Method for nucleic acid detection by guiding through a nanopore

Patent metadata

Field	Value
Publication number	US-11085078-B2
Application number	US-201716338399-A
Country	US
Kind code	B2
Filing date	Sep 29, 2017
Priority date	Sep 29, 2016
Publication date	Aug 10, 2021
Grant date	Aug 10, 2021

Abstract

The invention provides a method of detecting a target polynucleotide in a sample comprising: (a) contacting the sample with a guide polynucleotide that binds to a sequence in the target polynucleotide and a polynucleotide-guided effector protein, wherein the guide polynucleotide and polynucleotide-guided effector protein form a complex with any target polynucleotide present in the sample; (b) contacting the sample with a membrane comprising a transmembrane pore; (c) applying a potential to the membrane; and (d) monitoring for the presence or absence of an effect resulting from the interaction of the complex with the transmembrane pore to determine the presence or absence of the complex, thereby detecting the target polynucleotide in the sample.

First claim

The invention claimed is:

1. A system for detecting a target polynucleotide in a sample comprising:
(i) a nanopore;
(ii) an RNA-guided effector protein comprising a guide-RNA binding domain; and
(iii) a guide-RNA comprising a crRNA that is complementary to a sequence in the target polynucleotide to be detected and a tracrRNA that is complementary to a sequence in the guide-RNA binding domain of the RNA-guided effector protein, wherein the guide RNA or the RNA-guided effector protein further comprises an adaptor comprising a leader sequence or an anchor capable of coupling to a membrane.

2. The system of claim 1 , further comprising a target polynucleotide and a membrane, wherein the nanopore is present in the membrane.

3. The system of claim 1 , wherein the target polynucleotide, guide-RNA and RNA-guided effector protein form a complex.

4. The system of claim 1 , wherein the target polynucleotide is coupled to a surface.

5. The system of claim 1 , wherein the crRNA and tracrRNA are present in a single guide-RNA.

6. The system according to claim 1 wherein (i) the anchor or (ii) the adaptor is present at the 5′ end of the tracrRNA, the 3′ end of the tracrRNA, the 3′ end of the crRNA, or when the tracrRNA and crRNA are comprised in a sgRNA, the 3′ end of the sgRNA or internally.

7. The system according to claim 1 wherein the anchor is hydrophobic.

8. The system according to claim 1 , wherein the RNA-guided effector protein is a RNA-guided endonuclease or a RNA-guided endonuclease wherein the nuclease activity of the RNA-guided endonuclease is disabled.

9. The system according to claim 1 , wherein one or more of the catalytic nuclease sites of the RNA-guided endonuclease are inactivated.

10. The system according to claim 1 , wherein the RNA-guided effector protein is Cas, Cas12a or Cas13a.

11. The system according to claim 1 , wherein the RNA-guided effector protein is Cas9.

12. A system according to claim 1 , wherein the target polynucleotide is double stranded or comprises a double stranded region and is DNA, a DNA/RNA hybrid or RNA.

13. A system according to claim 1 , wherein the adaptor further comprises a barcode sequence.

Assignees

• Oxford Nanopore Tech Ltd

Inventors

• Heron Andrew John
• Graham James Edward
• Gutierrez Richard Alexander
• Bowen Rebecca Victoria
• White James
• Brown Clive Gavin
• Fordham Daniel George

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N33/48721

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title