Method for nanopore RNA characterisation

Patent metadata

Field	Value
Publication number	US-11021747-B2
Application number	US-201916575610-A
Country	US
Kind code	B2
Filing date	Sep 19, 2019
Priority date	Oct 17, 2014
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

Abstract

The invention relates to a new method of characterising a target RNA polynucleotide by taking one or more measurements as the target RNA polynucleotide moves with respect to a transmembrane pore. The movement is controlled by a DNA helicase. The invention also relates to a modified RNA construct wherein the RNA polynucleotide has been modified to increase DNA helicase binding thereto.

First claim

The invention claimed is:

1. A method of characterising a target RNA polynucleotide comprising:
(a) providing (i) an RNA polynucleotide that is modified to comprise a single stranded DNA polynucleotide and (ii) a helicase enzyme, wherein the single stranded DNA polynucleotide provides a binding site for the helicase enzyme;
(b) contacting the RNA polynucleotide and helicase enzyme provided in (a) with a transmembrane pore such that the helicase enzyme controls the movement of the RNA polynucleotide through the transmembrane pore;
(c) taking one or more measurements as the RNA polynucleotide moves with respect to the transmembrane pore, wherein the one or more measurements are indicative of one or more characteristics of the RNA polynucleotide, and
thereby characterising the target RNA polynucleotide.

2. A method according to claim 1 , wherein the single stranded DNA polynucleotide comprises a DNA adaptor.

3. A method according to claim 1 , wherein the single stranded DNA polynucleotide comprises a leader sequence.

4. A method according to claim 1 , wherein the single stranded DNA polynucleotide comprises a leader sequence that preferentially threads into the transmembrane pore.

5. A method according to claim 1 , wherein the single stranded DNA polynucleotide is covalently attached to the RNA polynucleotide.

6. A method according to claim 1 , wherein the single stranded DNA polynucleotide is ligated to the RNA polynucleotide.

7. A method according to claim 1 , wherein the single stranded DNA polynucleotide is hybridised to the RNA polynucleotide.

8. A method according to claim 1 , wherein the one or more characteristics are selected from (i) length of the RNA polynucleotide, (ii) identity of the RNA polynucleotide, (iii) the sequence of the RNA polynucleotide, (iv) the secondary structure of the RNA polynucleotide, and (v) whether the RNA polynucleotide is modified.

9. A method according to claim 1 , wherein the one or more measurements are electrical and/or optical measurements.

10. A method according to claim 1 , wherein step c) comprises measuring the current passing through the transmembrane pore as the RNA polynucleotide moves with respect to the transmembrane pore wherein the current is indicative of one or more characteristics of the RNA polynucleotide and thereby characterising the RNA polynucleotide.

11. A method according to claim 1 , wherein the RNA polynucleotide comprises modification by methylation, modification by oxidation, modification by damage, modification with one or more proteins, modification with base analogues or modification with one or more labels, tags or spacers.

12. A method according to claim 1 , wherein the RNA polynucleotide is coupled to the transmembrane pore using one or more anchors.

13. A method according to claim 1 , wherein the transmembrane pore is a protein pore or a solid state pore.

14. A method according to claim 1 , wherein the transmembrane protein pore is a protein pore, wherein the protein pore is derived from a group selected from hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, CsgG, lysenin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) or WZA.

15. A method of characterising an RNA polynucleotide comprising:
(a) providing (i) an RNA polynucleotide that is modified to comprise a single stranded DNA polynucleotide, (ii) a helicase enzyme, and (iii) one or more molecular brakes that are derived from helicases and are modified such that they bind the polynucleotide but do not function as a helicase;
(b) contacting the modified RNA polynucleotide and helicase enzyme provided in (a) with a transmembrane pore such that the helicase controls the movement of the RNA polynucleotide through the transmembrane pore;
(c) taking one or more measurements as the modified RNA polynucleotide moves with respect to the transmembrane pore, wherein the one or more measurements are indicative of one or more characteristics of the RNA polynucleotide, and
thereby characterising the RNA polynucleotide.

16. A method according to claim 15 , wherein the transmembrane pore is a protein pore or a solid state pore.

17. A method according to claim 15 , wherein the transmembrane protein pore is a protein pore, wherein the protein pore is derived from a group selected from hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, CsgG, lysenin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) or WZA.

18. A method according to claim 15 , wherein the single stranded DNA polynucleotide comprises a DNA adaptor.

19. A method according to claim 15 , wherein the single stranded DNA polynucleotide comprises a leader sequence that preferentially threads into the transmembrane pore.

Assignees

• Oxford Nanopore Tech Ltd

Inventors

• Garalde Daniel Ryan
• Heron Andrew John
• Jayasinghe Lakmal
• Turner Daniel John
• White James

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2521/513

  Winding/unwinding enzyme, e.g. helicase · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q2525/197

  incorporating a spacer/coupling moiety · CPC title

• C12Q2525/121

  incorporating both deoxyribonucleotides and ribonucleotides · CPC title