Enzymatic circuits for molecular sensors

Patent metadata

Field	Value
Publication number	US-12247251-B2
Application number	US-202017138314-A
Country	US
Kind code	B2
Filing date	Dec 30, 2020
Priority date	Apr 25, 2017
Publication date	Mar 11, 2025
Grant date	Mar 11, 2025

Abstract

In various embodiments a molecular circuit is disclosed. The circuit comprises a negative electrode, a positive electrode spaced apart from the negative electrode, and an enzyme molecule conductively attached to both the positive and negative electrodes to form a circuit having a conduction pathway through the enzyme. In various examples, the enzyme is a polymerase. The circuit may further comprise molecular arms used to wire the enzyme to the electrodes. In various embodiments, the circuit functions as a sensor, wherein electrical signals, such as changes to voltage, current, impedance, conductance, or resistance in the circuit, are measured as substrates interact with the enzyme.

First claim

We claim:

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.

Assignees

• Roswell Biotechnologies Inc
• Semicon Bio

Inventors

• Merriman Barry L
• Alagarswamy Govindaraj Venkatesh
• Mola Paul
• Geiser Tim

Classifications

• C12Q1/005

  involving specific analytes or enzymes (including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence) · CPC title

• G01N2333/9126

  DNA-directed DNA polymerase (2.7.7.7) · CPC title

• G01N33/5438

  Electrodes · CPC title

• C12Q1/6825

  Nucleic acid detection involving sensors · CPC title

• C12Q1/485

  involving kinase · CPC title