IP Library › Granted Patent US 12,173,333
Granted Patent B2
US 12,173,333 · App. 17/858,801 · Granted Dec 24, 2024

Variants of family a DNA polymerase and uses thereof

Inventors: Thomas Ybert (Paris, FR); Elise Champion (Paris, FR); Omar Vivar (Le Kremlin-Bicêtre, FR); Ahmed Said (Le Kremlin-Bicêtre, FR)
Assignee: DNA Script
C12N9/1252C12Y207/07007
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Quick Facts
Patent No.
US 12,173,333
App. No.
17/858,801
Granted
Dec 24, 2024
Kind
B2
Abstract

The present invention relates to variant of Family A polymerases able to synthesize a nucleic acid fragment without template and to incorporate a reversible modified terminator nucleotide during the nucleic acid fragment synthesis. The present invention further relates to uses thereof for enzymatic synthesis of nucleic acid molecules.

Claims (15)

1. A variant of a DNA polymerase of family A, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, with at least one amino acid modification as compared to SEQ ID NO: 2, wherein the variant comprises a substitution at a position corresponding to E2335, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, and wherein the variant comprises an E2335G mutation.

2. The variant of claim 1 , wherein the variant further comprises at least one substitution in the amino acid sequence as set forth in SEQ ID NO: 3, selected from the group consisting of D2330E/R/H/K/T/V/A/G, Y2331F/W/P/H/M/L/V/A, S2332T/N/Q/V/A/G, Q2333N/T/S/A/G/V, L2334M/E/N/F/K/D/A/G, L2336M/E/N/F/K/D/A/G, R2337H/K/D/E/A/G/M/F, I2338V/A/G/L/T/S/D/K/M, L2339M/E/N/F/K/D/A/G/I, and/or wherein the variant comprises at least one substitution in the amino acid sequence as set forth in SEQ ID NO: 4 selected from the group consisting of P2322A/V/I/L/G/C, G2323C/P/A/V/K/D, G2324C/P/A/V/K/D, S2325L/N/M/V/T/A/G/D/K, I2326V/A/G/L/T/S/D/K/M, L2327M/E/N/F/K/D/A/G/I/V, A2328V/T/G, A2329V/T/G, and/or wherein the variant comprises at least one substitution in the amino acid sequence as set forth in SEQ ID NO: 5 selected from the group consisting of D2376E/R/H/K/T/V/A/G/N, D2377E/R/H/K/T/V/A/G/N, L2378M/E/N/F/K/D/A/G/I, R2379H/K/D/E/A/G/M/F, Q2380N/T/S/A/G/V, Q2381N/T/S/A/G/V, A2382V/T/G, K2383R/H/D/E/Q/N/C/A/G/S/T, Q2384N/T/S/G/V, I2385V/A/G/L/T/S/D/K/M, C2386G/P/A/V/S/N/Q/D/K, Y2387F/W/P/H/M/L/V/A, G2388C/P/A/V/K/D, I2389V/A/G/L/T/S/D/K/M, I2390V/A/G/L/T/S/D/K/M, Y2391F/W/P/H/M/L/V/A, and/or wherein the variant comprises at least one substitution in the amino acid sequence as set forth in SEQ ID NO: 6 selected from the group consisting of E2199G/A/N/T/S/D/K, W2200Y/F/P/L/I/V/A/G/E, R2201H/K/D/E/A/G/M/F/S/P, R2202H/K/D/E/G/M/F/S/P, I2203V/A/G/L/T/S/D/K/M/P, T2204S/N/Q/C/G/M/K/D, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

3. The variant of claim 1 , wherein the variant further comprises at least one amino acid modification at position(s) corresponding to residues selected from D2330, D2540 or E2541, excluding D2540N/A or E2541Q/A, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

4. The variant of claim 3 , wherein the at least one amino acid modification comprises one or more substitutions selected from D2330E/R/H/K/T/V/A/G, D2540E/K/R/H/Q/S/T/C and E2541D/R/H/K/N/S/T/C.

5. The variant of claim 1 , wherein the variant further comprises at least one amino acid modification at position(s) corresponding to residues selected from K2181, R2315, F2359, Y2391 and selected from wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

6. The variant of claim 5 , wherein the at least one amino acid modification comprises one or more substitutions selected from K2181R/H/D/E/Q/N/C/G/S/T, R2315H/K/D/E/A/G/M/F, F2359M/L/I/V/A/G/P/T/K/D and A2477V/T/G.

7. The variant of claim 1 , wherein the variant comprises at least a substitution or a combination of substitutions selected from the group consisting of:

L2336A+A2328V+Y2387F+E2335G+P2322A+L2334M, L2336A+A2328V+Y2387F+E2335G+P2322A+L2334G, L2336A+A2328V+Y2387F+E2335G+P2322A, L2336A+A2328V+Y2387F+E2335G+P2322V+L2334M, L2336A+A2328V+Y2387F+E2335G+P2322V+L2334G, L2336A+A2328V+Y2387F+E2335G+P2322V, L2336A+A2328V+Y2387F+E2335G+L2334M, L2336A+A2328V+Y2387F+E2335G+L2334G, L2336A+A2328V+Y2387F+E2335G, L2336A+A2328V+Y2387F, L2336A+A2328V+E2335G+P2322A+L2334M, L2336A+A2328V+E2335G+P2322A+L2334G, L2336A+A2328V+E2335G+P2322A, L2336A+A2328V+E2335G+P2322V+L2334M, L2336A+A2328V+E2335G+P2322V+L2334G, L2336A+A2328V+E2335G+P2322V, L2336A+A2328V+E2335G+L2334M, L2336A+A2328V+E2335G+L2334G, L2336A+A2328V+E2335G, L2336A+Y2387F+E2335G+P2322A+L2334M, L2336A+Y2387F+E2335G+P2322A+L2334G, L2336A+Y2387F+E2335G+P2322A, L2336A+Y2387F+E2335G+P2322V+L2334M, L2336A+Y2387F+E2335G+P2322V+L2334G, L2336A+Y2387F+E2335G+P2322V, L2336A+Y2387F+E2335G+L2334M, L2336A+Y2387F+E2335G+L2334G, L2336A+Y2387F+E2335G, L2336A+E2335G+P2322A+L2334M, L2336A+E2335G+P2322A+L2334G, L2336A+E2335G+P2322A, L2336A+E2335G+P2322V+L2334M, L2336A+E2335G+P2322V+L2334G, L2336A+E2335G+P2322V, L2336A+E2335G+L2334M, L2336A+E2335G+L2334G, L2336A+E2335G, A2328V+Y2387F+E2335G+P2322A+L2334M, A2328V+Y2387F+E2335G+P2322A+L2334G, A2328V+Y2387F+E2335G+P2322A, A2328V+Y2387F+E2335G+P2322V+L2334M, A2328V+Y2387F+E2335G+P2322V+L2334G, A2328V+Y2387F+E2335G+P2322V, A2328V+Y2387F+E2335G+L2334M, A2328V+Y2387F+E2335G+L2334G, A2328V+Y2387F+E2335G, A2328V+E2335G+P2322A+L2334M, A2328V+E2335G+P2322A+L2334G, A2328V+E2335G+P2322A, A2328V+E2335G+P2322V+L2334M, A2328V+E2335G+P2322V+L2334G, A2328V+E2335G+P2322V, A2328V+E2335G+L2334M, A2328V+E2335G+L2334G, A2328V+E2335G, Y2387F+E2335G+P2322A+L2334M, Y2387F+E2335G+P2322A+L2334G, Y2387F+E2335G+P2322A, Y2387F+E2335G+P2322V+L2334M, Y2387F+E2335G+P2322V+L2334G, Y2387F+E2335G+P2322V, Y2387F+E2335G+L2334M, Y2387F+E2335G+L2334G, Y2387F+E2335G, E2335G+P2322A+L2334M, E2335G+P2322A+L2334G, E2335G+P2322A, E2335G+P2322V+L2334M, E2335G+P2322V+L2334G, E2335G+P2322V, E2335G+L2334M, and E2335G+L2334G, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

8. A nucleic acid molecule encoding a variant of a DNA polymerase of family A as defined in claim 1 .

9. An expression vector comprising the nucleic acid molecule of claim 8 .

10. A host cell comprising the nucleic acid molecule of claim 8 .

11. A process for producing a variant of a DNA polymerase of family A as defined in claim 1 , wherein a host cell comprising a nucleic acid molecule encoding a variant of a DNA polymerase of family A as defined in claim 1 is cultivated under culture conditions allowing for expression of the nucleic acid encoding said variant, and wherein the variant is optionally retrieved.

12. A kit for performing a nucleotide incorporation reaction comprising a DNA polymerase of family A as defined in claim 1 , and one or more nucleotides.

13. The kit of claim 12 , wherein the kit comprises one or more 3′-O-modified nucleotides.

14. The kit of claim 12 , wherein the kit further comprises at least one nucleic acid primer.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 6, 2023
From: YBERT, THOMAS; CHAMPION, ELISE; SAID, AHMED
To: DNA SCRIPT
Reel/Frame 064812/0064 →
EMPLOYMENT CONTRACT OF UNLIMITED DURATION Recorded Sep 6, 2023
From: VIVAR, OMAR
To: DNA SCRIPT
Reel/Frame 064818/0495 →
Priority Claims (1)
EP 17306052 · Aug 7, 2017 · regional
Continuity (2)
Continuation 16636875
Related Publication 20230193222A1 · Jun 22, 2023
Cited By (1)
US 12,606,856