Deterministic stepping of polymers through a nanopore

Patent metadata

Field	Value
Publication number	US-11035847-B2
Application number	US-201816022594-A
Country	US
Kind code	B2
Filing date	Jun 28, 2018
Priority date	Jun 29, 2017
Publication date	Jun 15, 2021
Grant date	Jun 15, 2021

Abstract

In a method p for controlling translocation of a target polymer molecule through a nanopore, a clamp is reversibly bound to a sequential plurality of polymer subunits along the target polymer molecule length and the molecule and clamp are disposed in an ionic solution that is in fluidic communication with the nanopore. A constant translocation force is applied across the nanopore to induce travel of the target polymer molecule into the nanopore, until the clamp abuts the nanopore aperture and stops further travel of the target polymer molecule into the nanopore. Then a voltage control pulse is applied across the nanopore and/or a thermal control pulse is applied at the nanopore, with a pulse duration that steps the clamp along the target polymer molecule by no more than one polymer subunit in a direction opposite that of travel into the nanopore. No fuel is provided to the clamp.

First claim

We claim:

1. A method for controlling translocation of a target polymer molecule through a nanopore, the target polymer molecule selected from nucleic acid polymer molecules and protein polymer molecules and including a sequential plurality of polymer subunits along a target polymer molecule length, comprising:
reversibly binding an enzyme clamp to a plurality of sequential polymer subunits along the target polymer molecule length;
disposing the target polymer molecule and reversibly bound clamp in an ionic solution that is in fluidic communication with the nanopore, the nanopore having an aperture diameter less than an outer diameter of the clamp;
applying a constant translocation force across the nanopore to induce travel of the target polymer molecule in the ionic solution into the nanopore until the clamp on the target polymer molecule abuts the nanopore aperture and stops further travel of the target polymer molecule into the nanopore;
applying a control pulse comprising at least one of a voltage control pulse across the nanopore and a thermal control pulse at the nanopore, the control pulse having a control pulse duration that steps the clamp along the target polymer molecule by no more than one polymer subunit in a direction opposite that of travel into the nanopore, with no fuel provided to the clamp, to step the target polymer molecule further into the nanopore by no more than one polymer subunit; and
repeatedly applying the control pulse to cause a sequential plurality of polymer subunits of the target polymer molecule to translocate through the nanopore.

2. The method of claim 1 wherein applying a constant translocation force across the nanopore comprises applying at least one of electrophoretic force, hydrostatic pressure, optical force, and magnetic force.

3. The method of claim 1 wherein repeatedly applying the control pulse to cause a sequential plurality of polymer subunits to translocate through the nanopore comprises repeating application of the control pulse until the target polymer molecule length fully translocates through the nanopore.

4. The method of claim 1 further comprising a step of acquiring a representative indication of a polymer subunit as the polymer subunit translocates through the nanopore.

5. The method of claim 1 wherein the applied control pulse has a pulse duration that is less than a length of time required for the constant translation force to induce the target polymer molecule to travel into the nanopore by an additional polymer subunit.

6. The method of claim 1 wherein the constant translocation force comprises an electrostatic force imposed by application of a voltage bias across the nanopore, and wherein the control pulse comprises a voltage control pulse having a control pulse amplitude that is greater than a voltage bias amplitude applied across the nanopore.

7. The method of claim 1 wherein the control pulse comprises a thermal control pulse that is generated in the ionic solution at the nanopore by applying laser energy to a laser-energy absorptive material disposed at the nanopore.

8. The method of claim 1 wherein the applied control pulse duration is no greater than about one millisecond.

9. The method of claim 1 further comprising measuring current through the nanopore while a sequential plurality of polymer subunits of the target polymer molecule translocates through the nanopore.

10. The method of claim 1 wherein the clamp comprises a helicase.

11. A method for characterizing a target polymer molecule, the target polymer molecule selected from nucleic acid polymer molecules and protein polymer molecules and having polymer subunits along a target polymer molecule length, comprising:
reversibly binding an enzyme clamp to a plurality of sequential polymer subunits along the target polymer molecule length;
disposing the target polymer molecule and reversibly bound clamp in an ionic solution that is in fluidic communication with the nanopore, the nanopore having an aperture diameter less than an outer diameter of the clamp;
applying a constant translocation force across the nanopore to induce travel of the target polymer molecule in the ionic solution into the nanopore until the clamp on the target polymer molecule abuts the nanopore aperture and stops further travel of the target polymer molecule into the nanopore;
applying a control pulse comprising at least one of a voltage control pulse across the nanopore and a thermal control pulse at the nanopore, the control pulse having a control pulse duration that steps the clamp along the target polymer molecule by no more than one polymer subunit in a direction opposite that of travel into the nanopore, with no fuel provided to the clamp, to step the target polymer molecule further into the nanopore by no more than one polymer subunit;
acquiring a characteristic indication of a polymer subunit when the polymer subunit is in the nanopore; and
repeatedly applying the control pulse to cause a sequential plurality of polymer subunits of the target polymer molecule to translocate through the nanopore into an ionic solution while acquiring a characteristic indication of each polymer subunit that translocates through the nanopore.

12. The method of claim 11 wherein a characteristic indication of a polymer subunit is acquired by measuring current flow through the nanopore when the polymer subunit is in the nanopore.

13. The method of claim 11 wherein repeatedly applying the control pulse comprises conducting one repetition of control pulse application after each acquisition of a characteristic indication of one polymer subunit in the sequential plurality of polymer subunits.

14. The method of claim 11 wherein acquiring a characteristic indication of a polymer subunit comprises at least one of counting the polymer subunit and identifying the polymer subunit.

15. The method of claim 11 wherein control pulse application and acquisition of a characteristic indication of a polymer subunit are repeated until the target polymer molecule length fully translocates through the nanopore.

16. The method of claim 15 wherein the acquisition of characteristic indications of sequential polymer subunits with repeated control pulse application comprises determining number of polymer subunits disposed along the target polymer molecule length.

17. The method of claim 15 wherein the acquisition of characteristic indications of sequential polymer subunits with repeated control pulse application comprises determining number of identical polymer subunits disposed along the target polymer molecule length.

18. The method of claim 11 wherein the constant translocation force comprises an electrostatic force imposed by application of a voltage bias across the nanopore, and wherein the control pulse comprises a voltage control pulse having a control pulse amplitude that is greater than a voltage bias amplitude applied across the nanopore.

19. The method of claim 11 wherein the control pulse comprises a thermal control pulse that is generated in the ionic solution at the nanopore by applying laser energy to a laser-energy absorptive material disposed at the nanopore.

20. The method of claim 11 wherein applying a constant translocation force across the nanopore comprises applying at least one of electrophoretic force, hydrostatic pressure, optical force, and magnetic force.

21. A nanopore system for characterizing a target polymer molecule selected from nucleic acid polymer molecules and protein polymer molecules and including a

Assignees

• Harvard College

Inventors

• Branton Daniel
• Fleming Stephen Jordan
• Golovchenko Jene A

Classifications

• G01N33/48721 Primary

  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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• B01D71/0211

  Graphene or derivates thereof · CPC title

• C12Y306/01039

  Thymidine-triphosphatase (3.6.1.39), i.e. T4 helicase · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title