Compositions and Methods for Labeling Modified Nucleotides in Nucleic Acids

A method comprising:

• (a) combining:
• i. a sample comprising hydroxymethylcytosine ribonucleotides (hmrC) or hydroxymethylcytosine deoxyribonucleotides (hmdC);
• ii. a hydroxymethylcytosine carbamoyltransferase (hmC-CT);
• and
• iii. a tagged carbamoyl phosphate, to product a reaction mixture and
• (b) incubating the reaction mixture to modify the hmrC or hmdC.

A method comprising:

• (a) combining:
• i. a pool of nucleoside triphosphates comprising hydroxymethylcytosine ribonucleotides (hmrC) or hydroxymethylcytosine deoxyribonucleotides (hmdC);
• ii. a hydroxymethylcytosine carbamoyltransferase (hmC-CT);
• iii. a carbamoyl phosphate substrate;
• iv. a nucleic acid template;
• and
• v. a polymerase, to product a reaction mix, and
• (b) incubating the reaction mix to produce a nucleic acid product that contains modified cytocines.

A composition comprising:

• a tagged carbamoyl phosphate having the formula
• wherein:
• (i) the R1 and R2 in Formula 1 independently of each other may be an H or a tag (T) comprising a chemically reactive group (C) a functional group (F) and/or a linking group (L) where the linking group may be positioned between the carbamoyl group and the chemically reactive group and /or between the chemically reactive group and the label;
• and
• (ii) wherein the chemically reactive group (C) is selected from a succinimidyl ester, a maleimide, an amine, a thiol, an alkyne, or an azide, a carbonyl;
• a carboxyl;
• an active ester, e.g., a succinimidyl ester;
• a maleimide;
• an amine;
• a thiol;
• an alkyne, an azide;
• an alkyl halide;
• an isocyanate;
• an isothiocyanate;
• an iodoacetamide;
• a 2-thiopyridine;
• a 3-arylproprionitrile;
• a diazonium salt;
• an alkoxyamine;
• a hydrazine;
• a hydrazide;
• a phosphine;
• an alkene;
• a semicarbazone;
• an epoxy;
• a phosphonate;
• and a tetrazine.

A method for distinguishing hydroxymethylcytosine (hmC) from methylcytosine (mC) in a nucleic acid molecule, comprising:

• (a) placing in a reaction mixture:
• the nucleic acid molecule;
• a hydroxymethylcytosine carbamoyltransferase (hmC-CT) and carbamoyl phosphate substrate;
• (b) modifying hmC in the nucleic acid molecule to form a carbamoyloxymethylcytosine (cmC) or tagged cmC;
• (c) detecting the cmC or tagged cmC in the nucleic acid molecule;
• and
• (d) distinguishing hmC from mC.

A method for obtaining nucleic acid modifying enzymes, comprising:

• (a) obtaining phage nucleic acid from an environmental sample from which phage particles have been enriched;
• (b) identifying whether the phage nucleic acid has modified nucleotides;
• (c) performing a contig analysis of the phage nucleic acid for sequences encoding enzymes capable of modifying the phage nucleic acid;
• and
• (d) obtaining nucleic acid modifying enzymes.

A method for determining the location of modified cytosines in a nucleic acid in a sample, comprising:

• (a) reacting an aliquot of the sample in which the nucleic acid is single stranded with a hydroxymethylcytosine carbamoyltransferase (hmC-CT) and carbamoyl phosphate;
• (b) reacting the oxidized carbamoyl nucleic acid with a complementary single strand nucleic acid to form a double stranded DNA for reacting with TET protein:
• (c) permitting any methylated cytosines in the nucleic acid sample to be modified by adding glucosyltransferase (GT);
• and
• (d) performing whole genome sequencing on double stranded nucleic acid to determine the location of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in the nucleic acid.