IP Library › Granted Patent US 11,390,856

Granted Patent B2

US 11,390,856 · App. 16/636,875 · Granted Jul 19, 2022

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

C12N9/1252 C12Y207/07007

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Quick Facts

Patent No.: US 11,390,856
App. No.: 16/636,875
Granted: Jul 19, 2022
Kind: B2

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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 (196)

1. A DNA polymerase theta variant comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:02, wherein the DNA polymerase theta variant comprises an amino acid sequence having at least one substitution or a combination of substitutions selected from:

L2336A, A2328V, Y2387F, E2335G, P2322A, and L2334M;

L2336A, A2328V, Y2387F, E2335G, P2322A, and L2334G;

L2336A, A2328V, Y2387F, E2335G, and P2322A;

L2336A, A2328V, Y2387F, E2335G, P2322V, and L2334M;

L2336A, A2328V, Y2387F, E2335G, P2322V, and L2334G;

L2336A, A2328V, Y2387F, E2335G, and P2322V;

L2336A, A2328V, Y2387F, E2335G, and L2334M;

L2336A, A2328V, Y2387F, E2335G, and L2334G;

L2336A, A2328V, Y2387F, E2335A, P2322A, and L2334M;

L2336A, A2328V, Y2387F, E2335A, P2322A, and L2334G;

L2336A, A2328V, Y2387F, E2335A, and P2322A;

L2336A, A2328V, Y2387F, E2335A, P2322V, and L2334M;

L2336A, A2328V, Y2387F, E2335A, P2322V, and L2334G;

L2336A, A2328V, Y2387F, E2335A, and P2322V;

L2336A, A2328V, Y2387F, E2335A, and L2334M;

L2336A, A2328V, Y2387F, E2335A, and L2334G;

L2336A, A2328V, Y2387F, P2322A, and L2334M;

L2336A, A2328V, Y2387F, P2322A, and L2334G;

L2336A, A2328V, Y2387F, and P2322A;

L2336A, A2328V, Y2387F, P2322V, and L2334M;

L2336A, A2328V, Y2387F, P2322V, and L2334G;

L2336A, A2328V, Y2387F, and P2322V;

L2336A, A2328V, Y2387F, and L2334M;

L2336A, A2328V, Y2387F, and L2334G;

L2336A, A2328V, E2335G, P2322A, and L2334M;

L2336A, A2328V, E2335G, P2322A, and L2334G;

L2336A, A2328V, E2335G, and P2322A;

L2336A, A2328V, E2335G, P2322V, and L2334M;

L2336A, A2328V, E2335G, P2322V, and L2334G;

L2336A, A2328V, E2335G, and P2322V;

L2336A, A2328V, E2335G, and L2334M;

L2336A, A2328V, E2335G, and L2334G;

L2336A, A2328V, E2335A, P2322A, and L2334M;

L2336A, A2328V, E2335A, P2322A, and L2334G;

L2336A, A2328V, E2335A, and P2322A;

L2336A, A2328V, E2335A, P2322V, and L2334M;

L2336A, A2328V, E2335A, P2322V, and L2334G;

L2336A, A2328V, E2335A, and P2322V;

L2336A, A2328V, E2335A, and L2334M;

L2336A, A2328V, E2335A, and L2334G;

L2336A, A2328V, P2322A, and L2334M;

L2336A, A2328V, P2322A, and L2334G;

L2336A, A2328V, and P2322A;

L2336A, A2328V, P2322V, and L2334M;

L2336A, A2328V, P2322V, and L2334G;

L2336A, A2328V, and P2322V;

L2336A, A2328V, and L2334M;

L2336A, A2328V, and L2334G;

L2336A, Y2387F, E2335G, P2322A, and L2334M;

L2336A, Y2387F, E2335G, P2322A, and L2334G;

L2336A, Y2387F, E2335G, and P2322A;

L2336A, Y2387F, E2335G, P2322V, and L2334M;

L2336A, Y2387F, E2335G, P2322V, and L2334G;

L2336A, Y2387F, E2335G, and P2322V;

L2336A, Y2387F, E2335G, and L2334M;

L2336A, Y2387F, E2335G, and L2334G;

L2336A, Y2387F, E2335A, P2322A, and L2334M;

L2336A, Y2387F, E2335A, P2322A, and L2334G;

L2336A, Y2387F, E2335A, and P2322A;

L2336A, Y2387F, E2335A, P2322V, and L2334M;

L2336A, Y2387F, E2335A, P2322V, and L2334G;

L2336A, Y2387F, E2335A, and P2322V;

L2336A, Y2387F, E2335A, and L2334M;

L2336A, Y2387F, E2335A, and L2334G;

L2336A, Y2387F, P2322A, and L2334M;

L2336A, Y2387F, P2322A, and L2334G;

L2336A, Y2387F, and P2322A;

L2336A, Y2387F, P2322V, and L2334M;

L2336A, Y2387F, P2322V, and L2334G;

L2336A, Y2387F, and P2322V;

L2336A, Y2387F, and L2334M;

L2336A, Y2387F, and L2334G;

L2336A, E2335G, P2322A, and L2334M;

L2336A, E2335G, P2322A, and L2334G;

L2336A, E2335G, and P2322A;

L2336A, E2335G, P2322V, and L2334M;

L2336A, E2335G, P2322V, and L2334G;

L2336A, E2335G, and P2322V;

L2336A, E2335G, and L2334M;

L2336A, E2335G, and L2334G;

L2336A, E2335A, P2322A, and L2334M;

L2336A, E2335A, P2322A, and L2334G;

L2336A, E2335A, and P2322A;

L2336A, E2335A, P2322V, and L2334M;

L2336A, E2335A, P2322V, and L2334G;

L2336A, E2335A, and P2322V;

L2336A, E2335A, and L2334M;

L2336A, E2335A, and L2334G;

L2336A, P2322A, and L2334M;

L2336A, P2322A, and L2334G;

L2336A and P2322A;

L2336A, P2322V, and L2334M;

L2336A, P2322V, and L2334G;

L2336A and P2322V;

L2336A and L2334M;

L2336A and L2334G;

A2328V, Y2387F, E2335G, P2322A, and L2334M;

A2328V, Y2387F, E2335G, P2322A, and L2334G;

A2328V, Y2387F, E2335G, and P2322A;

A2328V, Y2387F, E2335G, P2322V, and L2334M;

A2328V, Y2387F, E2335G, P2322V, and L2334G;

A2328V, Y2387F, E2335G, and P2322V;

A2328V, Y2387F, E2335G, and L2334M;

A2328V, Y2387F, E2335G, and L2334G;

A2328V, Y2387F, E2335A, P2322A, and L2334M;

A2328V, Y2387F, E2335A, P2322A, and L2334G;

A2328V, Y2387F, E2335A, and P2322A;

A2328V, Y2387F, E2335A, P2322V, and L2334M;

A2328V, Y2387F, E2335A, P2322V, and L2334G;

A2328V, Y2387F, E2335A, and P2322V;

A2328V, Y2387F, E2335A, and L2334M;

A2328V, Y2387F, E2335A, and L2334G;

A2328V, Y2387F, P2322A, and L2334M;

A2328V, Y2387F, P2322A, and L2334G;

A2328V, Y2387F, and P2322A;

A2328V, Y2387F, P2322V, and L2334M;

A2328V, Y2387F, P2322V, and L2334G;

A2328V, Y2387F, and P2322V;

A2328V, Y2387F, and L2334M;

A2328V, Y2387F, and L2334G;

A2328V, E2335G, P2322A, and L2334M;

A2328V, E2335G, P2322A, and L2334G;

A2328V, E2335G, and P2322A;

A2328V, E2335G, P2322V, and L2334M;

A2328V, E2335G, P2322V, and L2334G;

A2328V, E2335G, and P2322V;

A2328V, E2335G, and L2334M;

A2328V, E2335G, and L2334G;

A2328V, E2335A, P2322A, and L2334M;

A2328V, E2335A, P2322A, and L2334G;

A2328V, E2335A, and P2322A;

A2328V, E2335A, P2322V, and L2334M;

A2328V, E2335A, P2322V, and L2334G;

A2328V, E2335A, and P2322V;

A2328V, E2335A, and L2334M;

A2328V, E2335A, and L2334G;

A2328V, P2322A, and L2334M;

A2328V, P2322A, and L2334G;

A2328V and P2322A;

A2328V, P2322V, and L2334M;

A2328V, P2322V, and L2334G;

A2328V and P2322V;

A2328V and L2334M;

A2328V and L2334G;

Y2387F, E2335G, P2322A, and L2334M;

Y2387F, E2335G, P2322A, and L2334G;

Y2387F, E2335G, and P2322A;

Y2387F, E2335G, P2322V, and L2334M;

Y2387F, E2335G, P2322V, and L2334G;

Y2387F, E2335G, and P2322V;

Y2387F, E2335G, and L2334M;

Y2387F, E2335G, and L2334G;

Y2387F, E2335A, P2322A, and L2334M;

Y2387F, E2335A, P2322A, and L2334G;

Y2387F, E2335A, and P2322A;

Y2387F, E2335A, P2322V, and L2334M;

Y2387F, E2335A, P2322V, and L2334G;

Y2387F, E2335A, and P2322V;

Y2387F, E2335A, and L2334M;

Y2387F, E2335A, and L2334G;

Y2387F, P2322A, and L2334M;

Y2387F, P2322A, and L2334G;

Y2387F and P2322A;

Y2387F, P2322V, and L2334M;

Y2387F, P2322V, and L2334G;

Y2387F and P2322V;

Y2387F and L2334M;

Y2387F and L2334G;

E2335G, P2322A, and L2334M;

E2335G, P2322A, and L2334G;

E2335G and P2322A;

E2335G, P2322V, and L2334M;

E2335G, P2322V, and L2334G;

E2335G and P2322V;

E2335G and L2334M;

E2335G and L2334G;

E2335A, P2322A, and L2334M;

E2335A, P2322A, and L2334G;

E2335A and P2322A;

E2335A, P2322V, and L2334M;

E2335A, P2322V, and L2334G;

E2335A and P2322V;

E2335A and L2334M;

E2335A and L2334G;

P2322A and L2334M;

P2322A and L2334G;

P2322A;

P2322V and L2334M;

P2322V and L2334G;

P2322V;

L2334M; and

L2334G;

wherein the amino acid positions are numbered by reference to the amino acid sequence of SEQ ID NO:01, and amino acid positions D2330, D2540, and E2541 do not have an amino acid substitution, wherein the DNA polymerase theta variant is able to synthesize a nucleic acid fragment without a template and is able to incorporate a 3′-O-amino reversible modified terminator nucleotide into a nucleic acid fragment;

wherein the variant has an increased ability to incorporate the 3′-O-amino reversible modified terminator nucleotide into a nucleic acid fragment as compared to a DNA polymerase comprising the sequence of SEQ ID NO:01.

2. The DNA polymerase theta variant of claim 1 , which has at least 95%, 97%, 98% or 99% identity to the full length amino acid sequence set forth in SEQ ID NO:01.

Assignments (3)

EMPLOYMENT CONTRACT OF UNLIMITED DURATION Recorded Jul 19, 2022

From: VIVAR, OMAR

To: DNA SCRIPT

Reel/Frame 060751/0338 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 24, 2022

From: YBERT, THOMAS; CHAMPION, ELISE; SAID, AHMED

To: DNA SCRIPT

Reel/Frame 059091/0292 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 8, 2021

From: YBERT, THOMAS

To: DNA SCRIPT SAS