Enzymatic circuits for molecular sensors
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.
1. A method of sequencing a DNA or RNA molecule, the method comprising:
initiating at least one of a voltage or a current through a circuit comprising: a first gold electrode; a second gold 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 bonded to the first electrode through a gold-thiol bond; a second arm molecule having a first end and a second end, the second end of the second arm molecule bonded to the second electrode through a gold-thiol bond; and an enzyme, wherein the first end of each of the first and second arm molecules connect to the enzyme at two distinct sites on the enzyme so that a portion of the enzyme between the two distinct sites is included as a conductive pathway in the circuit;
exposing the circuit to a solution comprising dNTPs and the DNA or RNA molecule to be sequenced;
measuring electrical signals through the circuit as the enzyme engages and processes the DNA or RNA molecule; and
processing the resulting electrical signals to obtain a sequence of the DNA or RNA molecule processed by the enzyme.
2. The method of claim 1 , wherein the enzyme comprises a polymerase.
3. The method of claim 1 , wherein the enzyme comprises a genetically modified form of an E. coli Pol I polymerase, a Bst polymerase, a Taq polymerase, a Phi29 polymerase, or a T7 polymerase.
4. The method of claim 1 , wherein the enzyme comprises a reverse transcriptase.
5. The method of claim 1 , wherein the enzyme comprises 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.
6. The method of claim 1 , wherein the enzyme comprises human immunodeficiency virus reverse transcriptase.
7. The method of claim 1 , wherein the electrical signals comprise perturbations in at least one of a current, voltage, impedance, conductivity, resistance or capacitance in the circuit.
8. The method of claim 1 , wherein the first arm molecule and the second arm molecule each comprise a peptide alpha-helix.
9. The method of claim 2 , wherein the polymerase enzyme comprises an E. coli Pol I Klenow Fragment.
10. The method of claim 2 , wherein the portion of the polymerase enzyme between the two distinct sites 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.
11. The method of claim 1 , wherein the electrical signals are modulated by the choice and content of divalent cations present in the solution.