Compositions and methods for the transfer of a hexosamine to a modified nucleotide in a nucleic acid

• US 9,200,260 B2
• Filed: 03/14/2013
• Issued: 12/01/2015
• Est. Priority Date: 03/15/2012
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Correa, Ivan R. Jr., Dai, Nan, Bitinaite, Jurate, Pradhan, Sriharsa, Chin, Hang-Gyeong
• Primary Examiner(s): KIM, ALEXANDER D
• Application Number:
  US13/804,804
• Publication Number:
  US 20140127677A1
• Time in Patent Office: 992 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

Nucleic acids comprising β-glucosaminyloxy-5-methylcytosine; compositions, kits and methods of producing the nucleic acids using a glycosyltransferase; and methods of using the nucleic acids are described.

Claims

1. A cell free composition comprising:

an isolated DNA β

-glucosyltransferase;

UDP-glucosamine (UDP-GlcN); and

a restriction enzyme capable of cleaving a nucleic acid at a site comprising β

-glucosyl-5-hydroxymethylcytosine but not a site comprising β

-2-glucosaminyl-5-hydroxymethylcytosine.

2. The composition of claim 1, further comprising a buffer having a pH between 6 and 8.

3. The composition of claim 2, wherein the pH is between 6.8 and 7.4.

4. A cell free kit comprising:

a DNA β

-glucosyltransferase;

UDP-GlcN; and

a restriction enzyme capable of cleaving a nucleic acid at a site comprising β

-glucosyl-5-hydroxymethylcytosine but not a site comprising β

-2-glucosaminyl-5-hydroxymethylcytosine.

5. A method for labeling a modified nucleotide in a nucleic acid, comprising:

(a) combining;

i. a nucleic acid having a modified nucleotide

ii. an isolated DNA β

-glucosyltransferase; and

iii. UDP-glucosamine (UDP-GlcN), to produce a mixture; and

(b) incubating the mixture under conditions permitting the glucosamine to become covalently attached to the modified nucleotide in the nucleic acid; and

(c) adding to the mixture;

iv. a restriction enzyme capable of cleaving a nucleic acid at a site comprising β

-glucosyl-5-hydroxymethylcytosine but not a site comprising β

-2-glucosaminyl-5-hydroxymethylcytosine.

6. The method according to claim 5, wherein the modified nucleotide is hydroxymethylated cytosine (hmC).

7. The method according to claim 6, further comprising enriching, detecting, isolating and/or identifying the position of the glucosamine-attached nucleotide in the nucleic acid.