Mapping cytosine modifications
View Patent ↗Methods, compositions and kits for selectively altering and detecting modified cytosine residues are provided.
1. A method for selectively altering any modified cytosine in a nucleic acid that is hydroxymethylcytosine (hmC) or methylcytosine (mC), comprising:
(a) reacting the nucleic acid with UDP-glucosamine in the presence of a glycosyltransferase to convert hmC in the nucleic acid to glucosaminylated cytosine, and
(b) reacting the nucleic acid with an oxidizing agent and UDP-glucose to convert mC to glucosylated methylcytosine wherein the oxidizing agent is methyl cytosine oxygenase or KRuO 4 .
2. The method according to claim 1 , wherein the reaction with UDP-glucose is catalyzed by a glycosyltransferase.
3. The method according to claim 1 , further comprising cleaving the nucleic acid with an endonuclease specific for the glucosylated nucleotides and not for glucosaminylated nucleotides.
4. The method according to claim 3 , wherein the endonuclease has an amino acid sequence at least 95% identical to SEQ ID NO: 3.
5. The method according to claim 3 , wherein the endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of SEQ ID NOs: 3-8 and SEQ ID NO: 10.
6. The method according to claim 3 , comprising: ligating an adapter to the cleaved end of the nucleic acid.
7. The method according to claim 1 , wherein the methylcytosine oxygenase is a TET enzyme.
8. The method according to claim 1 , wherein the methylcytosine oxygenase is mYOX1.
9. The method according to any one of claims 1 - 6 , 7 and 8 , comprising differentiating pre-existing 5-hydroxymethylcytosine from newly formed 5-hydroxymethylcytosine in a nucleic acid where 5-methylcytosine is a precursor of 5-hydroxymethylcytosine.