Compositions, devices, systems, for using a Nanopore

• US 20100035260A1
• Filed: 06/26/2009
• Published: 02/11/2010
• 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: Olasagasti, Felix, Benner, Seico, Lieberman, Kate, Akeson, Mark A.
• Granted Patent: US 8,679,747 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 in the absence of requiring a terminating nucleotide. 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 drug discovery, molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.

Claims

1. A method for controlling movement of a polynucleotide using voltage feedback control, the method resulting in identifying the sequence of a polynucleotide, the method comprising the steps of:

providing two separate adjacent chambers comprising a medium, an interface between the two chambers, the interface comprising a material having at least channel therethrough and wherein one chamber is on the cis-side of the interface and the other chamber is on the trans-side of the interface, the 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 blocking oligomer;

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

providing a complimentary oligomer, wherein the complimentary oligomer is complementary to a portion of the single stranded polynucleotide;

providing a substrate;

introducing the polynucleotide complex into one of the two chambers;

introducing the blocking oligomer into the same chamber;

allowing the blocking oligomer to bind to the polynucleotide complex;

introducing the enzyme into the same chamber;

introducing the complementary oligomer into the other chamber;

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 thereby stripping the blocking oligomer from the polynucleotide complex;

measuring the electrical current through the channel thereby detecting the polynucleotide;

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

allowing the complementary oligomer to bind to the single-stranded polynucleotide;

reversing the potential difference, thereby creating a third polarity;

providing conditions to allow the enzyme to bind to the polynucleotide complex;

providing conditions to allow the enzyme to incorporate substrate into the polynucleotide, thereby increasing length of the double-stranded portion;

reversing the potential difference a second time;

measuring the electrical current through the channel, thereby detecting a polynucleotide having incorporated substrate or a polynucleotide bound to the enzyme;

repeating any one of the steps, thereby controlling the synthesis of the polynucleotide.

2. The method of claim 1 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.

3. The method of claim 1 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.

4. The method of claim 1 wherein the step of allowing the blocking oligomer to bind to the polynucleotide complex is performed prior to introducing the polynucleotide complex and the blocking oligomer into the same chamber, and is followed by a step of introducing the polynucleotide complex and the blocking oligomer into the chamber.

5. The method of claim 1 where there are a plurality of channels.

6. The method of claim 1 wherein the blocking oligomer is selected from the group consisting of an oligonucleotide having partial complementarity to a portion of the polynucleotide complex.

7. The method of claim 6 wherein the oligonucleotide having partial complementarity to a portion of the polynucleotide complex further comprises a duplex structure at one end of the oligonucleotide and a blocking moiety at the other end of the oligonucleotide.

8. The method of claim 6 wherein the oligonucleotide having partial complementarity to a portion of the polynucleotide complex further comprises a hairpin loop structure at one end of the oligonucleotide.

9. The method of claim 7 wherein the blocking moiety is selected from the group consisting of acridine, a peptide nucleic acid, a 2′

-O-methyl group, a fluorescent compound, DAPI, an anthocyanin, green fluorescent protein (GFP), β

-glucuronidase, luciferase, Cy3, Cy5, a derivatized nucleotide, and a nucleotide isomer.

10. The method of claim 1 wherein the complementary oligomer is selected from the group consisting of an oligonucleotide complementary to the polynucleotide, a peptide nucleic acid, a locked nucleic acid, a derivatized nucleotide, a nucleotide isomer, and a DNA aptamer.

11. The method of claim 1 wherein the enzyme is selected from the group consisting of DNA polymerase, RNA polymerase, endonuclease, exonuclease, DNA ligase, DNase, uracil-DNA glycosidase, topoisomerase, telomerase, DNA-repair enzyme;

DNA-handling enzyme, helicase, primase, gyrase, kinase, phosphatase, methylase, acetylase, histone, transcription factor, and ribosome.

12. The method of claim 1 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.

13. The method of claim 12, wherein the reagent is a cofactor.

14. The method of claim 13, wherein the cofactor is selected from the group consisting of Mg²⁺, Mn²⁺, Ca²⁺, ATP, NAD⁺, NADP⁺, and S-adenosylmethionine.

15. The method of claim 1, wherein the medium is electrically conductive.

16. The method of claim 1, wherein the medium is an aqueous medium.

17. The method of claim 1 wherein the substrate comprises a ribonucleotide selected from the group consisting of dATP, dGTP, TTP, dCTP, UTP, dUTP, ATP, GTP, TTP, CTP, and tRNA.

18. A polynucleotide sequencing system comprising (a) a structure comprising an ion-permeable passage connecting a first chamber and a second chamber, wherein a polynucleotide to be sequenced is placed with a blocking oligomer in the first chamber;

(b) an enzyme having a binding affinity for a polynucleotide of at least 10⁶ M<sup>−

1</sup>·

s<sup>−

1</sup> (c) a electronic power source for creating a potential difference between the two chambers;

(d) a detection system operative to detect a property of a the polynucleotide moving relative to the ion-permeable passage.

19. The polynucleotide sequencing system of claim 18, wherein the structure further comprises a lipid bilayer.

20. The polynucleotide sequencing system of claim 18, wherein the blocking oligomer binds to the polynucleotide to be sequenced under stringent conditions.

21. The polynucleotide sequencing system of claim 18, wherein the enzyme is selected from the group consisting of DNA polymerase, RNA polymerase, endonuclease, exonuclease, DNA ligase, DNase, uracil-DNA glycosidase, topoisomerase, telomerase, DNA-repair enzyme;

DNA-handling enzyme, helicase, primase, gyrase, kinase, phosphatase, methylase, acetylase, histone, transcription factor, and ribosome.

22. The polynucleotide sequencing system of claim 18, wherein the property of the polynucleotide is that of base identity at the 3′

end of the double-stranded portion of the polynucleotide.

23. A blocking oligomer, wherein the blocking oligomer comprises a single stranded oligonucleotide, the single stranded oligonucleotide comprising a duplex structure at one end of the oligonucleotide and a blocking moiety at the other end of the oligonucleotide.

24. The blocking oligomer of claim 23 wherein a portion of the blocking oligomer comprises a non-oligonucleotide composition wherein the non-oligonucleotide composition is selected from the group consisting of a polymer, a monomer, a dimer, a multimer, an acid, a base, an organic compound, and an inorganic compound.

25. The blocking oligomer of claim 23 wherein the blocking moiety is selected from the group consisting of acridine, a peptide nucleic acid, a 2′

-O-methyl group, a fluorescent compound, DAPI, an anthocyanin, green fluorescent protein (GFP), β

-glucuronidase, luciferase, Cy3, Cy5, a derivatized nucleotide, and a nucleotide isomer.

26. The blocking oligomer of claim 23, wherein the blocking oligomer binds to a polynucleotide to be sequenced under stringent conditions.

27. The blocking oligomer of claim 23, wherein the blocking oligomer comprises between about 5 and 30 nucleotides.

28. The blocking oligomer of claim 23, wherein the blocking oligomer comprises between about 15 and 25 nucleotides.