Enzyme stalling method

• US 10,221,450 B2
• Filed: 01/22/2014
• Issued: 03/05/2019
• Est. Priority Date: 03/08/2013
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Heron, Andrew John, Alves, David Antoni, Clarke, James Anthony, Crawford, Marion Louise, Garalde, Daniel Ryan, Hall, Graham, Turner, Daniel John, White, James
• Primary Examiner(s): Crow, Robert T.
• Application Number:
  US14/773,164
• Publication Number:
  US 20160010147A1
• Time in Patent Office: 1,868 Days
• Field of Search: None
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/513 Winding/unwinding enzyme, e.g. helicase
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C12Y 306/04012 DNA helicase (3.6.4.12)
• CPC Class Codes: G01N 27/4473 by electric means
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

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 characterizing target polynucleotides using helicases.

Claims

1. A method comprising:

(a) providing a complex comprising one or more helicases bound, in an active mode, to a polynucleotide, wherein the polynucleotide comprises one or more spacers that are configured such that movement of the one or more helicases relative to the polynucleotide is stalled;

(b) contacting a transmembrane pore, which is present in a membrane, with the complex, wherein the transmembrane pore comprises an opening that permits passage of the polynucleotide, but not the one or more helicases, across the membrane; and

(c) applying a potential difference across the membrane such that the polynucleotide moves through the transmembrane pore and such that the one or more helicases pass across the one or more spacers of the polynucleotide.

2. A method of comprising:

(a) providing a target polynucleotide comprising one or more spacers that are configured such that movement of a helicase relative to the target polynucleotide is stalled;

(b) contacting the target polynucleotide with one or more helicases such that the one or more helicases, in an active mode, bind to the target polynucleotide and stall at the one or more spacers;

(c) contacting a transmembrane pore, which is present in a membrane, with the target polynucleotide and the one or more stalled helicases, wherein the transmembrane pore comprises an opening that permits passage of the target polynucleotide, but not the one or more helicases, across the membrane; and

(d) applying a potential difference across the membrane such that the target polynucleotide moves through the transmembrane pore; and

such that the one or more helicases pass across the one or more spacers of the target polynucleotide and control the movement of the target polynucleotide through the transmembrane pore.

3. A method according to claim 2, further comprising

taking one or more measurements as the target polynucleotide moves with respect to the transmembrane pore wherein the measurements are indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.

4. A method according to claim 3, wherein the one or more characteristics are selected from the group consisting of:

(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.

5. A method according to claim 3, wherein the one or more characteristics of the target polynucleotide are measured by an electrical measurement and/or optical measurement.

6. 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 or ribonucleic acid and the one or more spacers comprise peptide nucleic acid, glycerol nucleic acid, threose nucleic acid, locked nucleic acid 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, one or more inverted dideoxy-thymidines, one or more dideoxy-cytidines, one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2′

-O-Methyl RNA bases, one or more Iso-deoxycytidines, one or more Iso-deoxyguanosines, one or more C3 groups, one or more photo-cleavable groups, one or more hexandiol groups, one or more 9-atom spacer groups, one or more 18-atom spacer groups, a polymer or one or more thiol connections, (d) the one or more spacers comprise a polypeptide or a polyethylene glycol, (e) the one or more spacers comprise one or more abasic nucleotides, (f) the one or more spacers comprise one or more chemical groups which cause the one or more helicases to stall, (g) the one or more spacers comprise one 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, digoxigenin and/or anti-digoxigenin or a dibenzylcyclooctyne group, (h) 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 (i) the one or more spacers are capable of stalling the one or more helicases in a salt concentration of about 100 mM or lower.

7. 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.

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

9. A method according to claim 8, 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 moiety.

10. A method according to claim 2, wherein the one or more helicases are selected from the group consisting of:

Hel308 helicases, RecD helicases, XPD helicases, Dda helicases, helicases derived from any of the foregoing helicases, and a combination of any of the foregoing helicases.

11. A method according to claim 2, wherein the transmembrane pore is:

(a) a protein pore or a solid state pore;

(b) a protein pore derived from one selected from the group consisting of;

a hemolysin, leucocidin, Mycobacterium smegmatis porin A, MspB, MspC, MspD, lysenin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria autotransporter lipoprotein and WZA;

or (c) formed of eight identical subunits as shown in SEQ ID NO;

2 or 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 activity of the transmembrane pore.

12. A method according to claim 5, wherein the one or more characteristics of the target polynucleotide are measured by an electrical measurement, wherein the electrical measurement is selected from the group consisting of:

a current measurement, an impedance measurement, a tunneling measurement, and a field effect transistor measurement.