Composition and methods of oxygenation of nucleic acids containing 5-methylpyrimidine

• US 9,040,239 B2
• Filed: 03/14/2013
• Issued: 05/26/2015
• Est. Priority Date: 03/15/2012
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Zheng, Yu, Saleh, Lana, Pais, June, Dai, Nan, Roberts, Richard J., Correa, Ivan R. Jr., Mabuchi, Megumu, Vaisvila, Romualdas
• Primary Examiner(s): Gebreyesus, Kagnew H
• Application Number:
  US13/827,087
• Publication Number:
  US 20140127683A1
• Time in Patent Office: 803 Days
• Field of Search: 435/6.11, 435/6.1, 435/377, 435/4, 435/6.14, 536/23.2, 514/19.3, 702/20, 506/26
• US Class Current: 435/6.11
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 19/18 produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• CPC Class Codes: C12P 19/30 Nucleotides
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• 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 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2523/115 oxidising agents
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2537/164 : Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts

Abstract

5-methylpyrimidine oxygenases and their use in the modification of nucleic acids are described.

Claims

1. A composition comprising:

(i) a buffer comprising Fe(II) and alpha-ketoglutarate; and

(ii) a polypeptide that has 5-methylpyrimidine oxygenase activity and has at least 90% identity to amino acids 154-304 of SEQ ID NO;

2.

2. A composition according to claim 1, wherein the buffer does not contain ATP.

3. A composition according to claim 1, wherein the buffer contains ATP.

4. A composition according to claim 1, wherein the buffer is at a pH in the range of pH 6 to pH 8.

5. A composition according to claim 4, wherein the buffer is at a pH in the range of pH 6 to pH 7.5.

6. A composition according to claim 1, further comprising a nucleic acid.

7. A kit comprising a composition according to claim 1 wherein the buffer and the polypeptide are in separate containers.

8. A kit according to claim 7, wherein the reaction buffer is at a pH in the range of pH 6 to pH 7.5.

9. A kit according to claim 7, wherein the reaction buffer contains ATP.

10. A kit according to claim 7, wherein the reaction buffer does not contain ATP.

11. A kit according to claim 7, further comprising a nucleic acid.

12. A kit according to claim 7, further comprising a reducing agent.

13. A kit according to claim 7, further comprising a β

-glycosyltransferase (BGT) and UDP-glucosamine.

14. A kit according to claim 7, further comprising a β

-glycosyltransferase (BGT) and UDP-glucose.

15. A kit according to claim 7, further comprising a DNA glycosylase.

16. A kit according to claim 7, further comprising an endonuclease.

17. A method for differentiating a 5-methylcytosine (5-mC) from 5-hydroxymethylcytosine (5-hmC) in a genome or genome fragment, comprising:

a. reacting the isolated genome or genome fragment containing 5-mC and 5-hmC with UDP-glucose or UDP-glucosamine;

a glycosyltransferase for transferring glucose or glucosamine to the 5hmC; and

a composition according to claim 1;

b. cleaving the glucosylated template with a modification-dependent endonuclease that recognizes at least one of the modified nucleotides; and

c. differentiating the 5-mC from the 5-hmC by an altered cleavage pattern.

18. A method according to claim 17, wherein the modification-dependent endonuclease is AbaSI.

19. A composition according to claim 1, wherein the polypeptide further comprises a binding domain.

20. A composition according to claim 19, wherein the binding domain is selected from the group consisting of:

a His-tag, a maltose-binding protein, a chitin binding domain, and a DNA binding domain.

21. The composition according to claim 20, wherein the binding domain comprises a zinc finger or transcription activator-like (TAL) effector domain.

22. The composition of claim 1, wherein the polypeptide is a fusion protein.

23. The composition of claim 1, wherein the polypeptide has at least 95% identity to amino acids 154-304 of SEQ ID NO:

2.

24. The composition of claim 1, wherein the polypeptide is identical at amino acids 154-304 of SEQ ID NO:

2.

25. The composition of claim 1, wherein the polypeptide is at least 90% identical to SEQ ID NO:

2.

26. The composition of claim 1, wherein the polypeptide is at least 95% identical to SEQ ID NO:

2.

27. The composition of claim 1, wherein the polypeptide is identical to SEQ ID NO:

2.