Methods for delivering an analyte to transmembrane pores

Patent metadata

Field	Value
Publication number	US-10760114-B2
Application number	US-201515519606-A
Country	US
Kind code	B2
Filing date	Oct 6, 2015
Priority date	Oct 17, 2014
Publication date	Sep 1, 2020
Grant date	Sep 1, 2020

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 an increased concentration of a polynucleotide analyte to a transmembrane pore in a membrane, comprising:
(a) providing the polynucleotide analyte attached to a microparticle;
(b) delivering the microparticle towards the membrane, thereby delivering the polynucleotide analyte to the transmembrane pore;
(c) allowing the polynucleotide analyte to interact with the transmembrane pore under conditions in which a polynucleotide binding protein, bound to the polynucleotide analyte, controls movement of the polynucleotide analyte, wherein the polynucleotide analyte is eluted from the microparticle and moves with respect to the transmembrane pore, wherein the concentration of the polynucleotide analyte delivered to the transmembrane pore is increased by at least about 10-fold; and
(d) taking measurements as the polynucleotide moves with respect to the pore, wherein the measurements are indicative of characteristics of the polynucleotide, and thereby characterising the polynucleotide.

2. The method of claim 1 , wherein step (b) comprises positioning the microparticle near to or adjacent to the membrane and allowing the microparticle to move towards the membrane.

3. The method of 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 of claim 1 , wherein 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1,000, 5,000, 10,000, 100,000, 1,000,000, or 5,000,000 polynucleotide analytes are attached to the microparticle.

5. The method of claim 1 , wherein the polynucleotide analyte comprises an anchor which are capable of coupling to the membrane.

6. The method of claim 5 , wherein the method further comprises coupling the polynucleotide analyte to the membrane using the the anchor.

7. The method of claim 5 , wherein the anchor comprises a polypeptide anchor and/or a hydrophobic anchor.

8. The method of claim 7 , wherein the hydrophobic anchor comprises a lipid, fatty acid, sterol, carbon nanotube or amino acid.

9. The method of claim 5 , wherein the anchor comprises a linker.

10. The method of claim 5 , wherein the polynucleotide analyte is coupled transiently to the membrane.

11. The method of claim 1 , wherein the polynucleotide analyte is attached to the microparticle via hybridisation.

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

13. The method of claim 1 , wherein the microparticle is formed from a ceramic, glass, silica, a polymer or a metal.

14. The method of claim 1 , wherein the microparticle is 500 μm in diameter or less.

15. The method of claim 1 , wherein the microparticle is magnetic.

16. The method of claim 1 , wherein the transmembrane pore is a transmembrane protein pore.

17. The method of claim 16 , wherein the transmembrane protein pore is derived from Mycobacterium smegmatis porin (Msp), α-hemolysin (α-HL) or lysenin.

18. The method of claim 1 , wherein the 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.

19. The method of claim 1 , wherein the characteristics of the polynucleotide are measured by electrical measurement and/or optical measurement.

20. The method of claim 19 , wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement.

21. The method of claim 1 , wherein the polynucleotide binding protein is derived from a helicase.

22. The method of claim 1 , wherein the method further comprises removing the microparticle from the membrane.

23. The method of claim 22 , wherein the method further comprises removing the microparticle from the membrane using a magnetic field or a flow-based method.

24. The method of claim 22 , wherein the method further comprises
(e) providing a second polynucleotide analyte in a second sample attached to a second microparticle after removal of the first microparticle from the membrane;
(f) delivering the second microparticle towards the membrane and thereby delivering the second polynucleotide analyte to the transmembrane pore.

25. The method of claim 24 , wherein the method further comprises, after step (f), allowing the second polynucleotide analyte to interact with the transmembrane pore and taking the measurements during the interaction, wherein the measurements are indicative of the presence, absence or characteristics of the second polynucleotide analyte.

Assignees

• Oxford Nanopore Tech Ltd

Inventors

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

Classifications

• C12Q2563/155

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

• C12Q1/6804 Primary

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• G01N33/48728

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

• C12Q2523/303

  Applying a physical force on a nucleic acid · CPC title

• C12Q2563/149

  Particles, e.g. beads · CPC title