Compositions and methods for the transfer of a hexosamine to a modified nucleotide in a nucleic acid
View Patent ↗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.
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.