DPO4 polymerase variants

Patent metadata

Field	Value
Publication number	US-12037577-B2
Application number	US-202217653441-A
Country	US
Kind code	B2
Filing date	Mar 3, 2022
Priority date	Nov 16, 2015
Publication date	Jul 16, 2024
Grant date	Jul 16, 2024

Abstract

Recombinant DPO4-type DNA polymerase variants with amino acid substitutions that confer modified properties upon the polymerase for improved single molecule sequencing applications are provided. Such properties may include enhanced binding and incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DPO4 variants and nucleotide analogs, as well as nucleic acids which encode the polymerases with the aforementioned phenotypes.

First claim

The invention claimed is:

1. An isolated recombinant DNA polymerase, which recombinant DNA polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:1, which recombinant polymerase comprises mutations at amino acid positions 76, 78, 79, 82, 83, and 86, wherein the identification of positions is relative to wildtype DPO4 polymerase (SEQ ID NO:1), which recombinant DNA polymerase further includes a deletion to remove the terminal 12 amino acids (the PIP box region) of the SEQ ID NO:1, and which recombinant DNA polymerase exhibits DNA polymerase activity.

2. The recombinant DNA polymerase of claim 1 , wherein the mutation at amino acid position 76 is selected from the group consisting of M76H, M76W, M76V, M76A, M76S, M76L, M76T, M76C, M76F, and M76Q.

3. The recombinant DNA polymerase of claim 1 , wherein the mutation at amino acid position 78 is selected from the group consisting of K78P, K78N, K78Q, K78T, K78L, K78V, K78S, K78F, K78E, K78M, K78A, K78I, K78H, K78Y, and K78G.

4. The recombinant DNA polymerase of claim 1 , wherein the mutation at amino acid position 79 is selected from the group consisting of E79L, E79M, E79W, E79V, E79N, E79Y, E79G, E79S, E79H, E79A, E79R, E79T, E79P, E79D, and E79F.

5. The recombinant DNA polymerase of claim 1 , wherein the mutation at amino acid position 82 is selected from the group consisting of Q82Y, Q82W, Q82N, Q82S, Q82H, Q82D, Q82E, Q82G, Q82M, Q82R, Q82K, Q82V, and Q82T.

6. The recombinant DNA polymerase of claim 1 , wherein the mutation at amino acid position 83 is selected from the group consisting of Q83G, Q83R, Q83S, Q83T, Q83I, Q83E, Q83M, Q83D, Q83A, Q83K, and Q83H.

7. The recombinant DNA polymerase of claim 1 , wherein the mutation at amino acid position 86 is selected from the group consisting of S86E, S86L, S86W, S86K, S86N, S86Q, S86V, S86M, S86T, S86G, S86R, S86A, and S86D.

8. The recombinant DNA polymerase of claim 1 , comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 2-7, 9-12, 14-19, and 21-46.

9. A composition comprising a recombinant DNA polymerase as set forth in claim 1 .

10. The composition of claim 9 , wherein the composition is present in a DNA sequencing system that comprises at least one non-natural nucleotide analog substrate.

11. An isolated recombinant DNA polymerase of claim 1 , wherein the recombinant DNA polymerase further comprises at least one mutation at an amino acid position selected from the group consisting of 5, 42, 56, 57, 62, 66, 141, 150, 152, 153, 155, 156, 184, 187, 188, 189, 190, 212, 214, 215, 217, 221, 226, 240, 241, 248, 289, 290, 291, 292, 293, 295, 297, 299, 300, 301, and 326.

12. The recombinant DNA polymerase of claim 11 , wherein the mutations at amino acid positions 76, 78, 79, 82, 83, and 86 are M76W, K78N, E79L, Q82W, Q83G, and S86E, respectively.

13. The recombinant DNA polymerase of claim 11 , wherein at least one of the following is satisfied:
the mutation at amino acid position 5 is F5Y
the mutation at amino acid position 42 is A42V;
the mutation at amino acid position 56 is K56Y;
the mutation at amino acid position 57 is A57P;
the mutation at amino acid position 62 is V62R;
the mutation at amino acid position 66 is K66R;
the mutation at amino acid position 141 is T141S;
the mutation at amino acid position 150 is F150L;
the mutation at amino acid position 152 is K152A, K152G, K152M, or K152P;
the mutation at amino acid position 153 is I153F, I153Q or I153W;
the mutation at amino acid position 155 is A155L, A155M, A155N, A155V, or A155G;
the mutation at amino acid position 156 is D156Y or D156W;
the mutation at amino acid position 184 is P184L;
the mutation at amino acid position 187 is G187W, G187D, G187P, or G187E;
the mutation at amino acid position 188 is N188Y;
the mutation at amino acid position 189 is I189W;
the mutation at amino acid position 190 is T190Y, T190D, or T190E;
the mutation at amino acid position 212 is K212V, K212L, or K212A;
the mutation at amino acid position 214 is K214S;
the mutation at amino acid position 215 is G215F;
the mutation at amino acid position 217 is I217V;
the mutation at amino acid position 221 is K221D, K221E, or K221Q;
the mutation at amino acid position 226 is I226F;
the mutation at amino acid position 240 is R240S or R240T;
the mutation at amino acid position 241 is V241N or V241R;
the mutation at amino acid position 248 is I248A or I248T;
the mutation at amino acid position 289 is V289W;
the mutation at amino acid position 290 is T290K or T290R;
the mutation at amino acid position 291 is E291S;
the mutation at amino acid position 292 is D292Y;
the mutation at amino acid position 293 is L293F or L293W;
the mutation at amino acid position 295 is I295Y;
the mutation at amino acid position 297 is S297H;
the mutation at amino acid position 299 is G299L;
the mutation at amino acid position 300 is R300E or R300V;
the mutation at amino acid position 301 is T301R; or
the mutation at amino acid position 326 is D326E.

14. A composition comprising a recombinant DNA polymerase as set forth in claim 11 .

15. The composition of claim 14 , wherein the composition is present in a DNA sequencing system that comprises at least one non-natural nucleotide analog substrate.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Kokoris Mark Stamatios
• Prindle Marc
• Nabavi Melud
• Ostrander Craig
• Lehmann Taylor
• Vellucci Samantha
• Kovarik Michael
• Chase Jack
• Busam Robert
• Lahman Miranda

Classifications

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12N15/10 Primary

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title