Composition and methods of 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 composition comprising:
(i) a buffer comprising Fe(II) and alpha-ketoglutarate; and
(ii) a polypeptide that has 5-methylpyrimidine oxygenase activity and has at least 90% identity to amino acids 154-304 of SEQ ID NO: 2.
2. A composition according to claim 1 , wherein the buffer does not contain ATP.
3. A composition according to claim 1 , wherein the buffer contains ATP.
4. A composition according to claim 1 , wherein the buffer is at a pH in the range of pH 6 to pH 8.
5. A composition according to claim 4 , wherein the buffer is at a pH in the range of pH 6 to pH 7.5.
6. A composition according to claim 1 , further comprising a nucleic acid.
7. A kit comprising a composition according to claim 1 wherein the buffer and the polypeptide are in separate containers.
8. A kit according to claim 7 , wherein the reaction buffer is at a pH in the range of pH 6 to pH 7.5.
9. A kit according to claim 7 , wherein the reaction buffer contains ATP.
10. A kit according to claim 7 , wherein the reaction buffer does not contain ATP.
11. A kit according to claim 7 , further comprising a nucleic acid.
12. A kit according to claim 7 , further comprising a reducing agent.
13. A kit according to claim 7 , further comprising a β-glycosyltransferase (BGT) and UDP-glucosamine.
14. A kit according to claim 7 , further comprising a β-glycosyltransferase (BGT) and UDP-glucose.
15. A kit according to claim 7 , further comprising a DNA glycosylase.
16. A kit according to claim 7 , further comprising an endonuclease.
17. 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 UDP-glucose or UDP-glucosamine; a glycosyltransferase for transferring glucose or glucosamine to the 5hmC; and a composition according to claim 1 ;
b. cleaving the glucosylated template 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.
18. A method according to claim 17 , wherein the modification-dependent endonuclease is AbaSI.
19. A composition according to claim 1 , wherein the polypeptide further comprises a binding domain.
20. A composition according to claim 19 , 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.
21. The composition according to claim 20 , wherein the binding domain comprises a zinc finger or transcription activator-like (TAL) effector domain.
22. The composition of claim 1 , wherein the polypeptide is a fusion protein.
23. The composition of claim 1 , wherein the polypeptide has at least 95% identity to amino acids 154-304 of SEQ ID NO: 2.
24. The composition of claim 1 , wherein the polypeptide is identical at amino acids 154-304 of SEQ ID NO: 2.
25. The composition of claim 1 , wherein the polypeptide is at least 90% identical to SEQ ID NO: 2.
26. The composition of claim 1 , wherein the polypeptide is at least 95% identical to SEQ ID NO: 2.
27. The composition of claim 1 , wherein the polypeptide is identical to SEQ ID NO: 2.