Enzyme stalling method

Patent metadata

Field	Value
Publication number	US-2016010147-A1
Application number	US-201414773164-A
Country	US
Kind code	A1
Filing date	Jan 22, 2014
Priority date	Mar 8, 2013
Publication date	Jan 14, 2016
Grant date	—

Abstract

The invention relates to new methods of moving helicases past spacers on polynucleotides and controlling the loading of helicases on polynucleotides. The invention also relates to new methods of characterising target polynucleotides using helicases.

First claim

1 . A method of moving one or more stalled helicases past one or more spacers in a polynucleotide, comprising contacting (a) the one or more stalled helicases and the polynucleotide with a transmembrane pore and (b) applying a potential across the pore and thereby moving the one or more helicases past the one or more spacers on the polynucleotide.

2 . A method of controlling the movement of a target polynucleotide through a transmembrane pore, comprising:
(a) providing the target polynucleotide with one or more spacers; (b) contacting the target polynucleotide with one or more helicases such that the one or more helicases stall at the one or more spacers; (c) contacting the target polynucleotide and the one or more stalled helicases with the pore; and (d) applying a potential across the pore such that the one or more helicases move past the one or more spacers and control the movement of the target polynucleotide through the pore.

3 . A method of characterising a target polynucleotide, comprising:
(a) carrying out the method of claim 2 ; and (b) taking one or more measurements as the polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the polynucleotide and thereby characterising the target polynucleotide.

4 . (canceled)

5 . A method according to claim 3 , wherein the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide, (v) whether or not the target polynucleotide is modified, and (vi) whether or not the target polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers.

6 . (canceled)

7 . A method according to claim 3 , wherein the one or more characteristics of the target polynucleotide are measured by (a) electrical measurement and/or optical measurement or (b) a current measurement, an impedance measurement a tunneling measurement or a field effect transistor (PET) measurement.

8 - 9 . (canceled)

10 . A method according to claim 2 , wherein (a) the one or more spacers have a different structure from the polynucleotide, (b) the polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and the one or more spacers comprise peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or a synthetic polymer with nucleotide side chains, (c) the one or more spacers comprise one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dTs), one or more inverted dideoxy-thymidines (ddTs), one or more dideoxy-cytidines (ddCs), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2′-O-Methyl RNA bases, one or more Iso-deoxycytidines (Iso-dCs), one or more Iso-deoxyguanosines (Iso-dGs), one or more C3 groups, one or more photo-cleavable (PC) groups, one or more hexandiol groups, one or more spacer 9 (iSp9) groups, one or more spacer 18 (iSp18) groups, polymer or one or more thiol connections, (d) the one or more spacers comprise a polypeptide or a polyethylene glycol (PEG), (e) the one or more spacers comprise one or more abasic nucleotides, (f) the one or more spacers comprise one or more abasic nucleotides, (g) the one or more spacers comprise one or more chemical groups which cause the one or more helicases to stall, (h) the one or more spacers comprise me or more chemical groups which cause the one or more helicases to stall and the one or more chemical groups are one or more fluorophores, streptavidin and/or biotin, cholesterol, methylene blue, a dinitrophenol (DNP), digoxigenin and/or anti-digoxigenin or a dibenzylcyclooctyne group, (i) the one or more spacers are capable of stalling the one or more helicases in the presence of free nucleotides and/or the presence of a helicase cofactor or (j) the one or more spacers are capable of stalling the one or more helicases in a salt concentration of about 100 mM or lower.

11 - 18 . (canceled)

19 . A method according to claim 2 , wherein the method comprises moving two or more stalled helicases past each spacer or comprises moving two or more helicases that are attached to one another or covalently attached to one another past each spacer.

20 - 22 . (canceled)

23 . A method according to claim 2 , wherein (a) at least a portion of the polynucleotide is double stranded or (b) at least a portion of the polynucleotide is double stranded and (i) the one or more spacers are included in a single stranded region or a non-hybridised region of the polynucleotide or (ii) the one or more spacers are included in a single stranded region of the polynucleotide and the single stranded region comprises a leader sequence which preferentially threads into the pore.

24 - 25 . (canceled)

26 . A method according to claim 23 , wherein (a) the two strands of the double stranded portion are linked using a bridging moiety or (b) the two strands of the double stranded portion are linked using a bridging moiety and the one or more spacers are included in the bridging moeity.

27 . (canceled)

28 . A method according to claim 2 , wherein the one or more helicases are (a) one or more Hel308 helicases, one or more RecD helicases, one or more XPD helicases or one or more Dda helicases (b) one or more helicases derived from any of the helicases in (a); or (c) a combination of any of the helicases in (a) and/or (b).

29 . A method according to claim 2 , wherein the pore is (a) a transmembrane protein pore or a solid state pore, (b) a transmembrane protein pore derived from a hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, lysenin, outer membrane porin F (OmpF), outer membrane porin (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) and WZA or (c) formed of eight identical subunits as shown in SEQ ID NO: 2 or (b) a variant thereof in which one or more of the eight subunits has at least 50% homology to SEQ ID NO: 2 based on amino acid identity over the entire sequence and retains pore activity.

30 - 33 . (canceled)

34 . A method of controlling the loading of one or more helicases on a target polynucleotide, comprising:
(a) providing the target polynucleotide with one or more spacers; and (b) contacting the target polynucleotide provided in (a) with the one or more helicases such that the one or more helicases bind to the target polynucleotide and stall at each spacer.

35 . (canceled)

36 . A method according to claim 34 , wherein the target polynucleotide is provided with (L-S)n or (S-L)n in the 5′ to 3′ direction, wherein L is a single stranded polynucleotide or a non-hybridised polynucleotide, S is a spacer and n is a whole number.

37 . (canceled)

38 . A method according to claim 37 , wherein step (a) comprises providing the target polynucleotide with (L 1 -S)n or (S-L 1 )n in the 5′ to 3′ direction, wherein L is a single stranded polynucleotide or a non-hybridised polynucleotide which is only long enough for one helicase to bind, S is a spacer and n is a whole number or wherein step (a) comprises providing the target polynucleotide with (L 2 -S)n or (S-L 2 )n in the 5′ to 3′ direct

Assignees

• Oxford Nanopore Tech Ltd

Inventors

• Heron Andrew John
• Alves David Antoni
• Clarke James Anthony
• Crawford Marion Louise
• Garalde Daniel Ryan
• Hall Graham
• Turner Daniel John
• White James

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12N9/14

  Hydrolases (3) · CPC title

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Y306/04012

  DNA helicase (3.6.4.12) · CPC title