Enzymatic circuits for molecular sensors

Patent metadata

Field	Value
Publication number	US-10508296-B2
Application number	US-201816015028-A
Country	US
Kind code	B2
Filing date	Jun 21, 2018
Priority date	Apr 25, 2017
Publication date	Dec 17, 2019
Grant date	Dec 17, 2019

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 circuit comprising:
a first electrode;
a second electrode spaced apart from the first electrode; and
a polymerase enzyme electrically connected to the first electrode by a first arm molecule, and electrically connected to the second electrode by a second arm molecule, to form a conductive pathway between the first electrode and the second electrode through the polymerase enzyme;
wherein each of the first and second arm molecules has a first end and a second end, wherein the first end of the first arm molecule is bonded to the polymerase enzyme and the second end of the first arm molecule is bonded to the first electrode, wherein the first end of the second arm molecule is bonded to the polymerase enzyme and the second end of the second arm molecule is bonded to the second electrode, and wherein the first end of each of the first and second arm molecules connect to the polymerase enzyme at two distinct sites on the polymerase enzyme so as to include a portion of the polymerase enzyme in the conductive pathway.

2. The circuit of claim 1 , wherein the first electrode is a positive electrode and the second electrode is a negative electrode, or wherein the first electrode is a negative electrode and the second electrode is a positive electrode.

3. The circuit 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.

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

5. The circuit 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.

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

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

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

9. The circuit of claim 8 , wherein the alpha-helix comprises 37 amino acids.

10. The circuit of claim 8 , wherein the two distinct sites are amino acid positions 508 and 548 in the polymerase enzyme.

11. The circuit of claim 1 , wherein the polymerase enzyme is engineered to have additional charge groups.

12. The circuit 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.

13. The circuit 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.

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

15. The circuit of claim 14 , 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.

16. The circuit of claim 1 , further comprising a gate electrode.

17. 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.

18. A method of sequencing a DNA molecule, comprising:
providing the circuit of claim 1 ;
initiating at least one of a voltage or a current through the circuit;
exposing the circuit to a solution having an ionic strength from dissolved ions and comprising at least one of primed single stranded DNA and dNTPs; and
measuring electrical signals through the circuit as the polymerase engages and extends a template,
wherein the electrical signals are processed to identify features that provide information on the underlying sequence of the DNA molecule when processed by the polymerase.

19. The method of claim 18 , wherein the ionic strength of the solution influences the conductive pathway as the polymerase processes the DNA molecule.

20. The method of claim 18 , wherein the circuit is usable to sense sequence information from a DNA template when processed by the polymerase.

21. The method of claim 18 , wherein the polymerase is 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 genetically modified reverse transcriptase.

22. The method of claim 18 , wherein the polymerase comprises an E. coli Pol I Klenow Fragment.

23. A method of molecular detection, comprising:
providing the circuit of claim 16 ;
initiating at least one of a voltage or a current through the circuit;
exposing the circuit to at least one of: a buffer of reduced ionic strength, a buffer comprising modified dNTPs, a buffer comprising altered divalent cation concentrations, specific applied voltage on the first or the second electrodes, a gate electrode voltage, or voltage spectroscopy or sweeping applied to the first and second electrodes or the gate electrode; and
measuring an electrical change in the circuit.

24. The method of claim 23 , wherein the electrical change is processed to identify features that provide information on the underlying sequence of a DNA molecule when processed by the polymerase.

25. The method of claim 23 , wherein the electrical change is at least one of current, voltage, impedance, conductivity, resistance or capacitance.

26. The method of claim 23 , wherein the circuit senses sequence information from a DNA template when processed by the polymerase.

27. The method of claim 23 , wherein the polymerase 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 genetically modified reverse transcriptase.

28. The method of claim 23 , wherein the polymerase comprises an E. coli Pol I Klenow Fragment.

29. The method of claim 23 , wherein the polymerase comprises a reverse transcriptase capable of directly acting on an RNA template.

30. The method of claim 29 , wherein the reverse transcriptase comprises a genetically modified form of a reverse transcriptase, a Moloney murine leukemia virus reverse transcriptase, a porcine endogenous retrovirus reverse transcriptase, a bovine leukemia virus reverse transcriptase, a mouse mammary tumor virus reverse transcriptase, a heterodimeric reverse transcriptase, or a Rous sarcoma virus reverse transcriptase.

Assignees

• Roswell Biotechnologies Inc

Inventors

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

Classifications

• C12Q1/001 Primary

  Enzyme electrodes · CPC title

• C12Q1/48

  involving transferase · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/002 Primary

  Electrode membranes · CPC title

• G01N33/5438

  Electrodes · CPC title