Compositions, devices, systems, and methods for using a nanopore

Patent metadata

Field	Value
Publication number	US-10208342-B2
Application number	US-201715713542-A
Country	US
Kind code	B2
Filing date	Sep 22, 2017
Priority date	Apr 4, 2007
Publication date	Feb 19, 2019
Grant date	Feb 19, 2019

Abstract

Devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore are provided. The devices and methods also determine (˜>50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore. The invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.

First claim

What is claimed is:

1. A method, comprising:
providing a device comprising:
a substrate having a pore therein, the substrate separating a first fluid-filled chamber from a second fluid-filled chamber, wherein the fluid of the first fluid-filled chamber comprises a polynucleotide comprising a macromolecule-binding region and a tethering region; and
a power source electrically coupled to electrodes, wherein the power source and electrodes are adapted to apply a potential difference between the first fluid-filled chamber and the second fluid-filled chamber;

threading a portion of the polynucleotide through the pore by applying a potential difference between the first fluid-filled chamber and the second fluid-filled chamber; and
tethering the polynucleotide to the pore via the tethering region.

2. The method according to claim 1 , wherein the tethering comprises hybridizing an oligonucleotide to the tethering region, the resulting duplex tethering the polynucleotide to the pore.

3. The method according to claim 1 , wherein the portion of the polynucleotide threaded through the pore comprises the tethering region.

4. The method according to claim 1 , wherein the macromolecule-binding region is a hairpin structure.

5. The method according to claim 1 , comprising binding a macromolecule to the macromolecule-binding region.

6. The method according to claim 5 , wherein the binding occurs prior to the threading.

7. The method according to claim 5 , wherein the binding occurs subsequent to the threading.

8. The method according to claim 5 , wherein the macromolecule is a protein.

9. The method according to claim 8 , wherein the protein is an enzyme.

10. The method according to claim 9 , wherein the enzyme is a polymerase, a nuclease, or a ligase.

11. The method according to claim 5 , comprising monitoring a function of the macromolecule.

12. The method according to claim 11 , wherein the macromolecule is an enzyme, and monitoring a function of the macromolecule comprises monitoring turnover of the enzyme at the pore.

13. The method according to claim 11 , wherein the macromolecule is an enzyme, and monitoring a function of the macromolecule comprises monitoring the binding of the enzyme to a ligand.

14. The method according to claim 11 , wherein the macromolecule is an enzyme, and monitoring a function of the macromolecule comprises monitoring a reaction product of the enzyme.

15. The method according to claim 11 , wherein the macromolecule is an enzyme, and monitoring a function of the macromolecule comprises monitoring the release of a reaction product from the enzyme.

16. The method according to claim 5 , further comprising sequencing a polynucleotide or a portion thereof using the pore.

17. The method according to claim 16 , wherein the sequencing comprises sequencing by synthesis.

18. The method according to claim 1 , wherein the device comprises a plurality of pores, and the method comprises tethering a polynucleotide to each of the plurality of pores.

19. The method according to claim 1 , further comprising, subsequent to the tethering, untethering the polynucleotide from the pore using a voltage force.

20. The method according to claim 1 , wherein the pore is a nanopore.

21. The method according to claim 1 , wherein the pore is a solid state pore.

22. The method according to claim 21 , wherein the solid state pore comprises a biological channel positioned therein.

23. The method according to claim 1 , wherein the pore is a biological pore.

Assignees

• Univ California

Inventors

• Dunbar William B
• Wilson Noah A
• Akeson Mark A
• Deamer David W
• Lieberman Kate

Classifications

• C12Q1/54

  involving glucose or galactose · CPC title

• G01N27/4166

  measuring a particular property of an electrolyte · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q2522/101

  Single or double stranded nucleic acid binding proteins · CPC title