Methods and compositions for discrimination between cytosine and modifications thereof, and for methylome analysis
View Patent ↗Compositions and methods are provided for discrimination between cytosine and modifications thereof using cytidine deaminases and/or oxygenases. Variants of wild type cytidine deaminases are described which show reduced bias with respect to adjacent nucleotides upstream of the cytosine. The methods provide a rapid and convenient use of enzymes to obtain methylomes.
1. A composition comprising a protein variant having at least 90% sequence identity to:
(a) SEQ ID NO:1 and comprising an Asp at the position corresponding to position 180 in SEQ ID NO:1 and:
i. an amino acid that is not Ser at the position corresponding to position 45 in SEQ ID NO:1,
ii. an amino acid that is not Glu at the position corresponding to position 109 in SEQ ID NO:1, and
iii. an amino acid that is not Arg at the position corresponding to position 123 in SEQ ID NO:1;
(b) SEQ ID NO:2 and comprising an Arg at the position corresponding to position 29 in SEQ ID NO:2, an Arg at the position corresponding to position 104 in SEQ ID NO:2, an Arg at the position corresponding to position 123 in SEQ ID NO:2 and:
i. an amino acid that is not a Thr at the position corresponding to position 23 in SEQ ID NO:2,
ii. an amino acid that is not Gly at the position corresponding to position 25 in SEQ ID NO:2, and
iii. an amino acid that is not Cys at the position corresponding to position 69 in SEQ ID NO:2, or
(c) SEQ ID NO:3 (activation induced cytidine deaminase (AID)) and comprising a deletion at a position corresponding to position 117 of SEQ ID NO: 3.
2. A composition according to claim 1 , further comprising at least one of a purified oxygenase, a polymerase, a polynucleotide and/or at least one primer and dNTPs.
3. An in vitro mixture comprising a protein variant of claim 1 and a purified oxygenase.
4. An in vitro mixture according to claim 3 , wherein the oxygenase is a methyl pyrimidine oxygenase or a 5-methylcytosine oxygenase.
5. An in vitro mixture according to claim 3 , wherein the protein variant has at least 90% sequence identity to:
(a) SEQ ID NO:1 and comprises an Asp at the position corresponding to position 180 in SEQ ID NO:1 and:
i. a Trp at the position corresponding to position 45 in SEQ ID NO:1,
ii. a Gln at the position corresponding to position 109 in SEQ ID NO:1, and
iii. aft His at the position corresponding to position 123 in SEQ ID NO:1, or
(b) SEQ ID NO:2 and comprises an Arg at the position corresponding to position 29 in SEQ ID NO:2, an Arg at the position corresponding to position 104 in SEQ ID NO:2, an Arg at the position corresponding to position 123 in SEQ ID NO:2 and:
i. an Asn at the position corresponding to position 23 in SEQ ID NO:2,
ii. a Val at the position corresponding to position 25 in SEQ ID NO:2, and
iii. an Arg at the position corresponding to position 69 in SEQ ID NO:2.
6. An in vitro mixture according to claim 4 , wherein the methyl pyrimidine oxygenase is mYOX.
7. An in vitro mixture according to claim 4 , wherein the 5-methylcytosine oxygenase is TETI.
8. An in vitro mixture, comprising a purified cytidine deaminase and a purified DNA polymerase, wherein the purified cytidine deaminase has at least 90% sequence identity to:
(a) SEQ ID NO:1 and comprises an Asp at the position corresponding to position 180 in SEQ ID NO:1 and:
i. an amino acid that is not Ser at the position corresponding to position 45 in SEQ ID NO:1,
ii. an amino acid that is not Glu at the position corresponding to position 109 in SEQ ID NO:1, and
iii. an amino acid that is not Arg at the position corresponding to position 123 in SEQ ID NO:1,
(b) SEQ ID NO:2 and comprises an Arg at the position corresponding to position 29 in SEQ ID NO:2, an Arg at the position corresponding to position 104 in SEQ ID NO:2, an Arg at the position corresponding to position 123 in SEQ ID NO:2 and:
i. an amino acid that is not Thr at the position corresponding to position 23 in SEQ ID NO:2,
ii. an amino acid that is not Gly at the position corresponding to position 25 in SEQ ID NO:2, and
iii. an amino acid that is not Cys at the position corresponding to position 69 in SEQ ID NO:2, or
(c) SEQ ID NO:3 and comprises a deletion at a position corresponding to position 117 of SEQ ID NO: 3.
9. An in vitro mixture according to claim 8 , further comprising a polynucleotide and at least one primer and dNTPs.
10. A method for determining for a cytidine deaminase, a cytosine preference according to an adjacent nucleotide, comprising:
(a) reacting a polynucleotide containing a cytosine with a protein variant of claim 1 to convert the cytosine to uracil where the cytosine may be adjacent to any of adenine, guanine, thymine, uracil or cytosine;
(b) reacting the product of (a) with a glycosylase and an AP endonuclease so as to cleave the polynucleotide at the uracil;
(c) detecting the cleavage product from (b) to determine the activity of the cytidine deaminase for the cytosine adjacent to any of adenine, guanine, thymine, uracil or cytosine.
11. A method according to claim 10 , wherein the polynucleotide is single stranded.
12. A method according to claim 11 , wherein the polynucleotide is labeled at one end.
13. The composition of claim 1 , wherein the protein has at least 90% sequence identity to SEQ ID NO:1, an Asp at the position corresponding to position 180 in SEQ ID NO:1 and:
i. an amino acid that is not Ser at the position corresponding to position 45 in SEQ ID NO:1,
ii. an amino acid that is not Glu at the position corresponding to position 109 in SEQ ID NO:1, and
iii. an amino acid that is not Arg at the position corresponding to position 123 in SEQ ID NO:1.
14. The composition of claim 1 , wherein the protein has at least 90% sequence identity to SEQ ID NO:2 and comprises an Arg at the position corresponding to position 29 in SEQ ID NO:2, an Arg at the position corresponding to position 104 in SEQ ID NO:2, an Arg at the position corresponding to position 123 in SEQ ID NO:2 and:
i. an amino acid that is not Thr at the position corresponding to position 23 in SEQ ID NO:2,
ii. an amino acid that is not Gly at the position corresponding to position 25 in SEQ ID NO:2, and
iii. an amino acid that is not Cys at the position corresponding to position 69 in SEQ ID NO:2.
15. The composition of claim 1 , wherein the protein has at least 90% sequence identity to SEQ ID NO:3 and comprises a deletion at a position corresponding to position 117 of SEQ ID NO: 3.