US 12,252,717 B2
Modified helicases
Andrew John Heron, Oxford (GB); James Anthony Clarke, Oxford (GB); Ruth Moysey, Oxford (GB); Elizabeth Jayne Wallace, Oxford (GB); Mark John Bruce, Oxford (GB); Lakmal Jayasinghe, Oxford (GB); Domenico Caprotti, Oxford (GB); Szabolcs Soeroes, Oxford (GB); Luke McNeill, Oxford (GB); David Antoni Alves, Oxford (GB); Rebecca Victoria Bowen, Oxford (GB); and John Milton, Oxford (GB)
Assigned to Oxford Nanopore Technologies PLC, Oxford (GB)
Filed by Oxford Nanopore Technologies PLC, Oxford (GB)
Filed on Oct. 18, 2022, as Appl. No. 18/047,589.
Application 18/047,589 is a continuation of application No. 17/064,329, filed on Oct. 6, 2020, granted, now 11,525,126.
Application 17/064,329 is a continuation of application No. 14/415,453, granted, now 10,808,231, previously published as PCT/GB2013/051925, filed on Jul. 18, 2013.
Claims priority of provisional application 61/774,862, filed on Mar. 8, 2013.
Claims priority of provisional application 61/673,452, filed on Jul. 19, 2012.
Prior Publication US 2023/0227799 A1, Jul. 20, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/14 (2006.01); C12N 9/90 (2006.01); C12Q 1/6827 (2018.01)
CPC C12N 9/14 (2013.01) [C12N 9/90 (2013.01); C12Q 1/6827 (2013.01); C12Y 306/04012 (2013.01); C12Y 599/01002 (2013.01); C12Y 599/01003 (2013.01); C07K 2319/80 (2013.01)] 13 Claims
 
1. A kit for characterising a target polynucleotide comprising (a) a pore and (b) a helicase comprising a polynucleotide binding domain, wherein two amino acid residues that are located on a surface of the helicase are covalently connected via a linkage between the two amino acid residues to form a covalently-closed structure, wherein the pore is a transmembrane pore, wherein the transmembrane pore is:
[i] derived from a hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) and WZA, or
[ii] a β-barrel pore or an α-helix bundle pore, wherein at least one amino acid of the two amino acid residues is substituted with cysteine, a non-natural amino acid or 4-azido-L-phenylalanine (Faz), and wherein the two amino acid residues that are located on the surface of the helicase are on one or more loop regions connecting α-helices and β-strands of the helicase and/or are spatially located proximal to the polynucleotide binding domain.