nucleic acid can be converted to a -6-azide-glucosyl-5-hydroxymethylcytosine in the presence of a 13- glycosyltransferase; Further derivatization of the azido moiety via azide-alkyne Huisgen cycloaddition using a copper(I) catalyst ("click chemistry") or via copper-free variants (for example, using strained cyclooctyne derivatives) can then optionally be used to label the nucleotide, such as with a biotin label, permitting the subsequent use of avidin to selectively isolate the labeled nucleotide.

s In other embodiments, following the conversion of hydroxymethylated nucleotides in the nucleic acid to glucosaminylated nucleotides, the nucleic acid is reacted with an oxidizing agent, a UDP-Glc derivative, and a glycosyltransferase to convert a second subset of modified nucleotides in the nucleic acid to glucosylated nucleotides. Suitable oxidizing agents include io those capable of oxidizing 5-mC to 5-hmC, such as mYOX1, a TET enzyme, or an inorganic oxidizing agent such as KRuO4. The reaction with UDP-Glc is typically conducted in the presence of a glycosyltransferase such as an a-glycosyltransferase or a 3-glycosyltransferase. These methods can, for example, differentiate pre-existing 5-hmC in a nucleic acid from 5-hmc is generated from a 5-mC precursor.

In any of these methods, whether incorporating a reducing agent or an oxidizing agent, an endonuclease can be used to characterize the reaction products. Typically, the endonuclease is specific for the glucosylated (or azidoglucosylated) nucleotides, and not for glucosaminylated nucleotides, La, the endonuclease has a higher enzymatic activity for a glucosylated or azidoglucosylated nucleic acid that it has for the same nucleic acid with an glucosaminylated nucleotide at the same position(s). Endonucleases that may be used include, for example, AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, and/or PxyI. An adapter molecule can then be ligated to the cleaved end of the endonuclease reaction product, facilitating the subsequent purification, amplification or sequencing of the nucleic acid.

Accordingly, an embodiment provides a method for differentiating a 5-mC from 5-hmC in a genome or genome fragment. The genome or genome fragment may, for example, be mammalian in origin, such as a human genome or genome fragment. The method includes reacting the isolated genome or genome fragment containing S-mC and 5-hmC with (i) UDP-GIcN in the presence of a glycosyltransferase catalyzing transfer of glucosamine to the 5-hmC; (ii) oxygenating any existing 5-mC residues to 5-hmC by the action of TEl or mYOX1; (iii) reacting the newly created 5-hmC sites with UDP-Glc in the presence of a glycosyltransferase catalyzing transfer of glucose to the 5-hmC; (iv) cleaving the glucosylated template with a modification-dependent endonuclease that recognizes at least one of the modified nucleotides; and (v) differentiating the original S-mC from the 5-hmC by an altered cleavage pattern.

In this embodiment, the oxygenation of any existing 5-mC residues to 5-hmC can be done in the presence of UDP-Glc and a glycosyltransferase to catalyze the transfer of glucose to 5-hmC as it is being formed from 5-mC.

Alternatively, reaction conditions such as pH of the oxygenation reaction can be selected to optimize the yield of 5-hmC. See, for example, Attorney Docket No. NEB-364-US, "Compositions and Methods for Oxygenation of Nucleic Acids Containing 5-Methylpyrimidine," filed on the same date as the present application and hereby incorporated by reference in its entirety.

In another embodiment, the invention provides a method for differentiating a 5-mC from one or more of its oxidation products in a genome or genome fragment containing 5-mC and 5-hmC. The method includes reacting the isolated genome or genome fragment with UDP-2-glucosamine in the presence of a 3-glycosyltransferase (BGT) catalyzing the transfer of 2-glucosamine to the 5-hmC; reacting the isolated genome or genome fragment with mYOXI. or TET or a chemical oxidizing agent and optionally with a reducing agent; cleaving the template with a modification dependent endonuclease that is capable of selectively cleaving a 5-hmC and not a 5-glucosaminated hydroxymethylcytosine; and differentiating the 5-mc from one or more of its oxidation products by an altered cleavage pattern.

Embodiments also provide preparations useful for converting methylcytosine or an oxidized nucleotide, such as 5-fC or 5-caC, or to a s glucosylated nucleotide. The preparations include a reducing agent, such as NaBH4, or an oxidizing agent, such as mYOX1, a TET enzyme, or an inorganic oxidizing agent such as KRuO4; a glycosyltransferase, such as an a-glycosyltransferase or a 3-glycosyltransferase; a UDP-GIcN or a UDP derivative, such as UDP-Glc or UDP-azido-glucose.

io Embodiments also provide preparations useful for modifying and selectively cleaving nucleic acids. The preparations include a glycosyltransferase and an endonuclease having an amino acid sequence at least 95% identical to an enzyme such as AbaSI, AbaAT, AbaCT, AbaDI, AbaTI, AbaUT, AcaPI, and/or PxyT. The preparations also include (a) UDP-GIc and an oxidizing agent, or (b) UDP-GIcN. Where UDP-GIcN is included, the glycosyltransferase may be a BGT to catalyze the transfer of glucose to hydroxymethylated pyrimidine residues. Where UDP-Glc and an oxidizing agent are included, the oxidizing agent, which may be a methylcytosine oxygenase such as mYOX1 or a TET enzyme or may be an inorganic oxidizing agent such as KRu04, promotes the conversion of methylcytosine residues in a nucleic acid to hydroxymethylcytosine, which can be glucosylated by the combination of UDP-Glc and the glycosyltransferase, and can be recognized by the endonuclease.

Embodiments also provide kits useful for making these preparations and practicing these methods. For example, kits for modifying formylcytosine or carboxycytosine residues in a nucleic acid can include a reducing agent, such as sodium borohydride, permitting the reduction of formylcytosine or carboxycytosine residues to hydroxymethylcytosine; a glycosyltransferase (such as a 3-glycosyltransferase); and a UDP derivative, such as LJDP-Glc and/or UDP-azidoglucose (such as UDP-6-azidoglucose), permitting the transfer of a sugar or modified sugar to the hydroxymethylcytosine. These kits may also include an oxidizing agent (e.g., a methylcytosine oxygenase such as mYOX1 or a TET enzyme or an inorganic oxidizing agent such as KRuO4) to promote the conversion of methylcytosine residues in a nucleic acid to hydroxymethylcytosine; UDP-GIcN; a restriction endonuclease (e.g., having an amino acid sequence at least 95% identical to AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, in and/or PxyI); or any combination of the above.

Kits for selectively modulating the susceptibility of modified nucleic acid residues to cleavage can include UDP-Glc; UDP-GIcN; a 3-glycosyltransferase; and a reducing (e.g. sodium borohydride) or oxidizing agent (e.g., a methylcytosine oxygenase such as mYOX1 or a TET enzyme or is an inorganic oxidizing agent such as KRuO4).

Kits useful for modifying and selectively cleaving nucleic acids can include, for example, a glycosyltransferase and an endonuclease having an amino acid sequence at least 95% identical to an enzyme such as AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUr, AcaPI, and/or PxyI. These kits can also include (a) UDP-Glc and an oxidizing agent, and/or (b) UDP-GIcN Some embodiments of the kits or preparations of the present invention include an optional nucleic acid, such as a nucleic acid that is to be modified, that is undergoing modification, and/or a control nucleic acid. Accordingly, a nucleic acid, if present, may include 5-hmC, 5-gmC, 5-gNmC, 5-fC, 5-caC, 5-mC, or any combination of the above.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows that AbaSI recognizes 5-(3-glucosyloxymethyl)cytosine (5-13-gmC) but not 5-(3-2-glucosaminyloxymethyl)cytosine (5-3-2-gNmC) with high specificity as compared to 5-mC and C. The different forms of cytosine modification (panels a-g) were digested by a 10-fold serial dilution of AbaSI enzyme (lane 1 to lane 7, lane 8 is undigested control).

Figure 2 depicts the relative selectivity of a variety of endonucleases for nucleic acid substrates containing 5-3-gmC ("3-glucosylated hmC"), 5-a- g mC ("a-glucosylated h mC"), 5-hydroxymethylcytosine ("hmC"), 5-methylcytosine ("mC"), or unmodified cytosine ("C").

io Figure 3 depicts selected cytosine modifications that can be achieved through the addition of glucose, modified glucose, or glucosamine residues by T4-BGT alone or in combination with 3-glucosyl-a-glycosyltransferase from bacteriophage T6 ("T6-BGAGT").

Figure 4 depicts one example of an assay for detecting and/or is mapping 5-mC.

Figure 5 depicts two examples of assays for detecting and/or mapping 5-fC. The assay in Figure SA includes a step involving glucosylating hydroxymethylcytosine residues newly-formed from the reduction of 5-fC.

The assay in Figure 56 includes a step involving azidoglucosylating hydroxymethylcytosine residues newly-formed from the reduction of 5-fC.

Figure 6 depicts an example of an assay for detecting and/or mapping 5-caC.

Figure 7 depicts LC-MS analyses of a nucleic acid containing 5-fC (top trace) and the same nucleic acid after treatment with 100 mM sodium borohydride (middle trace) or with sodium borohydride, UDP-Glc, and 14-BGT (bottom trace).

DETAILED DESCRIPTION OF EMBODIMENTS

Endonucleases have been identified from bacteria and more will undoubtedly be discovered using routine BLAST searches based on the s present disclosure that are capable of preferentially cleaving 5--gmC compared to C, 5-mC and 5-3-2-gNmC. In one example, the ZZYZ family members for example AbaSI (see for example WO 2011/091146), isoschizomers and mutants thereof preferentially cleave 5-3-gmC compared to C, 5-mC and 5-3-2-gNmC. For example, AbaSI has cleavage activity at 5-3-gmC that is 500 fold greater than 5-mC and 5-a-gmC. 5-3-gmC is the product of BGT mediated transfer of glucose from UDP-Glc to 5-hmC.

The specificity of AbaSI is demonstrated in the data shown in Figure 1.

AbaSI cleaves recognizes nucleic acids containing certain cytosine modifications, cleaving them a short distance from those modifications.

is Ten-fold serial dilutions of AbaSI were combined with a PCR product containing cytosine ("C," panel a), 5-methylcytosine ("5-mC," panel 6), 5-hydroxymethylcytosine ("5-hmC," panel C), 5-(-glucosyloxymethyl)cytosine ("5-j3-gmC," panel D), 5-(j3-2-glucosami nyloxymethyl)cytosine ("5-13-2-gNmC," panel E), 5-(3-6-glucosaminyloxymethyl)cytosine ("5-13-6-gNmC," panel F), or 5-( 3-6-azidoglucosyloxymethyl)cytosine ("5-3-6-N3gmC," panel C). The PCR products containing 5-3-gmC, 5-3-2-gNmC, 5-3-6-gNmC, or 5- 13-6-N3gmC were generated by treatment of 5-hmC PCR DNA (panel c) with T4-13-glucosyltransferase (BGT) and the corresponding UDP-sugar (uridine diphospho-glucose (UDP-Glc), UDP-2-glucosamine (UDP-2-GIcN), UDP-6-glucosamine (UDP-6-GIcN), and UDP-6-N3-Glc, respectively). The results show that AbaSI does not digest C and 5-mC even at high concentrations (panels a and b) whereas AbaSI can digest 5-13-gmC even at very low concentrations (panel d). With respect to the three glucosylated or glucosaminated cytosines, AbaSI can digest 5--6-gNmC and 5--6-N3gmC (panels f and g) more efficiently than it digests 5-hmC (although less efficiently than it digests 5--gmC. In contrast, it does not digest 5--2-gNmC (panel e).

s Other endonucleases that differentiate between various forms of modified cytosine are also available, such as other ZZYZ family members described in WO 2011/091146 and enzymes described in Borgaro eta!.

(2013) "Characterization of the 5-hydroxymethylcytosine-specific DNA restriction endonucleases," Nucleic Acids Research, doi: 10.1093/nar/gktlO2, the entire disclosures of each of which are incorporated herein by reference. Several endonucleases that discriminate among various cytosine modifications are described in Figure 2. For example, PvuRtslI cleaves nucleic acids containing hydroxymethylcytosine or glucosyloxymethylcytosine far more efficiently than it cleaves nucleic acids containing only methylcytosine or unmodified cytosine. AbaSI, as described in the preceding paragraph, cleaves nucleic acids containing 5-3-gmC more efficiently than it cleaves nucleic acids containing 5-hmC, which are nevertheless cleaved more efficiently than those nucleic acids containing only 5-mC or unmodified cytosine. Like AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, and PxyI all demonstrate increased specificity for nucleic acids containing 5--gmC compared to nucleic acids containing only 5-hmC.

Accordingly, for any of these enzymes 3-glucosylation of hydroxymethylcytosine can be used as an "on switch" to promote cleavage near those positions, whereas 3-glucosylation could be used as an "off switch" for enzymes such as PvuRtslI, AbaBGI, BbiDI, BmeDI, or PatTI. On the other hand, a-glucosylation of 5-hmC generates a more efficient substrate for PvuRtslI, and could be used as an "on switch" to target cleavage events to those locations in a nucleic acid.

It is expected that the use of endonucleases with preferential specificity for a specific modified nucleotide over other modified and unmodified nucleotides can be detected using the method described herein for 5-mC, 5-hmc, 5-fC, and 5-cac. In combination with the cofactor LJDP-GIc, this system enables sequencing different epigenetic states of 5-mC and greatly enhances the ability to determine the epigenetic modification at a single base resolution level.

An embodiment of the method relies on modifying the non-target modified base (for example, 5-hmC) chemically and/or enzymatically in such a way that its reactivity to a endonuclease is completely or partially blocked, and then chemically or enzymatically reacting the target modified base to convert it into a newly formed 5-hmC (e.g., with a reducing or oxidant agent) which can be then be reacted with a glycosyltransferase to form 5- gmC which in turn can be cleaved by an endonuclease that recognizes 5-is gmC preferentially in a positive identification.

By the appropriate choice of the substrate for modifying the non-target and target modified bases, this invention provides an on-off switch assay which is determined by enzyme specificity. For example, if in the first step UDP-2-GIcN is used to label pre-existing 5-hmC sites (resulting in an off-signal for AbaSI cleavage), and in the second step the sample is treated with a methylpyrimidine oxygenase (mYOX), BGT and UDP-Glc, an "on-signal" for AbaSI cleavage will be generated exclusively for 5-mC sites which underwent oxidation by mYOX. If UDP-Glc is used to label pre-existing 5-hmC sites (on-signal for AbaSI cleavage), and the sample is treated with an mYOX, BGT and UDP-2-GIcN, then an "off-signal" will be generated for all 5-mc sites which were converted into 5-hmC by mYOX-mediated oxidation.

The 5-mC may be chemically or enzymatically converted to 5-hmC by reacting the 5-mC with mYOX1 (see for example, "nMCO1" described in US Provisional Application No. 61/723,427), TEl enzymes or chemical oxidizing agents. Similarly, oxidation of 5-mG to 5-hmC to 5-fc to 5-GaG can be achieved by chemical or enzymatic oxidation using mYOX1 or lET. Specific chemical oxidation of 5-hmC to 5-fC in synthetic nucleotide oligomer single strand (55DNA) containing 5-hmC can be achieved with potassium perruthenate, KRu04 (Booth, et al. Science, 336:934-937 (2012)). KRuO4 can oxidize 5-hmC in double-stranded DNA (d5DNA), with an initial denaturing step before the addition of the oxidant, resulting in quantitative conversion of 5-hmC to 5-fC. Other oxidants known in art, such as Osmium (VIII)-based oxidants, Cerium (IV)-based oxidants, and Chromium (VI)-based oxidants may be used for the oxidation of 5-hmC to 5-fC.

A variety of sugars and modified sugars can be used to alter the propensity of a 5-hmC to trigger an endonuclease-mediated cleavage event.

Some of these sugars and modified sugars are depicted in Figure 3. For example, T4-BGT can be used to add glucose, 2-glucosamine, 6-glucosamine, or 6-azidoglucose to 5-hmC. T4-BGT can also be used in combination with a 3-glucosyl-a-glycosyltransferase, such as the one from bacteriophage 16, to generate disaccharidyl cytosine modifications as shown in Figure 3. By controlling the modifications made to a particular nucleobase, the properties of that nucleobase can be changed in a manner facilitating its discrimination, whether through changes in its reactivity with an endonuclease; changes in the kinetics of synthesis of a complementary nucleic acid; or directly measured changes in size, shape, or charge density.

The reduction of 5-fC and its conversion to 5-3-gmC was demonstrated in the experiment depicted in Figure 7. Specifically, Figure 7 is an LG-MS analysis of a nucleic acid originally including 5-fC (top trace). In the presence of sodium borohydride (NaBH4), the 5fC is converted to 5-hmC (middle trace). When UDP-Glc and a BGT such as T4-BGT were also provided, the 5-fC was converted all the way to 5-3-gmC, confirming that the modified forms of cytosine can be interconverted, facilitating their subsequent detection and discrimination.

In one embodiment the method comprises one or more of the following steps; (a) Genomic DNA is treated with BGT and UDP-2-GIcN, so that all 5-hmC residues are converted to 5-3-2-gNmC.

(b) (i) The resulting DNA is treated with mYOX1, a Tet enzyme or a chemical oxidant agent (converts 5-mC to 5-hmC), BGT and UDP-Glc, so that all existing 5-mC residues are converted to 5-13-gmC; or (ii) The resulting DNA is treated with a reducing agent (e.g., NaBH4, converts 5-fC to 5-hmC), BGT and UDP-Glc to generate 5-13-gmC; or (iii) The resulting DNA is treated with a reducing agent (e.g., NaBH4, converts 5-fC to 5-hmC), BGT and UDP-2-GIcN, so that all 5-fC residues are converted to 5-13-2-gNmC. Then, the resulting DNA is treated with a second reducing agent (a different reducing agent or the same reducing agent but in the presence of certain additives that converts 5-caC to 5-hmC), BGT, and UDP-Glc to generate 5-13-gmC.

(c) (i) The DNA is digested with a 5-13-gmC-dependent endonuclease such as AbaSI, which cleaved at a fixed distance from 5-13-gmC and left a sticky end (2-base 3-overhang). Since the endonuclease does not recognize C or 5-13-2-gNmC no cleavage associated with these sites occurs. The only sticky ends created are those resulting from 5-3-gmC residues, which in turn are exclusively associated to 5-mC sites; or (ii) The DNA is digested with endonuclease, which cleaves 5-13-gmC exclusively associated to 5-fCs sites, but not 5-3-2-gNmC, s 5-mC, or C, leaving a sticky end (2-base 3-overhang).

(Hi) the DNA is digested with endonuclease, which cleaves 5-13- gmC exclusively associated to CaC sites, but not 5-3-2-gNmC, 5-mc, or C, leaving a sticky end (2-base 3'-overhang).

(d) A first adaptor (e.g., biotinylated adaptor A) is then ligated onto the cleaved ends.

(e) The ligated DNA is then subjected to random fragmentation to about 200 bp.

(f) Beads may be used to pull out the fragments with the ligated adaptor. For example, avidin beads may be used to pull out the biotin labeled adaptor (adaptor A). A person of ordinary skill in the art will recognize other affinity systems and immobilization matrices that can be used in place of biotin/avidin beads.

(g) After polishing the ends, adaptor P is then ligated onto the DNA fragments on the beads.

(h) The adaptor-specific PCR and the adapter ligated DNA enters the library preparation pipeline for specific sequencing platform where the end-sequencing is done from the adaptor A. Reducing agents and conditions can be used herein that can convert the carboxylic acids into alcohols, e.g., NaBH4, CoCI2, i-Pr2NH, EtOH/H20 (]agdale, et al. SynthesIs, 660-664 (2009)); EDC, HOEt, NaBH4, THF/H20 (Morales-Serna, et al. Synthesis, 1375-1382 (2011)); and cyanuric chloride, NaBH4, NMM/H20 (Falorni, et al. Tetrahedron Lett., 4395-4396 (1999)) as well as other reducing agents known to a person of ordinary skilled in the art.

Indeed, many water-soluble metal or metalloid hydrides are able to reduce aldehydes and/or carboxylic acids to alcohols. Examples of such s reducing agents are sodium borohydride and related compounds where from 1 to 3 of the hydrogens are replaced by other moieties, such as cyano and alkoxy containing up to about 5 carbon atoms. Examples of substituted borohydrides, all of which are sodium, potassium, or lithium salts, include cyanoborohydride, dicyanoborohydride, methoxyborohydride, dimethoxyborohydride, trimethoxyborohydride, ethoxyborohydride, diethoxyborohydride, triethoxyborohydride, propoxyborohydride, dipropoxyborohydride, tripropoxyborohydride, butoxyborohydride, dibutoxyborohydride, tributoxyborohydride, and so forth. Examples of other water-soluble metal hydrides include lithium borohydride, potassium borohydride, zinc borohydride, aluminum borohydride, zirconium borohydride, beryllium borohydride, and sodium bis(2-methoxyethoxy)aluminium hydride. Sodium borohydride can also be used in combination with a metal halide, such as cobalt(II), nickel(II), copper(II), zinc(II), cadmium (II), calcium (II), magnesium(II), aluminum(III), titanium (IV), hafnium(IV), or rhodium(III), each of which can be provided as a chloride, bromide, iodide, or fluoride salt. Alternatively, sodium borohydride can be used in combination with iodine, bromine, boron trifluoride diethyl etherate, trifluoroacetic acid, catechol-trifluoroacetic acid, sulfuric acid, or diglyme. Particular reducing strategies include the combination of potassium borohydride with lithium chloride, zinc chloride, magnesium chloride, or hafnium chloride; or the combination of lithium borohydride and chlorotrimethylsilane. Other reducing strategies include the use of borane, borane dimethyl sulfide complex, borane tetrahydrofuran complex, borane-ammonia complex, borane morpholine complex, borane dimethylamine complex, borane trimethylamine complex, borane N,N-diisopropylethylamine complex, borane pyridine complex, 2-picoline borane complex, borane 4-methylmorpholine complex, borane tert-butylamine complex, borane triphenylphosphine complex, borane N,N-diethylaniline complex, borane di(tert-butyl)phosphine complex, borane diphenylphosphine complex, borane ethylenediamine complex, or lithium ammonia borane. Alternative reducing strategies include the reduction of carboxylic acids via the formation of hydroxybenzotriazole esters, carboxy methylenimi nium chlorides, carbonates, 0-acylisoureas, acyl fluorides, cyanurates, mixed anhydrides, arylboronic anhydrides, acyl imidazolide, acyl azides, or N-acyl benzotriazoles, followed by reaction with sodium borohydride to give the corresponding alcohols.

In one embodiment, 5-hmc sequencing can use BGT and UDP-Glc to generate AbaSI-active 5-3-gmC sites, and T4-o-glucosyltransferase (ACT) is and UDP-Glc to generate AbaSI-inactive 5-o-gmC sites as a negative control.

In another embodiment, 5-hmC sequencing can use BGT and UDP-Glc to generate the AbaSI-active 5-3-gmC sites, and BGT and LJDP-2-GIcN to generate AbaSI-inactive 5-3-2-gNmC as a negative control for 5-hmC.

In one embodiment, newly generated 5-hmC sites can be differentiated from pre-existing 5-hmC sites by sequentially transferring distinct sugar moieties from UDP-2-GIcN or native UDP-Glc using BGT.

In a further embodiment of the invention, UDP-Glc modified by any of keto, thiol, chloro, fluoro, bromo, iodo, nitro, boron, and other substituents may be transferred onto 5-hmC using BGT and may block AbaSI activity.

Keto, thiol, chloro, fluoro, bromo, iodo, nitro, boron, and other substituents modifying glucose containing 5-hmC residues in a nucleic acid may facilitate cytosine modification mapping and inhibit AbaSI cleavage.

All references cited herein, as well as U.S. Application No. 61/611,295, filed March 15, 2012; U.S. Application No. 61/722,968, filed November 6, 2012; U.S. Application No. 61/723,427, filed November 7, 2012; U.S. Application No. 61/724,041, filed November 8, 2012; U.S. Application No. 13/804,804, filed March 14, 2013; U.S. Application No. 13/826,395, filed March 14, 2013; U.S. Application No. 13/827,087, filed March 14, 2013, and U.S. Application No. 13/827,885, filed March 14, 2013, are incorporated by reference.

EXAMPLES

Example 1: Mapping of 5-hmC in a nucleic acid sequence The locations of 5-hmC in a nucleic acid sample can be determined using differential cleavage.

A BGT can transfer glucose ("GIc") from UDP-Glc to 5-hmC to form the glucoylated residue 5-3-2-gmC. Glucosylation enhances the sensitivity of is the nucleic acid to a glucosylation-sensitive restriction enzyme such as AbaSI (see Figure 2). Accordingly, the identification of 5-hmC sites in a sample can be facilitating by glucosylating the nucleic acid, followed by identifying the locations of AbaSI cleavage sites.

Example 2: Exclusive 5-mC methvlome mapøinci An exemplary process for mapping 5-mC residues in a nucleic acid is depicted in Figure 4.

As shown in Figure 4, genomic DNA is treated with a BGT and UDP-2-glucosamine, converting 5-hmC residues to 5-3-2-gNmC. The resulting DNA is treated with a methylpyrimidine oxygenase from Neisseria (mYOX1), TET or a chemical oxidizing agent, BGT and UDP-Glc, converting existing 5-mC residues to 5-3-gmC. The DNA is digested with a 5-3-gmC-dependent restriction enzyme, such as AbaSI, cleaving at a fixed distance from 5-13-gmC and leaving a sticky end (2-base 3'-overhang). Since AbaSI does not recognize C or 5-i3-2-gNmC, the only sticky ends created are those resulting s from 5-3-gmC residues, which in turn are exclusively associated to 5-mC sites. A biotinylated adaptor A is then ligated onto the cleaved ends. The ligated DNA is then subjected to random fragmentation to an average size of about 200 bp. Avidin beads are used to pull out the fragments with the ligated adaptor A. After polishing the ends, adaptor P is then ligated onto the DNA fragments on the beads. The adaptor-specific PCR and the adapter ligated DNA enters the library preparation pipeline for specific sequencing platform where the end-sequencing is done from the adaptor A. Bioinformatic analysis of the sequencing reads is facilitated by the presence of adapter A which marks the enzyme cleavage sites. After is mapping the read back to the reference genome, the modified cytosine can be mapped at fixed distance away from the cleavage sites.

Example 3: Exclusive 5-fC and/or 5-caC mapping Exemplary processes for identifying 5-fC or 5-caC residues in a nucleic acid are depicted in Figures 5A and 56.

Example 3A: Glucosamination As shown in Figure SA, a BGTcan be used to catalyze the addition of 2-glucosamine from LJDP-2-GIcN to a 5-hmC residue, converting those residues to 5-13-2-gNmC. A reducing agent such as NaBH4 (optionally in the presence of additives), can be used to reduce 5-fC and/or 5-caC to 5-hmC.

The newly formed 5-hmC when combined with BGT and IJDP-Glc can be subsequently converted to 5-13-gmC.

As described in Example 2, the DNA is digested with a S-3-gmC-dependent restriction enzyme, such as AbaSI, cleaving at a fixed distance from 5-j3-gmC and leaving a sticky end (2-base 3-overhang). Since AbaSI does not recognize C or 5-3-2-gNmC, the only sticky ends created are those s resulting from 5-3-gmC residues, which in turn are exclusively associated to S-fC and/or 5-caC sites. A biotinylated adaptor A is then ligated onto the cleaved ends. The ligated DNA is then subjected to random fragmentation to an average size of about 200 bp. Avidin beads are used to pull out the fragments with the ligated adaptor A. After polishing the ends, adaptor P is then ligated onto the DNA fragments on the beads. The adaptor-specific PCR and the adapter ligated DNA enters the library preparation pipeline for specific sequencing platform where the end-sequencing is done from the adaptor A. Bioinformatic analysis of the sequencing reads is facilitated by the is presence of adapter A which marks the enzyme cleavage sites. After mapping the read back to the reference genome, the modified cytosine can be mapped at fixed distance away from the cleavage sites.

Example 3B: 6-azido-glucose Another exemplary process for mapping the locations of 5-fC and/or 5-caC is shown in Figure SB. The process depicted in Figure SB, like the process depicted in Figure 5A, begins with the addition of 2-glucosamine from UDP-2-GIcN to a 5-hmC residue, converting those residues to 5-13-2- gNmC in a reaction catalyzed by a BGT, and the subsequent reduction of 5-fC and/or 5-caC residues to newly-generated 5-hmC residues. In the method depicted in Figure SB, UDP-6-azido-glucose (UDP-6-N3-Glc) is added to those newly-generated 5-hmC residues by a BGT. The DNA is digested with a 5-13-gmC-dependent restriction enzyme, such as AbaSI, cleaving at a fixed distance from 5-13-gmC and leaving a sticky end (2-base 3'-overhang).

Since AbaSI does not recognize C or 5--2-gNmC, the only sticky ends created are those resulting from the azidoglucosylated residues, which in turn are exclusively associated to 5-fC and/or 5-caC sites.

The sticky ends are ligated to an adapter A. The resulting DNA is s fragmented and ligated to a second adapter P1. The azido moiety can derivatized with a biotin label via azide-alkyne Huisgen cycloaddition using a copper(I) catalyst ("click chemistry") or via copper-free variants (for example, using strained cyclooctyne derivatives) and avidin beads can then be used to selectively purify the fragments containing the azidoglucosylated in residues.

Example 4: Exclusive 5-caC mapping An exemplary process for mapping the locations of 5-caC in a nucleic acid sample is provided in Figure 6.

As shown in Figure 6, genomic DNA is treated with BGT and UDP-2-GIcN, so that substantially all 5-hmC residues are converted to 5-3-2-gNmC.

The resulting DNA is treated with a reducing agent (e.g., NaBH4, converts 5-fC to 5-hmC), BGT and UDP-2-GIcN, so that all 5-fC residues are converted to 5--2-gNmC. Then, the resulting DNA is treated with a second reducing agent (a different reducing agent or the same reducing agent but in the presence of certain additives converts 5-caC to 5-hmC), BGT, and UDP-Glc to generate 5-3-gmC. The DNA is digested with a restriction endonuclease such as AbaSI, which cleaves 5-3-gmC exclusively associated to 5-caC sites but not 5--2-gNmC, 5-mC or C to leave a sticky end (2-base 3'-overhang).

These sites are then identified through ligation to a biotinylated adapter, fragmentation and purification using avidin-associated beads, ligation and analysis as described in Example 2.

CLAUSES

1. A preparation comprising: (i) a glycosyltransferase; (ii) an oxidizing agent and/or a reducing agent; and (iii) UDP-glucosamine and/or a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose.

2. A preparation according to Clause 1, comprising: (i) a glycosyltransferase; (ii) an oxidizing agent and/or a reducing agent; and (iii) a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose.

3. A preparation according to Clause 2, wherein the UDP derivative is UDP-azidoglucose.

4. A preparation according to Clause 3, wherein the UDP-azidoglucose is U D P-6-azi dog I u cose.

5. A preparation according to Clause 3 or 4, further comprising UDP-glucose.

6. A preparation according to Clause 1, comprising: (i) a glycosyltransferase; (ii) an oxidizing agent and/or a reducing agent; and (iii) UDP-glucosamine.

7. A preparation according to Clause 6, further comprising a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose.

8. A preparation according to any of Clauses 1-7, wherein (ii) is an oxidizing agent.

9. A preparation according to any of Clauses 1-7, wherein (ii) is a reducing agent.

10. A preparation according to Clause 8, wherein the oxidizing agent is a methylcytosine oxygenase, such as a TEl enzyme or mYOX1.

11. A preparation according to Clause 8, wherein the oxidizing agent is an inorganic oxidizing agent, such as KRuO4.

12. A preparation according to Clause 9, comprising: (i) a glycosyltransferase; (ii) a reducing agent; and (iii) UDP-glucose.

13. A preparation according to Clause 9 or 12, wherein the reducing agent is NaBH4.

14. A preparation according to any of Clauses 1-13, wherein the glycosyltra nsferase is a 3-glycosyltransferase.

15. A preparation according to any of Clauses 1-14, further comprising an endon ucl ease.

16. A preparation according to Clause 15, wherein the endonuclease has an amino acid sequence at least 9S% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, and PxyI.

17. A preparation according to Clause 16, comprising: (i) a glycosyltransferase; (ii) an oxidizing agent; (iii) UDP-glucose; and (iv) an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, and PxyI.

18. A preparation according to Clause 17, further comprising UDP-glucosamine.

19. A preparation comprising: (i) a j3-glycosyltransferase; (ii) UDP-glucosamine and/or a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose; and (iii) an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, and PxyI.

20. A preparation according to any one of Clauses 1-19, further comprising a nucleic acid.

21. A preparation according to Clause 20, wherein the nucleic acid comprises one or more modified nucleotides selected from 5- hydroxymethylcytosine, glucosyl-5-hydroxymethylcytosine, 13-glucosaminyloxy-5-methylcytosine, 5-formylcytosine, 5-carboxycytosine, and 5-methylcytosine.

22. A preparation according to Clause 20 or 21, wherein the nucleic acid comprises DNA.

23. A preparation according to Clause 20 or 21, wherein the nucleic acid comprises RNA.

24. A kit for making the preparation of any of Clauses 1-14, comprising (i), (ii) and (iH) as defined in any of Clauses 1-14.

25. A kit according to Clause 24, comprising: (i) a glycosyltransferase; (ii) a reducing agent; and (iii) a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose.

26. A kit according to Clause 24, comprising: (i) a 13-glycosyltransferase; (ii) an oxidizing agent; and (iii) UDP-glucosamine and UDP-glucose.

27. A kit according to any one of Clauses 24-26, further comprising a restriction endonuclease.

28. A kit according to Clause 27, wherein the restriction endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, and PxyI.

29. A kit for making the preparation of Clause 19, comprising (i), (ii) and (iii) as defined in Clause 19.

30. A kit according to any one of Clauses 24-29, further comprising a nucleic acid.

31. A kit according to Clause 30, wherein the nucleic acid comprises one or more modified nucleotides selected from 5-hydroxymethylcytosine, glucosyl- 5-hydroxymethylcytosine, 3-glucosaminyloxy-5-methylcytosine, 5-formylcytosine, 5-carboxycytosine, and 5-methylcytosi ne.

32. A method for selectively altering modified nucleotides in a nucleic acid containing modified nucleotides, using a preparation according to any one of Clauses 1-14, wherein the method comprises: (a) reacting a first subset of the modified nucleotides in the nucleic acid with UDP-glucosamine in the presence of a glycosyltransferase to convert hydroxymethylated nucleotides in the nucleic acid to glucosaminylated nucleotides; and (b) subsequently reacting the nucleic acid with: (i) a reducing agent and a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose; or (ii) an oxidizing agent and a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose; to convert a second subset of nucleotides in the nucleic acid to glucosylated or azidoglucosylated nucleotides.

33. A method according to Clause 32, wherein the reaction with the LJDP derivative in step (b) is catalyzed by a glycosyltransferase.

34. A method according to Clause 32 or 33, wherein step (b) comprises reacting the nucleic acid with (i), and wherein the second subset of nucleotides comprises 5-formylcytosine.

35. A method according to Clause 32 or 33, wherein step (b) comprises reacting the nucleic acid with (i), and wherein the second subset of nucleotides comprises 5-carboxycytosine.

36. A method according to Clause 34 or 35, wherein the reducing agent is NaBH4.

37. A method according to Clause 32 or 33, wherein step (b) comprises reacting the nucleic acid with (ii), and wherein the second subset of nucleotides comprise 5-methylcytosines.

38. A method according to Clause 37, wherein the oxidizing agent is a methylcytosine oxygenase, such as a TEl enzyme or mYOX1.

39. A method according to Clause 37, wherein the oxidizing agent is an inorganic oxidizing agent, such as KRuO4.

40. A method according to any one of Clauses 32-39, wherein the UDP derivative is UDP-glucose.

41. A method according to any one of Clauses 32-39, wherein the UDP derivative is UDP-azidoglucose.

42. A method according to Clause 41, wherein the UDP-azidoglucose is U D P-6-azi dog I u cose.

43. A method according to Clause 42, wherein the second subset of nucleotides in the nucleic acid are converted to 3-6-azide-glucosyl-5-hydroxymethylcytosine.

44. A method according to any one of Clauses 32-43, further comprising differentiating between: (i) pre-existing 5-hydroxymethylcytosine in the nucleic acid; and (ii) newly formed 5-hydroxymethylcytosine in the nucleic acid, formed by the reduction of the second subset of nucleotides in the nucleic acid in step (b)(i) or by the oxidation of the second subset of nucleotides in the nucleic acid in step (b)(ii).

45. A method according to any one of Clauses 32-44, further comprising cleaving the nucleic acid with an endonuclease specific for the glucosylated or azidoglucosylated nucleotides and not for glucosaminylated nucleotides.

46. A method according to Clause 45, wherein the endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, and PxyI.

47. A method according to Clause 45 or 46, comprising ligating an adapter to the cleaved end of the nucleic acid.

48. A method according to any one of Clauses 32-47, further comprising labeling the glucosylated or azidoglucosylated nucleotides with biotin.

49. A method for differentiating a 5-methylcytosine from 5-hydroxymethylcytosine in a genome or genome fragment, using a preparation according to any one of Clauses 1-8, 10-11 or 14-15, wherein the method comprises: (a) reacting the isolated genome or genome fragment containing 5-methylcytosine and 5-hydroxymethylcytosine with: (i) a first UDP derivative selected from UDP-glucosamine, UDP-glucose and UDP-azidoglucose in the presence of a glycosyltransferase catalyzing transfer of said glucosamine, glucose or azido-glucose to the 5-hydroxymethylcytosine; (ii) an oxidizing agent to convert 5-methylcytosine to 5-hydroxymethylcytosine; and (Hi) a second UDP derivative selected from UDP-glucosamine, IJDP-glucose and UDP-azidoglucose, wherein said second UDP derivative is different from the first UDP derivative, in the presence of a glycosyltransferase catalyzing transfer of said glucosamine, glucose or azido-glucose to the 5-hydroxymethylcytosine generated in step (ii); (b) cleaving the isolated genome or genome fragment obtained in step (a) with an endonuclease that recognizes either the modified 5- hydroxymethylcytosine generated in (i) or the modified 5-hydroxymethylcytosine generated in (Hi); and (c) differentiating the 5-methylcytosine from the 5-hydroxymethylcytosine by an altered cleavage pattern.

50. A method according to Clause 49, wherein the first UDP derivative is UDP-glucosamine and wherein the second IJDP derivative is selected from UDP-glucose and UDP-azidoglucose.

51. A method according to Clause 49 or 50, wherein steps (a)(ii) and (a)(iii) are performed concurrently, such that step (a)(i) is followed by simultaneous exposure to the oxidizing agent, the glycosyltransferase, and the IJDP derivative; thereby transferring said glucosamine, glucose or azido-glucose to the 5-hydroxymethylcytosine as it is being generated.

52. A method according to Clause 49, wherein in step (a)(i) the UDP derivative and glycosyltransferase are IJDP-2-glucosamine and a 13-glycosyltransferase; and wherein step (a)(iii) is omitted; and wherein in step (b) the modification dependent endonuclease is capable of selectively cleaving a nucleic acid at a 5-hydroxymethylcytosine and not at a 5-glucosaminylated hydroxymethylcytosine.

53. A method for differentiating 5-(3-glucosyloxymethyl) cytosine from 5- (13-2-glucosaminyloxymethyl) cytosine in a nucleic acid, comprising: reacting the nucleic acid with an endonuclease capable of cleaving a nucleic acid at a glucosylated nucleotide but not at an glucosaminylated nucleotide.

54. A method according to Clause 53, wherein the endonuclease has an amino acid sequence at least 9S% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaLJI, AcaPI, and PxyI.

55. A method for placing off switches and on switches for endonuclease cleavage of a nucleic acid containing modified nucleotides, the method comprising reacting the nucleic acid alternately with (i) LJDP-glucosamine and (ii) LJDP-glucose.

Claims

What is claimed is: 1. A preparation comprising: (i) a 3-glycosyltransferase; (ii) UDP-glucosamine and/or a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose; and (iii) an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaST, AbaAI, AbaCI, AbaDI, AbalI, AbaUl, AcaPI, and PxyI.
2. A preparation according to Claim 1, comprising: (i) a 3-glycosyltransferase; (ii) a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose; and (iii) an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUT, AcaPI, and PxyI.
3. A preparation according to Claim 2, wherein the UDP derivative is UDP-glucose.
4. A preparation according to Claim 2, wherein the UDP derivative is UDP-azidoglucose.
5. A preparation according to Claim 1, comprising: (i) a 3-glycosyltransferase; (ii) UDP-glucosamine; and (iii) an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaST, AbaAI, AbaCI, AbaDI, AbalI, AbaUl, AcaPI, and PxyI.
6. A preparation according to Claim 5, further comprising a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose.
7. A preparation according to any of Claims 1-6, further comprising an oxidizing agent and! or a reducing agent.
8. A preparation according to Claim 7, wherein the oxidizing agent is a methylcytosine oxygenase, such as a TET enzyme or mYOX1; or an inorganic oxidizing agent, such as KRuO4.
9. A preparation according to Claim 7 or 8, wherein the reducing agent is Na5H4.
10. A preparation according to any one of Claims 1-9, further comprising a nucleic acid.
11. A preparation according to Claim 10, wherein the nucleic acid comprises one or more modified nucleotides selected from 5- hyd roxymethylcytosine, glucosyl-5-hyd roxymethylcytosine, 13-g lucosami nyloxy-5-methylcytosi ne, 5-formylcytosine, 5-carboxycytosi ne, and 5-methylcytosine.
12. A preparation according to Claim 10 or 11, wherein the nucleic acid comprises DNA.
13. A preparation according to Claim 10 or 11, wherein the nucleic acid comprises RNA.
14. A kit for making the preparation of any one of Claims 1-9, comprising (i), (ii) and (iii) as defined in Claims 1-9.
15. A kit according to Claim 14, further comprising a nucleic acid.
16. A kit according to Claim 15, wherein the nucleic acid comprises one or more modified nucleotides selected from 5-hyd roxymethylcytosine, glucosyl-5-hydroxymethylcytosine, 13-glucosaminyloxy-5-methylcytosine, 5-formylcytosine, 5-ca rboxycytosine, and 5-methylcytosine.
17. A method for differentiating 5-(3-glucosyloxymethyl) cytosine from 5-(3-2-glucosaminyloxymethyl) cytosine in a nucleic acid, comprising: reacting the nucleic acid with an endonuclease capable of cleaving a nucleic acid at a glucosylated nucleotide but not at an glucosaminylated nucleotide.
18. A method according to Claim 17, wherein the endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUl, AcaPI, and PxyI.
19. A method for placing off switches and on switches for endonuclease cleavage of a nucleic acid containing modified nucleotides, the method comprising reacting the nucleic acid alternately with (i) UDP-glucosamine and (ii) UDP-glucose.