Selective oxidation of 5-methylcytosine by TET-family proteins

• US 10,533,213 B2
• Filed: 11/02/2016
• Issued: 01/14/2020
• 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
• Inventors: Rao, Anjana, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Primary Examiner(s): Dauner, Joseph G.
• Application Number:
  US15/341,344
• Publication Number:
  US 20170191119A1
• Time in Patent Office: 1,168 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) oxidizing the 5-methylcytosine residue in the nucleic acid to generate a modified nucleic acid, wherein the oxidizing comprises contacting the nucleic acid with TET1, TET2, TET3, CXXC4, a catalytic fragment of any of these, or any combination thereof; and

(b)-detecting the modified nucleic acid, wherein detection of the modified nucleic acid is indicative of a presence of the 5-methylcytosine residue in the nucleic acid.

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

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

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

5. The method of claim 1, further comprising adding a glucose molecule or a glucose-derivative donor substrate to a 5-hydroxymethylcytosine residue in the nucleic acid.

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

7. The method of claim 5, further comprising identifying the 5-hydroxymethylcytosine residue by sequencing.