Selective oxidation of 5-methylcytosine by tet-family proteins

Patent metadata

Field	Value
Publication number	US-2018298431-A1
Application number	US-201816012510-A
Country	US
Kind code	A1
Filing date	Jun 19, 2018
Priority date	Sep 26, 2008
Publication date	Oct 18, 2018
Grant date	—

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.

First claim

We claim:

1 . A method comprising:
contacting a mammalian nucleic acid sequence with an enzyme that utilizes a labeled glucose or a labeled glucose-derivative donor substrate to add a labeled glucose molecule or a labeled glucose-derivative to a 5-hydroxymethylcytosine in said mammalian nucleic acid sequence to generate a labeled glucosylated-5-hydroxymethylcytosine, wherein said enzyme comprises a glucosyltransferase and wherein said labeled glucose or said labeled glucose-derivative donor substrate comprises a uridine diphosphate glucose (UDPG).

2 . The method of claim 1 , wherein said glucosyltransferase comprises a beta glucosyltransferase or an alpha glucosyltransferase, or a homologue or a derivative thereof.

3 . The method of claim 1 , further comprising detecting said 5-hydroxymethylcytosine.

4 . The method of claim 1 , wherein said mammalian nucleic acid sequence further comprises a 5-methylcytosine.

5 . The method of claim 4 , further comprising distinguishing said 5-methylcytosine from said 5-hydroxymethylctosine.

6 . The method of claim 1 , wherein said labeled glucose or said labeled glucose-derivative donor substrate comprises a radioactive label.

7 . The method of claim 6 , wherein said radioactive label comprises 14 C or 3 H.

8 . The method of claim 1 , further comprising sequencing said mammalian nucleic acid sequence.

9 . The method of claim 1 , wherein said mammalian nucleic acid sequence comprises a 5-methylcytosine.

10 . The method of claim 1 , further comprising contacting said mammalian nucleic acid sequence with a moiety that recognizes glucose, said labeled glucose or said labeled glucose-derivative.

11 . The method of claim 10 , wherein said moiety is a protein.

12 . The method of claim 11 , wherein said protein is an antibody or a fragment thereof.

13 . The method of claim 10 , wherein said moiety is modified with a tag.

14 . The method of claim 13 , wherein said tag comprises biotin or a fluorescent label.

Assignees

• Childrens Medical Ct Corp
• The Us Secretary Department Of Health & Human Services

Inventors

• Rao Anjana
• Tahiliani Mamta
• Koh Kian Peng
• Agarwal Suneet
• Iyer Aravind

Classifications

• C12Q1/6827 Primary

  for detection of mutation or polymorphism · CPC title

• C12N2501/70

  Enzymes · CPC title

• C12Q2521/531

  Glycosylase · CPC title

• C12Q1/26

  involving oxidoreductase · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title