Granted Patent
B2
US 12,258,591 · App. 18/047,019 · Granted Mar 25, 2025
Modified helicases
Inventors:
Mark Bruce (Oxford, GB); Andrew John Heron (Oxford, GB); Ruth Moysey (Oxford, GB); Szabolcs Soeroes (Oxford, GB); Elizabeth Jayne Wallace (Oxford, GB); James White (Oxford, GB)
Assignee:
Oxford Nanopore Technologies PLC
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Abstract
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Dda helicase. The helicase controls the movement of the target polynucleotide through the pore. The invention also relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.
Claims (22)
1. A kit for characterising a target polynucleotide comprising:
(a) a pore and
(b) a helicase comprising:
(i) a first polypeptide comprising the pin domain and the 1A domain from a Dda helicase and not comprising any other domains from a Dda helicase, wherein at least one cysteine residue and/or at least one non-natural amino acid have been introduced into the pin domain and/or the 1A domain, and
(ii) a second polypeptide comprising the 2A domain, the tower domain and the hook domain from a Dda helicase and not comprising any other domains from a Dda helicase, wherein at least one cysteine residue and/or at least one non-natural amino acid have been introduced into the tower domain, wherein the helicase has the ability to control the movement of a polynucleotide;
wherein two or more of the introduced cysteines and/or non-natural amino acids are connected to one another;
wherein said first polypeptide is covalently attached to said second polypeptide; and
wherein the pore is (1) a transmembrane protein pore, (2) a solid state pore, or (3) an oligomer pore.
2. The kit of claim 1 , wherein the at least one non-natural amino acid in the first polypeptide and/or the at least one non-natural amino acid in the second polypeptide is 4-azido-L-phenylalanine (Faz).
3. The kit of claim 1 , wherein the helicase comprises at least one introduced cysteine residue and at least one introduced non-natural amino acid.
4. The kit of claim 3 , wherein the at least one introduced cysteine residue is connected to the at least one introduced non-natural amino acid.
5. The kit of claim 1 , wherein the first polypeptide is covalently attached to the second polypeptide via an amino acid.
6. The kit of claim 1 , wherein the helicase is further modified to increase or decrease its surface charge.
7. The kit of claim 1 , wherein the helicase is further modified by the removal of one or more native cysteine residues.
8. The kit of claim 7 , wherein the one or more cysteine residues are removed by substitution.
9. The kit of claim 1 , wherein the helicase is covalently attached to the pore.
10. The kit of claim 1 , wherein the pore is derived from Msp.
11. The kit of claim 1 , wherein the pore is a MspA pore.
12. The kit of claim 1 , further comprising an additional polynucleotide binding moiety, wherein the helicase is attached to the polynucleotide binding moiety.
13. The kit of claim 1 , wherein the kit comprises two or more of the helicases.
14. The kit of claim 1 , further comprising an additional polynucleotide binding moiety, wherein the pore is attached to the polynucleotide binding moiety.
15. The kit of claim 1 , wherein the kit further comprises a chip comprising a amphiphilic membrane.
Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jan 18, 2023
From: BRUCE, MARK; HERON, ANDREW JOHN; MOYSEY, RUTH; SOEROES, SZABOLCS; WALLACE, ELIZABETH JAYNE; WHITE, JAMES
To: OXFORD NANOPORE TECHNOLOGIES LTD.
Reel/Frame 062406/0293 →
CHANGE OF NAME
Recorded Jan 18, 2023
From: OXFORD NANOPORE TECHNOLOGIES LIMITED
To: OXFORD NANOPORE TECHNOLOGIES PLC
Reel/Frame 062409/0109 →
Priority Claims (3)
GB 1318464
· Oct 18, 2013
· national
GB 1404718
· Mar 17, 2014
· national
GB 1406151
· Apr 4, 2014
· national
Continuity (4)
Division
15028651
Division
PCTGB2014050175
· Jan 22, 2014
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