Compositions, devices, systems, and methods for using a nanopore
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.
1. A method for controlling movement of a polynucleotide using voltage control, the method resulting in identifying a sequence of a polynucleotide, the method comprising the steps of:
a) providing two separate adjacent chambers comprising a medium, an interface between the two chambers, the interface comprising a material having at least one 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;
b) providing an enzyme having binding activity for a polynucleotide, said enzyme being a polymerase; 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 complementary oligomer, wherein the complementary oligomer is complementary to a portion of the single stranded polynucleotide; providing a substrate;
c) 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;
d) introducing the enzyme into the same chamber; introducing the complementary oligomer into the other chamber;
e) 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;
f) measuring the electrical current through the channel after removal of the blocking oligomer;
g) providing a potential difference allowing the complementary oligomer to bind to the single-stranded polynucleotide;
h) reversing the potential difference, thereby creating a reverse 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;
i) reversing the potential difference a second time;
j) measuring the electrical current through the channel, thereby detecting a polynucleotide having incorporated substrate or a polynucleotide bound to the enzyme;
k) repeating any one of steps h) through j), thereby controlling movement of the polynucleotide in order to determine at least a portion of the sequence of the polynucleotide.
2. The method of claim 1 further comprising comparing the electrical current value obtained at step f) with the current value obtained at step j).
3. The method of claim 1 further comprising comparing the electrical current value obtained at step j) 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 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, formed with a portion of the polynucleotide complex that is single-stranded, 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, and RNA polymerase.
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 Mg 2+ , Mn 2+ , Ca 2+ , 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.