SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

• US 20170191119A1
• Filed: 11/02/2016
• Published: 07/06/2017
• Est. Priority Date: 09/26/2008
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation, United States Department Of Health And Human Services
• Original Assignee: Children's Medical Center Corporation, The United States of America As Represented By The Secretary of Agriculture
• Inventors: Rao, Anjana, Iyer, Aravind, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet
• Granted Patent: US 10,533,213 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/71 Oxidoreductases (EC 1.)
• CPC Class Codes: C12N 2501/724 Glycosyltransferases (EC 2.4.)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2522/10 Nucleic acid binding proteins
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.

Claims

1. A method for detecting a 5-methylcytosine residue in a nucleic acid, the method comprising:

a) providing the nucleic acid comprising the 5-methylcytosine residue;

b) converting the 5-methylcytosine residue into a modified base by one or more oxidation reactions of the 5-methycytosine residue beyond hydroxymethylation to generate a modified nucleic acid, wherein the converting comprises contacting the nucleic acid with one or more catalytically active TET family enzymes, functional TET family derivatives, TET catalytic fragments thereof, DNA encoding one or more catalytically active TET family enzymes, functional TET family derivatives, or TET catalytic fragments thereof; and

c) detecting the 5-methylcytosine residue in the modified nucleic acid by detecting the modified base.

2. The method of claim 1, wherein the one or more oxidation steps beyond hydroxymethylation generate a modified nucleic acid comprising an aldehyde or an acid.

3. The method of claim 1, wherein the nucleic acid comprises a plasmid DNA.

4. The method of claim 1, wherein the nucleic acid is an RNA or DNA molecule.

5. The method of claim 1, further comprising profiling a pattern of 5-methylcytosine residues in the modified nucleic acid.

6. The method of claim 1, wherein the modified nucleic acid further comprises a 5-hydroxymethylcytosine residue.

7. The method of claim 6, further comprising adding a glucose molecule or a glucose-derivative donor substrate to the 5-hydroxymethylcytosine residue.

8. The method of claim 7, further comprising identifying the 5-hydroxymethylcytosine residue to which the glucose molecule or the glucose-derivative donor substrate is added.

9. The method of claim 6, further comprising identifying the 5-hydroxymethylcytosine residue in the modified nucleic acid by sequencing.