Methods for delivering an analyte to transmembrane pores

Patent metadata

Field	Value
Publication number	US-11613771-B2
Application number	US-202016902306-A
Country	US
Kind code	B2
Filing date	Jun 16, 2020
Priority date	Oct 17, 2014
Publication date	Mar 28, 2023
Grant date	Mar 28, 2023

Abstract

The invention relates to a new method of delivering an analyte to a transmembrane pore in a membrane. The method involves the use of microparticles.

First claim

The invention claimed is:

1. A method for delivering a polynucleotide to a transmembrane pore in a membrane and characterising the polynucleotide, the method comprising:
(a) providing the polynucleotide attached to a microparticle;
(b) delivering the microparticle towards the membrane and thereby delivering the polynucleotide to the transmembrane pore, wherein the method comprises the microparticle moving (i) along the membrane; and/or (ii) in parallel with the membrane;
(c) allowing the polynucleotide to interact with the transmembrane pore such that the polynucleotide moves through the pore, and using a polynucleotide binding protein to control the movement of the polynucleotide through the pore; and
(d) 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 polynucleotide;
wherein the polynucleotide detaches from the microparticle when interacting with the pore.

2. The method according to claim 1 , wherein the method is for delivering an increased concentration of a polynucleotide to a transmembrane pore in a membrane, wherein the concentration of the polynucleotide delivered to the transmembrane pore is preferably increased by at least about 10 fold.

3. The method according to claim 1 , wherein the method comprises (a) allowing the microparticle to move along an electrochemical gradient, diffusion gradient, hydrophilic gradient or hydrophobic gradient (b) allowing the microparticle to move within a magnetic field; (c) allowing the microparticle to move within an electrical field; (d) allowing the microparticle to move under pressure; or (e) allowing the microparticle to move with gravity.

4. The method according to claim 1 , wherein 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, 5,000 or more, 10,000 or more, 100,000 or more, 1000,000 or more or 5000,000 or more polynucleotides are attached to the microparticle.

5. The method according to claim 1 , wherein the polynucleotide comprises one or more anchors which are capable of coupling to the membrane.

6. The method according to claim 5 , wherein the anchors comprise a polypeptide anchor and/or a hydrophobic anchor.

7. The method according to claim 1 , wherein the polynucleotide is attached to the microparticle via hybridisation and/or wherein the polynucleotide comprises one or more anchors which are capable of coupling to the membrane and the one or more anchors are linked to the polynucleotide via hybridisation.

8. The method according to claim 1 , wherein the microparticle is 500 μm in diameter or less and is formed from a ceramic, glass, silica, a polymer or a metal or is magnetic.

9. The method according to claim 1 , wherein the transmembrane pore is a transmembrane protein pore.

10. The method according to claim 9 , wherein the transmembrane protein pore is derived from Mycobacterium smegmatis porin (Msp), α-hemolysin (a-HL) or lysenin.

11. The method according to claim 1 , wherein the method further comprises removing the microparticle from the membrane.

12. The method according to claim 11 , wherein removing the microparticle from the membrane comprises using a magnetic field or flow-based method.

13. The method according to claim 1 , wherein the method comprises:
(a) providing a first polynucleotide in a first sample attached to a first microparticle;
(b) delivering the first microparticle towards the membrane and thereby delivering the first polynucleotide to the transmembrane pore;
(c) removing the first microparticle from the membrane and optionally removing the first polynucleotide;
(d) providing a second polynucleotide e in a second sample attached to a second microparticle; and
(e) delivering the second microparticle towards the membrane and thereby delivering the second polynucleotide to the transmembrane pore.

14. The method according to claim 13 , wherein:
(A) the method further comprises (i) between steps (b) and (c) allowing the first polynucleotide to interact with the transmembrane pore and taking one or more measurements during the interaction, wherein the measurements are indicative of the presence, absence or one or more characteristics of the first polynucleotide and/or (ii) after step (e) allowing the second polynucleotide to interact with the transmembrane pore and taking one or more measurements during the interaction, wherein the measurements are indicative of the presence, absence or one or more characteristics of the second polynucleotide; or
(B) the first and second analytes are polynucleotides and the method further comprises (i) between steps (b) and (c) allowing the first polynucleotide to interact with the transmembrane pore such that the first polynucleotide moves through the pore and taking one or more measurements as the first polynucleotide moves with respect to the pore, wherein the measurements are indicative of one or more characteristics of the first polynucleotide, and thereby characterising the first polynucleotide and/or (ii) after step (e) allowing the second polynucleotide to interact with the transmembrane pore such that the second polynucleotide moves through the pore and taking one or more measurements as the second polynucleotide moves with respect to the pore, wherein the measurements are indicative of one or more characteristics of the second polynucleotide, and thereby characterising the second polynucleotide.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Brown Clive Gavin
• Garalde Daniel Ryan
• Heron Andrew John
• Turner Daniel John
• White James

Classifications

• G01N33/48728

  Investigating individual cells, e.g. by patch clamp, voltage clamp (investigating individual particles in general G01N15/10) · CPC title

• C12N15/1013

  by using magnetic beads · CPC title

• C12Q2563/155

  Particles of a defined size, e.g. nanoparticles · CPC title

• G01N33/6872

  Intracellular protein regulatory factors and their receptors, e.g. including ion channels · CPC title

• C12N9/52

  derived from bacteria {or Archaea} · CPC title