SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

• US 20170168043A1
• Filed: 02/23/2017
• Published: 06/15/2017
• 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, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Granted Patent: US 10,031,131 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 distinguishing 5-hydroxymethylcytosine from 5-methylcytosine in a nucleic acid, said method comprising:

contacting said nucleic acid with an enzyme that utilizes modified glucose or glucose-derivative donor substrate to add a modified glucose molecule or a glucose-derivative to a 5-hydroxymethylcytosine residue in said nucleic acid to generate a glucosylated-5-hydroxymethylcytosine or a gentibiose-containing-5-hydroxymethylcytosine.

2. The method of claim 1, wherein said enzyme is an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase.

3. The method of claim 2, wherein said modified glucose or glucose-derivative donor substrate comprises a labeled glucose or glucose-derivative donor substrate.

4. The method of claim 3, wherein said labeled glucose or glucose-derivative donor substrate is radioactively labeled.

5. The method of claim 4, wherein said radioactive label comprises with ¹⁴C or ³H.

6. The method claim 1, wherein said glucose or glucose-derivative donor substrate comprises a uridine diphosphate glucose (UDPG).

7. The method of claim 1, wherein said 5-hydroxymethylcytosine is naturally occurring.

8. The method of claim 1, further comprising contacting said glucosylated-5-hydroxymethylcytosine or gentibiose-containing-5-hydroxymethylcytosine with a protein that recognizes a glucose molecule, glucose-derivative or gentibiosyl molecule.

9. The method of claim 8, wherein the protein is lectin.

10. The method of claim 8, wherein said protein is an antibody or antigen-binding fragment thereof.

11. The method of claim 10, wherein said antibody or antigen-binding fragment thereof is modified with a tag.

12. The method of claim 11, wherein said tag is a biotin molecule, a bead, a gold particle, or a fluorescent molecule.

13. The method of claim 12, further comprising detecting a presence of said antibody or antigen-binding fragment thereof.

14. The method of claim 1, wherein said nucleic acid is treated with a TET enzyme or a catalytically active fragment thereof prior to contacting said nucleic acid with said enzyme.

15. The method of claim 1, further comprising treating said nucleic acid with a TET enzyme or a catalytically active fragment thereof.

16. The method of claim 15, wherein said TET enzyme is TET1, TET2, TET3, or CXXC4.

17. A kit comprising:

a) an enzyme, wherein said enzyme is an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase; and

b) a modified;

glucose or glucose-derivative donor substrate.

18. The kit of claim 17, wherein said modified glucose or glucose-derivative donor substrate comprises a labeled glucose or glucose-derivative donor substrate.

19. The kit of claim 17, wherein said glucose or glucose-derivative donor substrate comprises uridine diphosphate glucose (UDPG).

20. The kit of claim 17, wherein said modification comprises a detectable moiety, a blocking moiety, or an isolation tag.