Detection of 5-hydroxymethylcytosine by glycosylation

• US 10,508,301 B2
• Filed: 06/19/2018
• Issued: 12/17/2019
• Est. Priority Date: 09/26/2008
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation, United States Department Of Health And Human Services (Government of the United States of America)
• 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:
  US16/012,510
• Publication Number:
  US 20180298431A1
• Time in Patent Office: 546 Days
• Field of Search: None
• 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:

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 beta-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, further comprising detecting said 5-hydroxymethylcytosine.

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

4. The method of claim 3, further comprising distinguishing said 5-methylcytosine from said 5-hydroxymethylcytosine.

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

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

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

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

9. The method of claim 8, wherein said moiety is a protein.

10. The method of claim 9, wherein said protein is an antibody or a fragment thereof.

11. The method of claim 8, wherein said moiety is modified with a tag.

12. The method of claim 11, wherein said tag comprises biotin or a fluorescent label.