SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

• US 20180291435A1
• Filed: 06/19/2018
• Published: 10/11/2018
• 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, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Granted Patent: US 10,337,053 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 comprising:

a. labeling covalently, a hydroxyl group on a hydroxymethylated residue in a nucleic acid to generate a labeled hydroxymethylated residue; and

b. sequencing said nucleic acid comprising said labeled hydroxymethylated residue or derivatives thereof.

2. The method of claim 1, wherein said nucleic acid is mammalian nucleic acid.

3. The method of claim 1, wherein said hydroxymethylated residue is a 5-hydroxymethylcytosine.

4. The method of claim 3, wherein said labeling comprises glycosylating said 5-hydroxymethylcytosine.

5. The method of claim 4, wherein said glycosylating said 5-hydroxymethylcytosine comprises glucosylating said 5-hydroxymethylcytosine with a modified glucose.

6. The method of claim 5, wherein said glucosylating is catalyzed by a glucosyltransferase.

7. The method of claim 6, wherein said glucosyltransferase comprises an alpha-glucosyltransferase or a beta-glucosyltransferase.

8. The method of claim 6, wherein said glucosyltransferase comprises said beta-glucosyltransferase.

9. The method of claim 2, wherein said nucleic acid further comprises a methylated cytosine residue.

10. The method of claim 9, wherein said methylated cytosine residue is a 5-methylcytosine.

11. The method of claim 10, further comprising distinguishing said 5-methylcytosine from said hydroxymethylated residues.

12. The method of claim 1, wherein said sequencing comprises high-throughput sequencing.