IP Library › Granted Patent US 10,752,887
Granted Patent B2
US 10,752,887 · App. 16/423,972 · Granted Aug 25, 2020

Variants of terminal deoxynucleotidyl transferase and uses thereof

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Quick Facts
Patent No.
US 10,752,887
App. No.
16/423,972
Filed
May 28, 2019
Granted
Aug 25, 2020
Kind
B2
Art Unit
1652
USPC
435/188
Abstract

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.

Claims (26)

1. 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.

2. The TdT variant of claim 1 , wherein the modified nucleotide is a 3′-O-modified nucleotide.

3. The TdT variant of claim 2 , wherein the 3′-O-modified nucleotide is a 3′-O—NH 2 -nucleoside triphosphate, a 3′-O-azidomethyl-nucleoside triphosphate, a 3′-O-allyl-nucleoside triphosphate, a 3′O-(2-nitrobenzyl)-nucleoside triphosphate, or a 3′-O-propargyl-nucleoside triphosphate.

4. The TdT variant of claim 1 , wherein the modified nucleotide is incorporated onto a free 3′-hydroxyl of a nucleic acid fragment.

5. The TdT variant of claim 1 , wherein the TdT variant incorporates the modified nucleotide at a rate greater than that of a wild type TdT.

6. The TdT variant of claim 1 , further comprising 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.

7. The TdT variant of claim 1 , further comprising 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.

8. The TdT variant of claim 1 , further comprising a substitution of arginine at position 208 with respect to SEQ ID NO:21.

9. The TdT variant of claim 1 , further comprising a substitution of arginine at position 324 with respect to SEQ ID NO:11 or SEQ ID NO:13.

10. The TdT variant of claim 1 , further comprising a substitution of arginine at position 331 with respect to SEQ ID NO:17.

11. The TdT variant of claim 1 , further comprising 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 TdT variant of claim 1 , further comprising a substitution of arginine at position 328 with respect to SEQ ID NO: 29.

13. The TdT variant of claim 1 , further comprising a substitution of glutamic acid at position 327 with respect to SEQ ID NO:11 or SEQ ID NO:13.

14. The TdT variant of claim 1 , further comprising a substitution of glutamic acid at position 334 with respect to SEQ ID NO: 17 or SEQ ID NO:21.

15. The TdT variant of claim 1 , further comprising a substitution of glutamic acid at position 331 with respect to SEQ ID NO:29.

16. The TdT variant of claim 1 , further comprising a substitution of glutamic acid at position 328 with respect to SEQ ID NO:2, SEQ ID NO:19, or SEQ ID NO:31.

17. The TdT variant of claim 1 , wherein the substitution of the cysteine is G, R, P, A, V, S, N, Q or D.

18. The TdT variant of claim 6 , wherein said substitution of said methionine is R, Q, G, A, V, D, N, H or E.

19. The TdT variant of claim 7 , wherein the substitution of the arginine is N, L, K, H, G, D, A or P.

20. The TdT variant of claim 13 , wherein the substitution of the glutamic acid is N, L, T or S.

21. The TdT variant 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.

22. The TdT variant 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.

23. A nucleic acid encoding a TdT variant as defined in claim 1 .

24. A method of producing a TdT variant comprising:

(a) culturing a host cell comprising a nucleic acid according to claim 23 under conditions suitable to express the nucleic acid encoding the TdT variant; and optionally

(b) recovering said TdT variant from the cell culture.