IP Library › Granted Patent US 10,760,063
Granted Patent B2
US 10,760,063 · App. 16/113,757 · Granted Sep 1, 2020

Modified template-independent enzymes for polydeoxynucleotide synthesis

Inventors: J. William Efcavitch (San Diego, CA); Julie L. Tubbs (San Diego, CA); Prem Sinha (San Diego, CA); Boguslaw Stec (San Diego, CA)
Assignee: Molecular Assemblies, Inc.
C12N9/1264C12P19/34C12Y207/07031
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Quick Facts
Patent No.
US 10,760,063
App. No.
16/113,757
Granted
Sep 1, 2020
Kind
B2
Abstract

The invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of binding nucleotides comprising removable 3′-O-blocking moieties to a nucleic acid initiator, without the use of a template. The invention further includes the identified polymerases, and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences.

Claims (25)

1. A modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation selected from the group consisting of E180D and W450H, M192E, E180K, E180K and R454A, R454K, M192K, M192K and E180K, Q455I, M192W, E180R, E180L, M192R, N474R, R461V, E180K and R454I, E180D and M192E, E180D and M192E and R454T, and E180K and W450H, said modified TdT capable of adding a nucleotide analog comprising a removable 3′-O-blocking moiety to the 3′-OH of a nucleic acid initiator in the absence of a nucleic acid template.

2. The modified TdT of claim 1 , wherein the modified TdT comprises N474R.

3. The modified TdT of claim 1 , wherein the modified TdT comprises R461V.

4. The modified TdT of claim 1 , wherein the modified TdT comprises R454K.

5. The modified TdT of claim 1 , wherein the modified TdT comprises E180K and R454A.

6. The modified TdT of claim 1 , wherein the modified TdT comprises M192K and E180K.

7. The modified TdT of claim 1 , wherein the modified TdT comprises E180K.

8. The modified TdT of claim 1 , wherein the modified TdT comprises M192K.

9. The modified TdT of claim 1 , wherein the modified TdT comprises E180L.

10. The modified TdT of claim 1 , wherein the modified TdT comprises E180D and W450H.

11. The modified TdT of claim 1 , wherein the modified TdT comprises M192E.

12. The modified TdT of claim 1 , wherein the modified TdT comprises Q455I.

13. The modified TdT of claim 1 , wherein the modified TdT comprises E180K and W450H.

14. The modified TdT of claim 1 , wherein the modified TdT comprises M192W.

15. The modified TdT of claim 1 , wherein the modified TdT comprises E180K and R454I.

16. The modified TdT of claim 1 , wherein the modified TdT comprises E180R.

17. The modified TdT of claim 1 , wherein the modified TdT comprises M192R.

18. The modified TdT of claim 1 , wherein the modified TdT comprises E180D and M192E.

19. The modified TdT of claim 1 , wherein the modified TdT comprises E180D, M192E, and R454T.

20. The modified TdT of claim 1 , wherein the modified TdT exhibits an increased rate of incorporation of modified nucleotides, compared to native TdT.

21. The modified TdT of claim 1 , wherein the modified TdT is capable of incorporating modified nucleotides at a reaction temperature of about 30° C.

22. The modified TdT of claim 1 , wherein the modified TdT is capable of incorporating modified nucleotides at a reaction temperature from 30° C. to 80° C.

23. The modified TdT of claim 1 , wherein the modified TdT is capable of incorporating modified nucleotides at a concentration of 1000 μM or less.

24. The modified TdT of claim 16 , wherein the modified TdT is capable of incorporating modified nucleotides at a concentration of 100 μM or less.

25. The modified TdT of claim 1 , wherein the modified TdT comprises a nucleic acid sequence being at least 90% identical to SEQ ID NOS. 2, 4, 6, 8 or 9.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 21, 2020
From: EFCAVITCH, J. WILLIAM; TUBBS, JULIE L.; SINHA, PREM; STEC, BOGUSLAW
To: MOLECULAR ASSEMBLIES, INC.
Reel/Frame 053269/0160 →
Continuity (3)
Continuation In Part 14918212 · Oct 20, 2015
Provisional Application 62065976 · Oct 20, 2014
Related Publication 20200190490A1 · Jun 18, 2020
Cited By (1)
US 12,371,680