Compositions and methods for oxygenation of nucleic acids containing 5-methylpyrimidine
View Patent ↗5-methylpyrimidine oxygenases and their use in the modification of nucleic acids are described.
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.