IP Library › Granted Patent US 12,071,638
Granted Patent B2
US 12,071,638 · App. 17/544,647 · Granted Aug 27, 2024

Variants of terminal deoxynucleotidyl transferase and uses thereof

Inventors: Elise Champion (Paris, FR); Mikhael Soskine (Franconville, FR); Thomas Ybert (Paris, FR); Marc Delarue (Versailles, FR)
Assignees: DNA Script; Institut Pasteur; Centre National de la Recherche Scientifique
C12N9/1264C12N15/70C12Y207/07031
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,071,638
App. No.
17/544,647
Filed
Dec 7, 2021
Granted
Aug 27, 2024
Kind
B2
Art Unit
1652
USPC
435/194
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 (25)

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 3′-O-modified nucleotide into the nucleic acid fragment, and

wherein the TdT variant incorporates the 3′-O-modified nucleotide at a rate greater than that of a wild type TdT.

2. The TdT variant of claim 1 , 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.

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

4. 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.

5. 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.

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

7. 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.

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

9. 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.

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

11. 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.

12. 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.

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

14. 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.

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

16. The TdT variant of claim 4 , wherein said substitution of said methionine is R, Q, G, A, V, D, N, H or E.

17. The TdT variant of claim 5 , wherein the substitution of the arginine is N, L, K, H, G, D, A or P.

18. The TdT variant of claim 11 , wherein the substitution of the glutamic acid is N, L, T or S.

19. The TdT variant of claim 1 , wherein the amino acid sequence 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.

20. The TdT variant of claim 1 , wherein the amino acid sequence 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.

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

22. A method of producing a TdT variant comprising:

(a) culturing a host cell comprising a nucleic acid according to claim 19 under conditions suitable to express the nucleic acid encoding the TdT variant; and optionally

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