Compositions, systems, and methods for detecting events using tethers anchored to or adjacent to nanopores

• US 10,364,463 B2
• Filed: 06/16/2017
• Issued: 07/30/2019
• Est. Priority Date: 06/03/2014
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Mandell, Jeffrey G., Gunderson, Kevin L., Gundlach, Jens H.
• Primary Examiner(s): Forman, Betty J
• Application Number:
  US15/625,100
• Publication Number:
  US 20170369942A1
• Time in Patent Office: 774 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/44743 Introducing samples
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

Compositions, systems, and methods for detecting events are provided. A composition can include a nanopore including a first side, a second side, and an aperture extending through the first and second sides; and a permanent tether including head and tail regions and an elongated body disposed therebetween. The head region can be anchored to or adjacent to the first or second side of the nanopore. The elongated body including a reporter region can be movable within the aperture responsive to a first event occurring adjacent to the first side of the nanopore. For example, the reporter region is translationally movable toward the first side responsive to the first event, then toward the second side, then toward the first side responsive to a second event. The first event can include adding a first nucleotide to a polynucleotide. The second event can include adding a second nucleotide to the polynucleotide.

Claims

1. A composition, comprising:

a nanopore including a first side, a second side, and an aperture extending through the first and second sides; and

a permanent tether including a head region, a tail region, and an elongated body disposed therebetween,

wherein the head region is anchored to a polymerase disposed adjacent to the first side of the nanopore, the tail region extends freely toward the second side of the nanopore, and the elongated body comprises a reporter region movable within the aperture and responsive to a first event occurring adjacent to the first side of the nanopore, wherein the first event includes the polymerase acting upon a first nucleotide, wherein the first nucleotide includes an elongated tag including a moiety that interacts with the tether, wherein the interaction of the moiety with the tether translationally moves the reporter region.

2. The composition of claim 1, wherein the reporter region is translationally movable toward the first side of the nanopore responsive to the first event.

3. The composition of claim 1, wherein the reporter region is translationally movable toward the second side after the first event.

4. The composition of claim 1, wherein the first nucleotide is identifiable based on a measured parameter selected from a magnitude of a first change in a current or flux through the aperture, a time duration of a first change in a current or flux through the aperture, a magnitude and time duration of a first change in a current or flux through the aperture, and a first optical signal, wherein the measured parameter is responsive to a translational movement of the reporter region responsive to a first conformational change of the polymerase.

5. The composition of claim 1, wherein the elongated body comprises a first oligonucleotide, wherein the moiety includes a second oligonucleotide that hybridizes to the first oligonucleotide, and wherein the hybridization of the second oligonucleotide to the first oligonucleotide shortens the tether by a first amount.

6. The composition of claim 5, wherein the first nucleotide is identifiable based on a measured parameter selected from a magnitude of change in a current or flux through the aperture, a time duration of change in a current or flux through the aperture, a magnitude and time duration of change in a current or flux through the aperture, and an optical signal, wherein the measured parameter is responsive to the shortening of the tether by the first amount.

7. The composition of claim 6, wherein the reporter region further is translationally movable toward the first side responsive to the polymerase acting upon a second nucleotide.

8. The composition of claim 7, wherein the second nucleotide includes a third oligonucleotide that hybridizes to the first oligonucleotide, wherein the hybridization of the third oligonucleotide to the first oligonucleotide shortens the tether by a second amount, wherein the second nucleotide is identifiable based on a measured parameter selected from a magnitude of a second change in the current or flux through the aperture, a time duration of a second change in the current or flux through the aperture, a magnitude and time duration of a second change in the current or flux through the aperture, and a second optical signal, wherein the measured parameter is responsive to the shortening of the tether by the second amount.

9. A composition of claim 1, comprising:

a nanopore including a first side, a second side, and an aperture extending through the first and second sides; and

a permanent tether including a head region, a tail region, and an elongated body disposed therebetween, wherein the head region is anchored to, or adjacent to, the first side of the nanopore, the tail region extends freely toward the second side of the nanopore, and the elongated body comprises a reporter region movable within the aperture and responsive to a first event occurring adjacent to the first side of the nanopore, wherein the elongated body includes a moiety;

a polymerase disposed adjacent to the first side of the nanopore; and

a first nucleotide including a first elongated tag, the first elongated tag including a first moiety that interacts with the moiety of the tether and is responsive to the polymerase acting upon the first nucleotide.

10. The composition of claim 9, further including first and second polynucleotides in contact with the polymerase, the polymerase configured to add the first nucleotide to the first polynucleotide based on a sequence of the second polynucleotide.

11. The composition of claim 9, wherein the first moiety and the moiety of the tether are configured to hybridize with one another so as to form a hairpin structure.

12. The composition of claim 9, the first elongated tag further comprising a second moiety, the composition further comprising a third moiety anchored to or adjacent to the first side or second side of the nanopore, the second moiety and the third moiety interacting responsive to addition of the first nucleotide to the first polynucleotide.

13. The composition of claim 9, wherein the tail region is movable between the first and second side of the nanopore responsive to an applied voltage.

14. The composition of claim 9, wherein the interaction between the first moiety and the moiety of the tether defines a duplex, wherein the nanopore further includes a constriction disposed between the first and second sides, and wherein the duplex is sufficiently large as to inhibit movement of the duplex through the constriction.

15. The composition of claim 9, wherein the first elongated tag of the first nucleotide further includes a first reporter region, wherein the first reporter region is configured to be disposed within the aperture responsive to the first moiety interacting with the moiety of the tether.

16. The composition of claim 9, wherein the reporter region is disposed at a predefined location relative to the first moiety responsive to the interaction of the first moiety with the moiety of the tether.

17. A composition comprising:

of claim 1,

a nanopore including a first side, a second side, and an aperture extending through the first and second sides; and

a permanent tether including a head region, a tail region, and an elongated body disposed therebetween, wherein the head region is anchored to, or adjacent to, the first side of the nanopore, the tail region extends freely toward the second side of the nanopore, and the elongated body comprises a reporter region movable within the aperture and responsive to a first event occurring adjacent to the first side of the nanopore, and wherein the head region is anchored to a polymerase disposed adjacent to the first side of the nanopore and the elongated body comprises a moiety; and

a first nucleotide including a first elongated tag, the first elongated tag including a first moiety that interacts with the moiety of the tether responsive to the polymerase acting upon the first nucleotide.

18. The composition of claim 17, wherein the tail region comprises a first nucleic acid.

19. The composition of claim 18, further comprising a second nucleic acid to which the first nucleic acid is hybridized.

20. The composition of claim 19, the head region being disposed on the first side of the nanopore, the tail region being disposed on the second side.

21. The composition of claim 20, wherein the interaction between the first nucleic acid and the second nucleic acid defines a duplex.

22. The composition of claim 21, wherein the nanopore further includes a constriction disposed between the first and second sides.

23. The composition of claim 22, wherein the duplex is sufficiently large as to inhibit movement of the duplex through the constriction.

24. The composition of claim 23, wherein the tether comprising the duplex inhibits separation of the polymerase from the nanopore.