Granted Patent B2

US 12,630,851 · App. 17/509,988 · Granted May 19, 2026

Inventors: David Jackson Stoddart (Oxford, GB); James White (Oxford, GB)

Assignee: Oxford Nanopore Technologies PLC

C12P19/34C12N9/1241C12Q1/68C12Q1/6869C12Y207/07

Abstract

Claims (23)

1 . A population of double stranded polynucleotide MuA substrates for modifying a template polynucleotide, wherein each substrate comprises a first strand comprising a 5′ overhang comprising universal nucleotides and a 5′ phosphate, and configured to repair a nick in the template polynucleotide created by a MuA transferase.

2 . The population of double stranded polynucleotide MuA substrates of claim 1 , wherein the universal nucleotide is selected from the group consisting of hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole, and phenyl (C6-aromatic ring).

3 . The population of double stranded polynucleotide MuA substrates of claim 1 , wherein the universal nucleotide is selected from the group consisting of 2′-deoxyinosine, inosine, 7-deaza-2′-deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 4-nitroindole 2′-deoxyribonucleoside, 4-nitroindole ribonucleoside, 5-nitroindole 2′-deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2′-deoxyribonucleoside, 6-nitroindole ribonucleoside, 3-nitropyrrole 2′-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, an acyclic sugar analogue of hypoxanthine, nitroimidazole 2′-deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole 2′-deoxyribonucleoside, 4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole 2′-deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside, 5-nitroindazole 2′-deoxyribonucleoside, 5-nitroindazole ribonucleoside, 4-aminobenzimidazole 2′-deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside, and phenyl C-2′-deoxyribosyl nucleoside.

4 . The population of double stranded polynucleotide MuA substrates of claim 1 , wherein the at least one 5′ overhang is five nucleotides in length.

5 . The population of double stranded polynucleotide MuA substrates of claim 1 , wherein

(a) each substrate comprises a 5′ overhang and a 5′ phosphate at one end and a hairpin loop at the other end;

(b) a proportion of the substrates in the population comprise 5′ overhang and a 5′ phosphate at one end and a hairpin loop at the other end and a proportion of the substrates in the population are Y substrates with a 5′ overhang and a 5′ phosphate at one end and a region that is not complementary at the other end; or

(c) each substrate is a Y substrate with a 5′ overhang and a 5′ phosphate at one end and a region that is not complementary at the other end.

6 . The population of double stranded polynucleotide MuA substrates of claim 1 , wherein each substrate comprises a selectable binding moiety.

7 . A kit for modifying a template polynucleotide, wherein the kit comprises:

(a) a population of double stranded MuA substrates of claim 1 ; and

(b) a MuA transposase.

8 . The kit of claim 7 , further comprising one or more of: components of a membrane, components of a transmembrane pore, and a polynucleotide binding protein.

9 . A population of double stranded polynucleotide MuA substrates for modifying a template polynucleotide, wherein each substrate comprises (i) at least one 5′ overhang and a 5′ phosphate, and (ii) at least one nucleotide in the same strand as the at least one overhang which comprises a nucleoside that is not present in the template polynucleotide.

10 . The population of double stranded polynucleotide MuA substrates of claim 9 , wherein the template polynucleotide comprises the nucleosides deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).

11 . The population of double stranded polynucleotide MuA substrates of claim 9 , wherein the nucleoside that is not present in the template polynucleotide is abasic, adenosine (A), uridine (U), 5-methyluridine (m5U), cytidine (C) or guanosine (G) or comprises urea, 5, 6 dihydroxythymine, thymine glycol, 5-hydroxy-5 methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihdrothimine, methyltartronylurea, 7, 8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methy-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine, 5-hydroxy-uracil, 3-methyladenine, 7-methylguanine, 1,N6-ethenoadenine, hypoxanthine, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil or a cis-syn-cyclobutane pyrimidine dimer.

12 . The population of double stranded polynucleotide MuA substrates of claim 9 , wherein the at least one nucleotide is 10 nucleotides or fewer from the at least one 5′ overhang.

13 . The population of double stranded polynucleotide MuA substrates of claim 9 , wherein the at least one nucleotide is the first nucleotide in the at least one 5′ overhang.

14 . The population of double stranded polynucleotide MuA substrates of claim 9 , wherein all of the nucleotides in the at least one 5′ overhang comprise a nucleoside that is not present in the template polynucleotide.

15 . The population of double stranded polynucleotide MuA substrates of claim 9 , wherein each substrate comprises a 5′ overhang and a 5′ phosphate at one end and a hairpin loop at the other end.

16 . The population of double stranded polynucleotide MuA substrates of claim 9 , wherein each substrate is a Y substrate with a 5′ overhang and a 5′ phosphate at one end and a region that is not complementary at the other.

17 . The population of double stranded polynucleotide MuA substrates of claim 9 , wherein a proportion of the substrates in the population comprise a 5′ overhang and a 5′ phosphate at one end and a hairpin loop at the other end and a proportion of the substrates in the population are Y substrates with a 5′ overhang and a 5′ phosphate at one end and a region that is not complementary at the other end.

18 . The population of double stranded polynucleotide MuA substrates of claim 9 , wherein the substrate comprises a selectable binding moiety.

Assignments (2)

Continuity (3)

References Cited (400)