Compositions and Methods for the Transfer of a Hexosamine to a Modified Nucleotide in a Nucleic Acid

• US 20140127677A1
• Filed: 03/14/2013
• Published: 05/08/2014
• 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
• Granted Patent: US 9,200,260 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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

1-26. -26. (canceled)

27. A composition comprising:

a β

-glycosyltransferase; and

UDP-glucosamine (UDP-GlcN).

28. The composition of claim 27, further comprising a buffer having a pH between 6 and 8.

29. The composition of claim 28, wherein the pH is between 6.8 and 7.4.

30. The composition of claim 27, further comprising 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.

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

combining a nucleic acid having a modified nucleotide with the composition of claim 27 under conditions permitting the glucosamine to become covalently attached to the modified nucleotide in the nucleic acid.

32. A method according to claim 31, further comprising reacting the covalently attached glucosamine with a label.

33. A method according to claim 31, wherein the modified nucleotide is hydroxymethylated cytosine (hmC).

34. A method according to claim 33, further comprising enriching, detecting, isolating and/or identifying the position of the glucosamine-attached nucleotide in the nucleic acid.

35. A method according to claim 34, where the nucleic acid is DNA.

36. A method for detecting a modified nucleotide in a genome or genomic fragment, comprising:

(a) combining a nucleic acid having a modified nucleotide with UDP-glucosamine (UDP-GlcN) and T4-β

-glucosyltransferase (BGT);

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

(c) detecting the modified nucleotide of (b) by means of a label or by modification-specific enzymatic cleavage.

37. A method according to claim 36, wherein the enzyme is capable of cleaving 5-hydroxymethylcytosine (5-hmC), β

-glucosyl-5-hydroxymethylcytosine (5-gmC), but not glucosamine-containing 5-hmC or labeled glucosamine-containing 5-hmC sites.

38. A method according to claim 36, wherein the enzyme is capable of cleaving cytosine, and/or 5-methylcytosine (5-mC), and/or 5-hydroxymethylcytosine (5-hmC), but not glucosamine-containing 5-hmC or labeled glucosamine-containing 5-hmC sites.

39. A method for detecting a modified nucleotide in a genome or genomic fragment, comprising:

(a) combining a nucleic acid having a modified nucleotide with UDP-glucosamine (UDP-GlcN) and T4-β

-glucosyltransferase (BGT);

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

(c) detecting the modified nucleotide of (b) by means of a nucleic acid modification-specific polymerase reaction.

40. A kit comprising a β

-glycosyltransferase and UDP-GlcN.

41. A nucleic acid comprising modified β

-glucosyl-5-hydroxymethylcytosine in which one or more of the hydroxyl groups at the 2- and/or 6-positions of the glucosyl moiety have been replaced with amino groups.

42. The nucleic acid of claim 41, wherein the modified β

-glucosyl-5-hydroxymethylcytosine is a β

-glucosaminyloxy-5-methylcytosine.

43. The nucleic acid of claim 42, wherein the β

-glucosaminyloxy-5-methylcytosine is β

-2-glucosaminyloxy-5-methylcytosine.

44. The nucleic acid of claim 42, wherein the modified β

-glucosyl-5-hydroxymethylcytosine is β

-6-glucosaminyloxy-5-methylcytosine.

45. A nucleic acid according to claim 41, further comprising a label.

46. A mammalian genome or genome fragment comprising a nucleic acid according to claim 41.

47. A method for labeling a modified nucleotide in a nucleic acid, comprising reacting a nucleic acid according to claim 41 with a label at a modified β

-glucosyl-5-hydroxymethylcytosine.

48. A method for detecting a modified nucleotide in a nucleic acid, comprising labeling a modified nucleotide in a nucleic acid according to the method of claim 47, and detecting the label.