Method for characterising a double stranded nucleic acid using a nano-pore and anchor molecules at both ends of said nucleic acid

Patent metadata

Field	Value
Publication number	US-10337060-B2
Application number	US-201515301471-A
Country	US
Kind code	B2
Filing date	Mar 31, 2015
Priority date	Apr 4, 2014
Publication date	Jul 2, 2019
Grant date	Jul 2, 2019

Abstract

The invention relates to a method for method of characterizing, such as sequencing, a target double stranded polynucleotide. The polynucleotide is coupled to a membrane using at least two adaptors with different strengths of coupling to the membrane.

First claim

The invention claimed is:

1. A method of characterising a target double stranded polynucleotide using a transmembrane pore in a membrane, comprising:
a) providing the target double stranded polynucleotide with a Y adaptor at one end and a hairpin loop adaptor at the other end, wherein the Y adaptor comprises one or more first anchors for coupling the polynucleotide to the membrane, wherein the hairpin loop adaptor comprises one or more second anchors for coupling the polynucleotide to the membrane and wherein the strength of coupling of the hairpin loop adaptor to the membrane is greater than the strength of coupling of the Y adaptor to the membrane;
b) contacting the polynucleotide provided in step a) with the transmembrane pore such that at least one strand of the polynucleotide moves through the pore; and
c) taking one or more measurements as the at least one strand of the polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the at least one strand of the polynucleotide and thereby characterising the double stranded target polynucleotide.

2. A method according to claim 1 , wherein (a) the hairpin loop adaptor comprises more anchors than the Y adaptor, (b) the strength of coupling of one or more second anchors to the membrane is greater than the strength of coupling of the one or more first anchors to the membrane (c) the strength of coupling of the one or more second anchors to the hairpin loop adaptor is greater than the strength of coupling of the one or more first anchors to the Y adaptor, (d) the one or more first anchors and one or more second anchors couple their respective adaptors to the membrane via hybridisation and the strength of hybridisation is greater in the one or more second anchors than in the one or more first anchors, (e) the one or more first anchors comprise one or more rigid linkers and the one or more second anchors comprise one or more flexible linkers or (f) any combination of (a) to (e).

3. A method according to claim 2 , wherein (i) the one or more second anchors couple to the membrane using cholesterol and the one or more first anchors couple to the membrane using palmitate, (ii) the one or more second anchors couple to the membrane using a mono-acyl species and the one or more first anchors couple to the membrane using a diacyl species, (iii) the one or more first anchors comprise one or more polynucleotide linkers and the one or more second anchors comprise one or more flexible linkers comprising one or more one or more spacer 9 (iSp9) groups or one or more spacer 18 (iSp18), (iv) a combination of (i) and (iii) or (v) a combination of (ii) and (iii).

4. A method according to claim 1 , wherein step a) comprises modifying the target double stranded polynucleotide so that it comprises the Y adaptor at one end and the hairpin loop adaptor at the other end.

5. A method according to claim 1 , wherein step b) comprises contacting the polynucleotide provided in step a) with a transmembrane pore such that both strands of the polynucleotide move through the pore and step c) comprises taking one or more measurements as the both strands of the polynucleotide move with respect to the pore wherein the measurements are indicative of one or more characteristics of the strands of the polynucleotide and thereby characterising the target polynucleotide.

6. A method according to claim 1 , wherein the one or more characteristics are selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified.

7. A method according to claim 1 , wherein the one or more characteristics of the polynucleotide are measured by electrical measurement and/or optical measurement.

8. A method according to claim 7 , wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement.

9. A method according to claim 1 , wherein step b) further comprises contacting the polynucleotide provided in step a) with a polynucleotide binding protein such that the protein controls the movement of the at least one strand of the polynucleotide through the pore.

10. A method according to claim 9 , wherein the method comprises:
b) contacting the polynucleotide provided in step a) with a transmembrane pore and a polynucleotide binding protein such that at least one strand of the polynucleotide moves through the pore and the protein controls the movement of the at least one strand of the polynucleotide through the pore; and
c) measuring the current passing through the pore as the at least one strand of the polynucleotide moves with respect to the pore wherein the current is indicative of one or more characteristics of the at least one strand of the polynucleotide and thereby characterising the double stranded target polynucleotide.

11. A method according to claim 9 , wherein the polynucleotide binding protein is derived from a helicase.

12. A method according to claim 1 , wherein the membrane is an amphiphilic layer or a solid state layer.

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

14. A method according to claim 13 , wherein the transmembrane protein pore is derived from Mycobacterium smegmatis porin (Msp), α-hemolysin (α-HL) or lysenin.

15. A method for modifying a target double stranded polynucleotide for characterisation using a transmembrane pore in a membrane, comprising
(a) ligating a Y adaptor to one end of the polynucleotide and ligating a hairpin loop adaptor to the other end of the polynucleotide; and
(b) attaching to the Y adaptor one or more first anchors for coupling the polynucleotide to the membrane, attaching to the hairpin loop adaptor one or more second anchors for coupling the polynucleotide to the membrane and thereby providing a modified target double stranded polynucleotide;
wherein the strength of coupling of the hairpin loop adaptor to the membrane is greater than the strength of coupling of the Y adaptor to the membrane.

16. A method according to claim 15 , wherein (a) the hairpin loop adaptor comprises more anchors than the Y adaptor, (b) the strength of coupling of one or more second anchors to the membrane is greater than the strength of coupling of the one or more first anchors to the membrane, (c) the strength of coupling of the one or more second anchors to the hairpin loop adaptor is greater than the strength of coupling of the one or more first anchors to the Y adaptor, (d) the one or more first anchors and one or more second anchors couple their respective adaptors to the membrane via hybridisation and the strength of hybridisation is greater in the one or more second anchors than in the one or more first anchors, (e) the one or more first anchors comprise one or more rigid linkers and the one or more second anchors comprise one or more flexible linkers, or (f) any combination of (a) to (e).

17. A method according to claim 10 , wherein the polynucleotide binding protein is derived from a helicase.

Assignees

• Oxford Nanopore Tech Ltd

Inventors

• Crawford Marion Louise
• White James

Classifications

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Y306/04012

  DNA helicase (3.6.4.12) · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12Q2563/157

  Nanotubes or nanorods · CPC title