Granted Patent B2

US 12,546,744 · App. 18/789,966 · Granted Feb 10, 2026

Inventors: Jens H. Gundlach (Seattle, WA); Ian Michael Derrington (Seattle, WA); Andrew Laszlo (Seattle, WA); Jonathan Craig (Seattle, WA); Henry Brinkerhoff (Seattle, WA)

Assignee: University of Washington

G01N27/44743G01N27/44791G01N33/48721G01N33/6818

Abstract

Claims (30)

1 . A method of characterizing a conformational state of a protein in a nanopore system, the nanopore system comprising: a nanopore disposed in a membrane that separates a first conductive liquid medium from a second conductive liquid medium, wherein the nanopore comprises a tunnel that provides liquid communication between the first conductive liquid medium and the second conductive liquid medium, and wherein the protein is physically associated with a polymer in the first conductive liquid medium, the method comprising:

(a) applying an electrical potential between the first conductive liquid medium and the second conductive liquid medium and causing the polymer to interact with the nanopore tunnel, wherein the protein is unable to pass through the nanopore tunnel;

(b) measuring an ion current through the nanopore during the interaction of the polymer with the nanopore tunnel to provide a first current pattern;

(c) determining a position and/or movement of at least one polymer subunit in the nanopore tunnel from the current pattern; and

(d) associating the position and/or movement of the at least one polymer subunit with the conformational state of the protein.

2 . The method of claim 1 , wherein the polymer is a nucleic acid, peptide nucleic acid (“PNA”), a peptide, or a combination thereof.

3 . The method of claim 2 , wherein the nucleic acid comprises an abasic residue.

4 . The method of claim 1 , wherein the protein is covalently coupled to the polymer.

5 . The method of claim 1 , wherein the conformational state of the protein is characterized by the position of the at least one polymer subunit within the nanopore.

6 . The method of claim 5 , wherein the position of the at least one polymer subunit within the nanopore is indicative of the distance between the protein and the at least one polymer subunit within the nanopore, and wherein a change in the distance is indicative of a change in conformational state of the protein.

7 . The method of claim 1 , wherein the protein is a natural protein.

8 . The method of claim 1 , wherein the protein is a mutant protein or a fusion protein.

9 . The method of claim 8 , wherein the fusion protein comprises two or more domains that mutually interact to cause a conformational change.

10 . The method of claim 1 , wherein the conformational state is characterized by measuring the movement of the polymer in the nanopore.

11 . The method of claim 10 , wherein measuring the movement of the polymer in the nanopore comprises measuring the frequency, duration, and distance of polymer movement.

12 . The method of claim 1 , wherein the protein is an enzyme, and wherein the polymer is a nucleic acid.

13 . The method of claim 12 , wherein the method characterizes conformational changes associated with the activity of the enzyme.

14 . The method of claim 13 , wherein the conformational change is in response to presence, absence, or difference in concentration of a putative agonist, antagonist, or co-factor in the first conductive liquid medium.

15 . The method of claim 12 , wherein the enzyme is a molecular motor.

16 . The method of claim 15 , wherein the molecular motor is a translocase, a polymerase, a helicase, an exonuclease, a viral packaging motor, or a topoisomerase.

17 . The method of claim 12 , wherein the enzyme does not process the nucleic acid, and wherein the enzyme is covalently linked to the nucleic acid.

18 . The method of claim 1 , wherein the nanopore is a solid-state nanopore, a protein nanopore, a hybrid solid state-protein nanopore, a biologically adapted solid-state nanopore, or a DNA origami nanopore.

19 . The method of claim 18 , wherein the protein nanopore is alpha-hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP), WZA, Nocardia farcinica NfpA/NfpB cationic selective channel, lysenin or a homolog or variant thereof.

20 . The method of claim 1 , wherein the electrical potential applied is between 10 mV and 1 V or between −10 mV and −1 V.

21 . A method of characterizing a conformational state of a protein in a nanopore system comprising a nanopore disposed in a membrane that separates a first conductive liquid medium from a second conductive liquid medium, wherein the nanopore comprises a tunnel that provides liquid communication between the first conductive liquid medium and the second conductive liquid medium, and wherein the protein is physically associated with a polymer in the first conductive liquid medium, the method comprising:

(a) applying an electrical potential between the first conductive liquid medium and the second conductive liquid medium to cause the polymer to interact with the nanopore tunnel, wherein the protein is unable to pass through the nanopore tunnel;

(b) measuring an ion current through the nanopore during the interaction of the polymer with the nanopore tunnel to provide a first current pattern;

(c) comparing the first current pattern to a reference current pattern;

(d) determining a change in position and/or movement of at least one polymer subunit in the nanopore tunnel from the position and/or movement of at least one polymer subunit in the nanopore tunnel determined from the reference current pattern; and

(e) associating the change in position and/or movement of the at least one polymer subunit in the nanopore tunnel with the conformational state of the protein.

Assignments (1)

Continuity (6)

References Cited (158)