Compositions and methods for oxygenation of nucleic acids containing 5-methylpyrimidine

• US 9,464,277 B2
• Filed: 04/20/2015
• Issued: 10/11/2016
• 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:
  US14/691,088
• Publication Number:
  US 20150218530A1
• Time in Patent Office: 540 Days
• Field of Search: None
• US Class Current: 1/1
• 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 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;

i. a UDP-associated sugar and a glucosyltransferase that transfers the sugar to the 5hmC, and

ii. a polypeptide that has 5-methylpyrimidine oxygenase activity and comprises an amino acid sequence that is at least 90% identical to amino acids 154-304 of SEQ ID NO;

2;

(b) cleaving the product of (a) 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.

2. The method of claim 1, wherein the modification-dependent endonuclease is AbaSI.

3. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to amino acids 154-304 of SEQ ID NO:

2.

4. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that identical to amino acids 154-304 of SEQ ID NO:

2.

5. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

2.

6. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

7. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is identical to SEQ ID NO:

2.

8. The method of claim 1, wherein the polypeptide further comprises a binding domain.

9. The method of claim 8, 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.

10. The method of claim 8, wherein the binding domain comprising a zinc finger or transcription activator-like (TAL) effector domain.

11. The method of claim 1, wherein the polypeptide is a fusion protein.

12. The method of claim 1, wherein the reacting step (a) is done in a buffer that does not contain ATP.

13. The method of claim 1, wherein the reacting step (a) is done in a buffer that contains ATP.

14. The method of claim 1, wherein the reacting step (a) is done at a pH in the range of pH 6 to pH 8.

15. The method of claim 1, wherein the reacting step (a) is done at a pH in the range of pH 6 to pH 7.5.

16. The method of claim 1, wherein the reacting step (a) is done in a buffer that comprises Fe(II) and α

-ketoglutarate.

17. The method of claim 1, wherein the UDP-associated sugar is UDP-glucose.

18. The method of claim 1, wherein the UDP-associated sugar is UDP-glucosamine.

19. The method of claim 1, wherein the glucosyltransferase is T4 DNA β

-glucosyltransferase.