Compositions, devices, systems, and methods for using a nanopore

• US 20110005918A1
• Filed: 04/04/2008
• Published: 01/13/2011
• Est. Priority Date: 04/04/2007
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Benner, Seico, Deamer, David W., Abu-Shumays, Robin, Dunbar, Wiliam B., Chen, Roger Jinteh Arrigo, Hurt, Nicholas, Lieberman, Kate, Akeson, Mark A., Wilson, Noah A.
• Application Number:
  US12/080,684
• Publication Number:
  US 20110005918A1
• Time in Patent Office: Days
• US Class Current: 204/165
• 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. A method for controlling binding of an enzyme to a partially double-stranded polynucleotide complex, the method comprising:

providing two separate, adjacent pools of a medium and an interface between the two pools, the interface having a channel so dimensioned as to allow sequential monomer-by-monomer passage from one pool to the other pool of only one polynucleotide at a time;

providing an enzyme having binding activity to a partially double-stranded polynucleotide complex;

providing a polynucleotide complex comprising a first polynucleotide and a second polynucleotide, wherein a portion of the polynucleotide complex is double-stranded, and wherein the first polynucleotide further comprises a moiety that is incompatible with the second polynucleotide;

introducing the polynucleotide complex into one of the two pools;

introducing the enzyme into one of the pools;

applying a potential difference between the two pools, thereby creating a first polarity;

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

reversing the potential difference a second time to create the first polarity, thereby controlling the binding of the enzyme to the partially double-stranded polynucleotide complex.

2. The method of claim 1 further comprising the steps of measuring the electrical current between the two pools;

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 pools;

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 moiety is selected from the group consisting of a peptide nucleic acid, a 2′

-O-methyl group, a fluorescent compound, a derivatized nucleotide, and a nucleotide isomer.

5. 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, kinase, phosphatase, methylase, acetylase, and ribosomes.

6. The method of claim 1 further comprising the steps of providing at least one reagent that initiates enzyme activity;

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

incubating the pool at a suitable temperature.

7. The method of claim 6, wherein the reagent is selected from the group consisting of a deoxyribonucleotide and a cofactor.

8. The method of claim 7, wherein the deoxyribonucleotide is introduced into the pool prior to introducing the cofactor.

9. The method of claim 7, wherein the cofactor is selected from the group consisting of Mg²⁺, Mn²⁺, Ca²⁺, ATP, NAD⁺, NADP⁺, and S-adenosylmethionine.

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

11. The method of claim 10, wherein the medium is an aqueous solution.

26. The device of claim 1 wherein the pore aperture is between about 0.5 and 8 nm in size.

27. The device of claim 26 wherein the pore aperture is between about 0.5 and 4 nm in size.

28. The device of claim 27 wherein the pore aperture is between about 1 and 2 nm in size.

12. A device for detecting binding of at least two compounds, the device comprising a mixed-signal semiconductor wafer, at least one electrochemical layer, the electrochemical layer comprising a semiconductor material, wherein the semiconductor material further comprises a surface modifier, wherein the electrochemical layer defines a plurality of orifices, the orifices comprising a chamber and a neck and wherein the chamber of the orifices co-localize with a metallization composition of the mixed-signal semiconductor wafer, wherein a portion of the orifice is plugged with a metal, wherein the metal is in electronic communication with the metallization composition, and wherein the orifice further comprises a thin film, the thin film forming a solvent-impermeable seal at the neck of the orifice, the thin film further comprising a pore, the pore further comprising a pore aperture.

13. The device of claim 12 wherein the compounds are biological compounds.

14. The device of claim 13 wherein the biological compounds are selected from the group consisting of polynucleotides, polypeptides, phospholipids, polysaccharides, polyketides, proteases, kinases, phosphatases, hydrolases, oxidoreductases, isomerases, transferases, methylases, acetylases, ligases, and lyases.

15. The device of claim 12 wherein the semiconductor material is silicon dioxide.

16. The device of claim 12 wherein the surface modifier is a hydrocarbon.

17. The device of claim 12 wherein the metallization composition is selected from the group consisting of nickel, gold, copper, and aluminum.

18. The device of claim 12 wherein the metal is silver.

19. The device of claim 12 wherein the thin film is a phospholipid bilayer.

20. The device of claim 12 wherein the orifice is between 0.5 and 3 μ

m in size.

23. The device of claim 12 wherein the pore is a biological molecule.

24. The device of claim 23 wherein the biological molecule is selected from the group consisting of an ion channel, a nucleoside channel, a peptide channel, a sugar transporter, a synaptic channel, a transmembrane receptor, and a nuclear pore.

25. The device of claim 23 wherein the biological molecule is α

-hemolysin.

21. The device of claim 21 wherein the orifice is between 1 and 2 μ

m in size.

22. The device of claim 21 wherein the orifice is between 1.25 and 1.5 μ

m in size.