SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

• US 20170176421A1
• Filed: 02/23/2017
• Published: 06/22/2017
• 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 9,816,986 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 5-hydroxymethylcytosine in a nucleic acid, the method comprising:

contacting said nucleic acid with an enzyme that utilizes a labeled glucose or a labeled glucose-derivative donor substrate to add one or more labeled glucose molecule or one or more labeled glucose-derivative to a 5-hydroxymethylcytosine residue to generate a glucosylated-5-hydroxymethylcytosine or a gentibiose-containing-5-hydroxymethylcytosine, wherein the enzyme is an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase, wherein the glucose or glucose-derivative donor substrate comprises a uridine diphosphate glucose.

2. The method of claim 1, wherein the labeled glucose or the labeled glucose-derivative donor substrate is radioactively labeled.

3. The method of claim 1, wherein the enzyme is an alpha-glucosyltransferase.

4. The method of claim 1, wherein the enzyme is a beta-glucosyltransferase.

5. The method of claim 1, wherein the enzyme is a beta-glucosyl-alpha-glucosyl-transferase.

6. A method comprising:

a) contacting said nucleic acid with an enzyme that utilizes a labeled glucose or a labeled glucose-derivative donor substrate to add one or more labeled glucose molecule or one or more labeled glucose-derivative to a 5-hydroxymethylcytosine residue to generate a glucosylated-5-hydroxymethylcytosine or a gentibiose-containing-5-hydroxymethylcytosine, wherein the enzyme is an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase, wherein the glucose or the glucose-derivative donor substrate comprises a uridine diphosphate glucose (UDPG), wherein the labeled glucose or the labeled glucose-derivative donor substrate is radioactively labeled; and

b) detecting said glucosylated-5-hydroxymethylcytosine or said gentibiose-containing-5-hydroxymethylcytosine.

7. The method of claim 6, wherein the enzyme is an alpha-glucosyltransferase.

8. The method of claim 6, wherein the enzyme is a beta-glucosyltransferase.

9. The method of claim 6, wherein the enzyme is a beta-glucosyl-alpha-glucosyl-transferase.