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

Patent metadata

Field	Value
Publication number	US-9464277-B2
Application number	US-201514691088-A
Country	US
Kind code	B2
Filing date	Apr 20, 2015
Priority date	Mar 15, 2012
Publication date	Oct 11, 2016
Grant date	Oct 11, 2016

Abstract

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

First claim

What is claimed:

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.

Assignees

• New England Biolabs Inc

Inventors

• Zheng Yu
• Saleh Lana
• Pais June
• Dai Nan
• Roberts Richard J
• Correa Jr Ivan R
• Mabuchi Megumu
• Vaisvila Romualdas

Classifications

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12N9/0069 Primary

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q2521/301

  Endonuclease · CPC title

• C07H21/00

  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 title