COMPOSITIONS, DEVICES, SYSTEMS, AND METHODS FOR USING A NANOPORE

• US 20110174625A1
• Filed: 04/04/2008
• Published: 07/21/2011
• Est. Priority Date: 04/04/2007
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Dunbar, William B., Benner, Seico, Deamer, David W., Abu-Shumays, Robin, Chen, Roger Jinteh Arrigo, Hurt, Nicholas, Lieberman, Kate, Akeson, Mark A., Wilson, Noah A., Branton, Daniel
• Granted Patent: US 8,500,982 B2
• Time in Patent Office: Days
• US Class Current: 204/601
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C25B 3/29 Coupling reactions
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 27/4166 measuring a particular property of an electrolyte
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

The invention herein disclosed provides for devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore. The devices and methods are also used to determine rapidly (˜>50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore. The invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.

Claims

1-28. -28. (canceled)

29. A method for controlling binding of an enzyme to a polynucleotide using voltage feedback control, the method resulting in repeated capture of and dissociation of the enzyme by the polynucleotide, the method comprising the steps of:

providing two separate adjacent chambers comprising a medium, an interface between the two chambers, the interface having a channel so dimensioned as to allow sequential monomer-by-monomer passage from the cis-side of the channel to the trans-side of the channel of only one polynucleotide strand at a time;

providing an enzyme having binding activity for a polynucleotide;

providing a protected deoxyribonucleotide;

providing a polynucleotide-binding compound;

providing a polynucleotide complex, wherein a portion of the polynucleotide complex is double-stranded and a portion is single-stranded;

introducing the polynucleotide complex into one of the two chambers;

applying a potential difference between the two chambers, thereby creating a first polarity, the first polarity causing the single stranded portion of the polynucleotide to transpose through the channel to the trans-side;

introducing the protected deoxyribonucleotide into the same chamber;

introducing the enzyme into the same chamber;

allowing the enzyme to bind to the polynucleotide;

allowing the protected deoxyribonucleotide to bind to the polynucleotide;

measuring the electrical current through the channel thereby detecting the binding of the enzyme and the protected deoxyribonucleotide to the polynucleotide;

introducing the polynucleotide-binding compound into the other of the two chambers;

decreasing the potential difference a first time, thereby creating a second polarity;

allowing the polynucleotide-binding compound to bind to the single-stranded polynucleotide;

reversing the potential difference, thereby creating a third polarity;

reversing the potential difference a second time;

measuring the electrical current through the channel, thereby detecting a polynucleotide alone or a polynucleotide bound to the enzyme and the protected deoxyribonucleotide;

repeating any one of the steps, thereby controlling the binding of the enzyme to the polynucleotide.

30. The method of claim 29 further comprising the steps of measuring the electrical current between the two chambers;

comparing the electrical current value obtained at the first time the first polarity was induced with the electrical current value obtained at the time the second time the first polarity was induced.

31. The method of claim 29 further comprising the steps of measuring the electrical current between the two chambers;

comparing the electrical current value obtained at the first time the first polarity was induced with the electrical current value obtained at a later time.

32. The method of claim 29 wherein the polynucleotide-binding compound is selected from the group consisting of an oligonucleotide complementary to the polynucleotide, a peptide nucleic acid, a locked nucleic acid, a derivatized nucleotide, and a nucleotide isomer.

33. The method of claim 29 wherein the enzyme is selected from the group consisting of DNA polymerase, RNA polymerase, endonuclease, exonuclease, DNA ligase, DNase, uracil-DNA glycosidase, kinase, phosphatase, methylase, acetylase, and ribosome.

34. The method of claim 29 further comprising the steps of providing at least one reagent that initiates enzyme activity;

introducing the reagent to the chamber comprising the polynucleotide complex; and

incubating the chamber at a temperature sufficient to maintain enzyme activity.

35. The method of claim 34, wherein the reagent is a cofactor.

36. The method of claim 35, wherein the cofactor is selected from the group consisting of Mg²⁺, Mn²⁺, Ca²⁺, ATP, NAD⁺, NADP⁺, and S-adenosylmethionine.

37. The method of claim 29, wherein the medium is electrically conductive.

38. The method of claim 29, wherein the medium is an aqueous medium.

39. The method of claim 29 wherein the protected deoxyribonucleotide comprises a deoxyribonucleotide selected from the group consisting of dATP, dGTP, TTP, dCTP, UTP, and dUTP.

40. The method of claim 34 wherein the reagent is selected from the group consisting of ddATP, ddGTP, ddTTP, ddCTP, and ddUTP.

41-52. -52. (canceled)

53. A finite state machine, the finite state machine comprising cis and trans chambers connected by an electrical communication means, the cis and trans chambers separated by a thin film comprising at least one pore or channel, wherein the pore or channel is shaped and sized having dimensions suitable for passaging a polymer, means for applying an electric field between the cis and the trans chambers, and means for detecting the current between the cis and the trans chambers.

54. The finite state machine of claim 53, wherein the thin film comprises a compound having a hydrophobic domain and a hydrophilic domain.

55. The finite state machine of claim 53 wherein the thin film comprises a phospholipid.

56. The finite state machine of claim 53 wherein the pore or channel accommodates a substantial portion of the polymer.

57. The finite state machine of claim 53 wherein the pore or channel has biological activity.