Recombinant polymerizing enzymes for sequencing reactions

Patent metadata

Field	Value
Publication number	US-12545901-B2
Application number	US-202217690920-A
Country	US
Kind code	B2
Filing date	Mar 9, 2022
Priority date	Dec 19, 2016
Publication date	Feb 10, 2026
Grant date	Feb 10, 2026

Abstract

Compositions comprising modified recombinant polymerizing enzymes are provided, along with nucleic acid molecules encoding the modified polymerizing enzymes. In some aspects, methods of using such polymerizing enzymes to synthesize a nucleic acid molecule or to sequence a nucleic acid template are provided.

First claim

What is claimed is:

1 . A recombinant polymerizing enzyme having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 and comprising one or more of:
(a) a sequence that is at least 90% identical to amino acids 1-51 from SEQ ID NO: 2 in place of amino acids corresponding to amino acids 1-54 of SEQ ID NO: 1; (b) a sequence that is at least 90% identical to amino acids 445-449 from SEQ ID NO: 5 in place of amino acids corresponding to amino acids 429-433 in SEQ ID NO: 1; (c) a sequence that is at least 90% identical to amino acids 271-375 from SEQ ID NO: 5 in place of amino acids corresponding to amino acids 260-359 in SEQ ID NO: 1; or (d) a sequence that is at least 90% identical to amino acids 72-89 from SEQ ID NO: 5 in place of amino acids corresponding to amino acids 75-91 in SEQ ID NO: 1.

2 . The recombinant polymerizing enzyme of claim 1 , further comprising an alanine to threonine mutation at a position corresponding to A444 in SEQ ID NO: 1.

3 . The recombinant polymerizing enzyme of claim 1 , wherein the recombinant polymerizing enzyme comprises (a)-(d).

4 . The recombinant polymerizing enzyme of claim 1 , comprising two or more amino acid segments corresponding to amino acids selected from the group consisting of: amino acids 1-51 from SEQ ID NO: 2, amino acids 271-375 from SEQ ID NO: 5, amino acids 72-89 from SEQ ID NO: 5, and amino acids 445-449 from SEQ ID NO: 5.

5 . The recombinant polymerizing enzyme of claim 1 , comprising at least one amino acid insertion, deletion, or substitution at a position corresponding to a position selected from positions: M8, V51, N62, I71, L107, K131, K135, L142, G197, Y224, E239, V250, L253, Y281, I288, T301, R306, R308, D325, D341, K354, T368, E375, A437, A444, E466, D476, A484, E508, D510, K512, E515, K539, D570, and T571 in SEQ ID NO: 1.

6 . The recombinant polymerizing enzyme of claim 5 , wherein the one or more amino acid substitutions are selected from M8R, V51A, N62D, I71V, L107I, K131E, K135Q, L142K, G197D, Y224K, E239G, V250A, V250I, L253A, L253H, Y281H, I288L, T301C, R306Q, R308L, D325E, D341E, K354R, T368F, E375Y, A437G, A444T, E466K, D476H, A484E, E508R, D510K, D510R, K512Y, E515Q, K539E, D570S, and T571V.

7 . The recombinant polymerizing enzyme of claim 1 , comprising one or more amino acid substitutions at positions corresponding to I71, L107, K135, L142, G197, Y224, E239, V250, L253, E375, A437, A444, E466, D476, A484, E508, D510, K512, E515, K539, D570, and T571 in SEQ ID NO: 1.

8 . The recombinant polymerizing enzyme of claim 7 , wherein the one or more amino acid substitutions are selected from I71V, L107I, K135Q, L142K, G197V, Y224K, E239G, V250I, L253A, E375Y, A437G, A444V, E466K, D476H, A484E, E508R, D510R, K512Y, E515Q, K539E, D570S, and T571V.

9 . The recombinant polymerizing enzyme of claim 1 , comprising one or more amino acid substitutions at positions corresponding to K131, K135, L142, Y148, Y224, E239, V250, L253, R306, R308, E375, A437, E466, D476, A484, E508, D510, K512, E515, K539, D570, and T571 in SEQ ID NO: 1.

10 . The recombinant polymerizing enzyme of claim 9 , wherein the one or more amino acid substitutions are selected from K131E, K135Q, L142K, Y148I, Y224K, E239G, V250A, V250I, L253A, L253H, R306Q, R308L, E375Y, A437G, E466K, D476H, A484E, E508R, D510K, D510R, K512Y, E515Q, K539E, D570E, D570S, and T571V.

11 . The recombinant polymerizing enzyme of claim 9 , wherein the one or more amino acid substitutions are selected from K131E, L142K, Y148I, Y224K, E239G, V250A, L253H, E375Y, A437G, A484E, E508R, D510K, K512Y, E515Q, and D570E in SEQ ID NO: 1.

12 . The recombinant polymerizing enzyme of claim 1 , further comprising a purification tag.

13 . The recombinant polymerizing enzyme of claim 1 , further comprising a coupling group.

14 . The recombinant polymerizing enzyme of claim 1 , wherein the coupling group is a biotinylation sequence.

15 . The recombinant polymerizing enzyme of claim 1 immobilized on a surface.

16 . An isolated nucleic acid molecule that encodes the recombinant polymerizing enzyme of claim 1 .

17 . A method of sequencing a nucleic acid, the method comprising contacting the recombinant polymerizing enzyme of claim 1 with a sequencing reaction mixture.

18 . The recombinant polymerizing enzyme of claim 1 , having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 and comprising a sequence that is at least 90% identical to amino acids 1-51 from SEQ ID NO: 2 in place of amino acids corresponding to amino acids 1-54 of SEQ ID NO: 1.

19 . The recombinant polymerizing enzyme of claim 1 , having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 and comprising a sequence that is at least 90% identical to amino acids 445-449 from SEQ ID NO: 5 in place of amino acids corresponding to amino acids 429-433 in SEQ ID NO: 1.

20 . The recombinant polymerizing enzyme of claim 1 , having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 and comprising a sequence that is at least 90% identical to amino acids 271-375 from SEQ ID NO: 5 in place of amino acids corresponding to amino acids 260-359 in SEQ ID NO: 1.

21 . The recombinant polymerizing enzyme of claim 1 , having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 and comprising a sequence that is at least 90% identical to amino acids 72-89 from SEQ ID NO: 5 in place of amino acids corresponding to amino acids 75-91 in SEQ ID NO: 1.

Assignees

• Quantum Si Inc

Inventors

• Reed Brian
• Bhuiya Mohammad Wadud
• Pandey Manjula
• Lackey Jeremy
• Rothberg Jonathan M
• Christian Thomas

Classifications

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12Y207/07007

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

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title