SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

• US 20190062817A1
• Filed: 11/05/2018
• Published: 02/28/2019
• 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, United States Department Of Health And Human Services (Government of the United States of America)
• Inventors: Rao, Anjana, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Granted Patent: US 10,767,216 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) obtaining a sample comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises a 5-hydroxymethylcytosine and a 5-methylcytosine;

(b) sequencing said nucleic acid sequence; and

(c) distinguishing said 5-hydroxymethylcytosine from said 5-methylcytosine.

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

3. The method of claim 1, wherein said sample comprises genomic DNA.

4. The method of claim 1, wherein said sample comprises an extracellular fluid.

5. The method of claim 1, wherein said sample comprises a blood sample.

6. The method of claim 1, wherein said sequencing is high-throughput sequencing.

7. The method of claim 1, wherein said sample is from a subject having or suspected of having cancer.

8. The method of claim 1, wherein said distinguishing comprises detecting said 5-hydroxymethylcytosine.

9. The method of claim 1, wherein said distinguishing comprises detecting said 5-methylcytosine.

10. The method of claim 1, further comprising glycosylating said 5-hydroxymethylcytosine.

11. The method of claim 10, further comprising associating a label with a glucose added to said 5-hydroxymethylcytosine by said glycosylating.

12. The method of claim 11, wherein said label comprises a J-binding protein.

13. The method of claim 10, further comprising contacting said nucleic acid sequence with a dioxygenase to convert a methylated cytosine in said nucleic acid sequence to a modified base.

14. The method of claim 13, wherein said modified base is formed by one or more oxidation reactions.

15. The method of claim 13, further comprising contacting said nucleic acid sequence with sodium bisulfite.

16. The method of claim 1, further comprising associating a label with an epigenetically modified base of said nucleic acid sequence.

17. The method of claim 1, wherein said epigenetically modified base is said 5-methylcytosine or said 5-hydroxymethylcytosine.

18. The method of claim 17, wherein said label comprises a J-binding protein.

19. The method of claim 17, wherein said label comprises a sugar.

20. The method of claim 17, wherein said label comprises a bead.