Compositions, devices, systems, 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 polynucleotide sequencing system comprising (a) a structure comprising an ion-permeable passage connecting a first pool of a medium and a second pool of a medium, wherein a polynucleotide to be sequenced and a blocking oligomer are present in the first pool; (b) an enzyme having binding affinity for said polynucleotide; (c) an electronic power source for creating a potential difference between the two pools; and (d) a detection system operative to detect a property of the polynucleotide, wherein the blocking oligomer inhibits an interaction between the enzyme and the polynucleotide.
2. The polynucleotide sequencing system of claim 1 , wherein the structure further comprises a thin film comprising a compound having a hydrophobic domain and a hydrophilic domain.
3. The polynucleotide sequencing system of claim 1 , wherein the blocking oligomer binds to the polynucleotide to be sequenced under stringent conditions.
4. The polynucleotide sequencing system 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.
5. The polynucleotide sequencing system of claim 1 , wherein the property of the polynucleotide is its identity.
6. The polynucleotide sequence system of claim 1 , wherein the property of the polynucleotide is its sequence.
7. The polynucleotide sequence system of claim 1 , wherein the property of the polynucleotide is the number of nucleotides in the polynucleotide.
8. The polynucleotide sequence system of claim 1 , wherein the property of the polynucleotide is that of the base identity at the 3′ end of a double-stranded portion of the polynucleotide.
9. The polynucleotide sequence system of claim 1 , wherein the ion-permeable passage is a nanopore.
10. The polynucleotide sequence system of claim 1 , wherein the ion-permeable passage is a biological nanopore.
11. The polynucleotide sequence system of claim 1 , wherein the blocking oligomer comprises a blocking moiety.
12. The polynucleotide sequence system of claim 1 , wherein the blocking oligomer comprises a duplex structure.