Variants of terminal deoxynucleotidyl transferase and uses thereof
The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.
1. A method of synthesizing a polynucleotide having a predetermined sequence, the method comprising the steps of:
a) providing an initiator having a 3′-terminal nucleotide having a free 3′-hydroxyl;
b) repeating cycles of:
(i) contacting under elongation conditions the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a terminal deoxynucleotidyl transferase (TdT) variant such that the initiator or elongated fragments are elongated by incorporation of a 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated fragments
wherein the TdT variant comprises an amino acid sequence at least 90% identical to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:29, or SEQ ID NO:31 with an amino acid substitution of cysteine at position 173, wherein the TdT variant (i) is capable of synthesizing a nucleic acid fragment without a template and (ii) is capable of incorporating a modified nucleotide into the nucleic acid fragment, and
(ii) deblocking the elongated fragments to form elongated fragments having free 3′-hydroxyls, until the polynucleotide is synthesized.
2. The method of claim 1 , wherein said 3′-O-blocked nucleoside triphosphate is a 3′-O—NH 2 -nucleoside triphosphate, a 3′-O-azidomethyl-nucleoside triphosphate, a 3′-O-allyl-nucleoside triphosphate, or a 3′-O-(2-nitrobenzyl)-nucleoside triphosphate.
3. The method of claim 1 , wherein the modified nucleotide is incorporated onto a free 3′-hydroxyl of a nucleic acid fragment.
4. The method of claim 1 , wherein the TdT variant incorporates the modified nucleotide at a rate greater than that of a wild type TdT.
5. The method of claim 1 , wherein the TdT variant further comprises a substitution of methionine at position 63 with respect to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:29.
6. The method of claim 1 , wherein the TdT variant further comprises a substitution of arginine at position 207 with respect to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:29 of SEQ ID NO:31.
7. The method of claim 6 , wherein the substitution of the arginine is N, L, K, H, G, D, A or P.
8. The method of claim 1 , wherein the TdT variant further comprises a substitution of arginine at position 208 with respect to SEQ ID NO:21.
9. The method of claim 1 , wherein the TdT variant further comprises a substitution of arginine at position 324 with respect to SEQ ID NO:11 or SEQ ID NO:13.
10. The method of claim 1 , wherein the TdT variant further comprises a substitution of arginine at position 331 with respect to SEQ ID NO:17.
11. The method of claim 1 , wherein the TdT variant further comprises a substitution of arginine at position 325 with respect to SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:31.
12. The method of claim 1 , wherein the TdT variant further comprises a substitution of arginine at position 328 with respect to SEQ ID NO: 29.
13. The method of claim 1 , wherein the TdT variant further comprises a substitution of glutamic acid at position 327 with respect to SEQ ID NO:11 or SEQ ID NO:13.
14. The method of claim 13 , wherein the substitution of the glutamic acid is N, L, T or S.
15. The method of claim 1 , wherein the TdT variant further comprises a substitution of glutamic acid at position 334 with respect to SEQ ID NO: 17 or SEQ ID NO:21.
16. The method of claim 1 , wherein the TdT variant further comprises a substitution of glutamic acid at position 331 with respect to SEQ ID NO:29.
17. The method of claim 1 , wherein the TdT variant further comprises a substitution of glutamic acid at position 328 with respect to SEQ ID NO:2, SEQ ID NO:19, or SEQ ID NO:31.
18. The method of claim 1 , wherein the TdT variant further comprises of the cysteine is G, R, P, A, V, S, N, Q or D.
19. The method of claim 1 , wherein the substitution of said methionine is R, Q, G, A, V, D, N, H or E.
20. The method of claim 1 , wherein the amino acid sequence is at least 95% identical to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:29, or SEQ ID NO:31.
21. The method of claim 1 , wherein the amino acid sequence is at least 97% identical to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:29, or SEQ ID NO:31.
22. A kit for performing a nucleotide incorporation reaction comprising
a terminal deoxynucleotidyl transferase (TdT) variant comprising an amino acid sequence at least 90% identical to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:29, or SEQ ID NO:31 with an amino acid substitution of cysteine at position 173, wherein the TdT variant (i) is capable of synthesizing a nucleic acid fragment without a template and (ii) is capable of incorporating a modified nucleotide into the nucleic acid fragment,
one or more nucleotides, and
a nucleic acid primer.