Granted Patent B2

US 12,247,251 · App. 17/138,314 · Granted Mar 11, 2025

Inventors: Barry L. Merriman (La Jolla, CA); Venkatesh Alagarswamy Govindaraj (San Diego, CA); Paul Mola (San Diego, CA); Tim Geiser (San Diego, CA)

Assignee: Semicon Bio

C12Q1/6869C07H21/02C12Q1/001C12Q1/002C12Q1/005C12Q1/26C12Q1/48C12Q1/485C12Q1/6825G01N33/5438G01N2333/9126

Abstract

Claims (26)

1. A method to obtain a sequence information of a target substrate, the method comprising:

forming at least one of a voltage or a current circuit, the circuit further comprising:

a first electrode;

a second electrode spaced apart from the first electrode;

a first arm molecule having a first end and a second end, the second end of the first arm molecule electrically coupled to the first electrode;

a second arm molecule having a first end and a second end, the second end of the second arm molecule electrically coupled to the second electrode; and

a polymerase enzyme,

wherein the first end of each of the first and second arm molecules are conjugated to two distinct sites on the polymerase enzyme so that a portion of the polymerase enzyme between the two distinct sites is included in the circuit;

exposing the circuit to a solution having an ionic strength from dissolved ions and the target substrate;

measuring electrical signals through the circuit as the polymerase enzyme engages the target substrate; and

processing the electrical signals to identify the sequence information of the target substrate when engaged by the polymerase enzyme;

wherein the target substrate comprises DNA or RNA.

2. The method of claim 1 , wherein each of the first and second arm molecules is selected from the group consisting of a double stranded oligonucleotide, a peptide nucleic acid duplex, a peptide nucleic acid-DNA hybrid duplex, a protein alpha-helix, a graphene-like nanoribbon, a natural polymer, a synthetic polymer, and an antibody Fab domain.

3. The method of claim 1 , wherein the portion of the polymerase enzyme included in the circuit comprise an internal structural element selected from the group consisting of an alpha-helix, a beta-sheet, and a multiple of such elements in series.

4. The method of claim 1 , wherein the two distinct sites on the polymerase enzyme are points on the polymerase enzyme capable of undergoing a conformational change or relative motion during polymerase enzyme function.

5. The method of claim 1 , wherein each of the first and second arm molecules comprises a molecule having tension, twist or torsion dependent conductivity.

6. The method of claim 1 , wherein the polymerase enzyme comprises an E. coli Pol I Klenow Fragment.

7. The method of claim 1 , wherein the portion of the polymerase enzyme in the circuit includes an alpha-helix passing through the center of the polymerase enzyme.

8. The method of claim 7 , wherein the alpha-helix comprises 37 amino acids.

9. The method of claim 7 , wherein the two distinct sites are amino acid positions 508 and 548 in the polymerase enzyme.

10. The method of claim 1 , wherein the polymerase enzyme is engineered to have at least one additional charge group.

11. The method of claim 1 , wherein the polymerase enzyme comprises a genetically modified form of an E. coli Pol I polymerase, a Bst polymerase, a Taq polymerase, a Phi29 polymerase, a T7 polymerase or a reverse transcriptase.

12. The method of claim 1 , wherein the two distinct sites in the polymerase enzyme comprise at least one of a native cysteine, a genetically engineered cysteine, a genetically engineered amino acid with a conjugation residue, or a genetically engineered peptide domain comprising a peptide that has a conjugation partner.

13. The circuit of claim 12 , wherein the genetically engineered cysteine is present at each of the two distinct sites on the polymerase enzyme.

14. The method of claim 13 , wherein the first end of each of the first and second arm molecules comprise a maleimide functionality, and wherein the first end of each of the first and second arm molecules connect to amino acid positions 508 and 548 , respectively, by maleimide-cysteine conjugation.

15. The circuit of claim 1 , further comprising a third arm molecule connecting the polymerase enzyme to either the first or the second electrode or to a substrate supporting the electrodes.

Assignments (5)

Continuity (6)

References Cited (400)