Method for attaching one or more polynucleotide binding proteins to a target polynucleotide

• US 10,385,389 B2
• Filed: 01/22/2015
• Issued: 08/20/2019
• Est. Priority Date: 01/22/2014
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Heron, Andrew John, Brown, Clive Gavin, Bowen, Rebecca Victoria, White, James, Turner, Daniel John, Lloyd, Joseph Hargreaves, Youd, Christopher Peter
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/113,207
• Publication Number:
  US 20170107569A1
• Time in Patent Office: 1,671 Days
• Field of Search: None
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2521/513 Winding/unwinding enzyme, e.g. helicase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C12Y 605/01001 DNA ligase (ATP) (6.5.1.1)

Abstract

The invention relates to new methods of attaching one or more polynucleotide binding proteins to a target polynucleotide. The invention also relates to new methods of characterizing target polynucleotides.

Claims

1. A method for attaching one or more polynucleotide binding proteins to a target polynucleotide, comprising:

(a) providing the one or more polynucleotide binding proteins bound to one or more loading moieties; and

(b) covalently attaching the one or more loading moieties to the target polynucleotide.

2. A method according to claim 1, wherein the method comprises before step (a) binding the polynucleotide binding proteins to the one or more loading moieties.

3. A method according to claim 1, wherein the one or more polynucleotide binding proteins are polynucleotide handling enzymes.

4. A method according to claim 3, wherein the one or more polynucleotide handling enzymes are one or more polymerases, exonucleases, helicases, topoisomerases or a combination thereof.

5. A method according to claim 4, wherein the one or more helicases are Hel308 helicases, RecD helicases, XPD helicases or Dda helicases.

6. A method according to claim 5, wherein the one or more helicases are modified to reduce the size of an opening in the polynucleotide binding domain through which in at least one conformational state the polynucleotide can unbind from the helicase.

7. A method according to claim 1, wherein the one or more polynucleotide binding proteins remain bound to the one or more loading moieties at the end of the step (b).

8. A method according to claim 1, wherein (i) the one or more loading moieties are synthetic;

and/or (ii) the one or more loading moieties comprise a loading polynucleotide; and

/or (iii) the one or more loading moieties comprise a single stranded polynucleotide to which the one or more polynucleotide binding proteins are bound.

9. A method according to claim 1, wherein the target polynucleotide is a double stranded polynucleotide.

10. A method according to claim 9, wherein the at least one of the one or more loading moieties is a Y adaptor.

11. A method according to claim 9, wherein at least one of the one or more loading moieties is a bridging moiety.

12. A method according to claim 11, wherein the bridging moiety is a hairpin loop.

13. A method according to claim 1, wherein the one or more loading moieties comprise one or more anchors that are capable of coupling to a membrane.

14. A method according to claim 1, wherein the one or more loading moieties is a loading polynucleotide and step (b) comprises attaching the one or more loading moieties to the target polynucleotide using a ligase.

15. A method according to claim 14, wherein the method further comprises (c) removing the ligase from method conditions.

16. A method according to claim 14, wherein step (b) is performed in the absence of ATP or using gamma-S-ATP (ATPγ

S) instead of ATP.

17. A method of characterising a target polynucleotide, comprising:

(a) carrying out a method according to claim 1;

(b) contacting the target polynucleotide having the one or more attached polynucleotide binding proteins as provided in step (a) with a transmembrane pore such that the one or more polynucleotide binding proteins control the movement of the polynucleotide with respect to the pore; and

(c) 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.

18. A method according to claim 17, 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 and (v) whether or not the target polynucleotide is modified.

19. A method according to claim 17, wherein the one or more characteristics of the target polynucleotide are measured by (i) electrical measurement and/or optical measurement or (ii) the electrical measurement which is a current measurement, an impedance measurement, a tunneling measurement or a field effect transistor (FET) measurement.

20. A kit for covalently attaching one or more polynucleotide binding proteins to a target polynucleotide, comprising (a) the one or more polynucleotide binding proteins bound to one or more loading moieties and (b) a ligase.