Methods for using a nanopore
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 are provided. The devices and methods also determine (˜>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.
1. A method for controlling the activity of an enzyme on a partially double-stranded polynucleotide complex, the method comprising:
(a) 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 passage from one pool to the other pool of only one single-stranded polynucleotide at a time;
(b) providing a partially double-stranded polynucleotide complex comprising a first polynucleotide, a second polynucleotide partially annealed to the first polynucleotide, and a blocking oligomer in one of the two pools;
wherein the blocking oligomer is annealed to a portion of the second polynucleotide not annealed to the first polynucleotide, and wherein the blocking oligomer comprises a non-hybridizing 3 ′ end;
(c) providing an enzyme having binding activity to the partially double-stranded polynucleotide complex in the same pool as the partially double-stranded polynucleotide complex;
(d) allowing the enzyme to bind to the partially double-stranded polynucleotide complex, wherein the blocking oligomer prevents the activity of the enzyme on the partially double-stranded polynucleotide complex; and
(e) applying a potential difference between the two pools, thereby creating a first polarity, to dissociate the blocking oligomer from the second polynucleotide; thereby controlling the activity of the enzyme on 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 time the first polarity was induced with the electrical current value obtained at a later time.
3. 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.
4. 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 the enzyme.
5. The method of claim 4 , wherein the at least one reagent is a cofactor.
6. The method of claim 5 , wherein a deoxyribonucleotide is introduced into the pool prior to introducing the cofactor.
7. The method of claim 6 , wherein the cofactor is selected from the group consisting of Mg 2+ , Mn 2+ , Ca 2+ , ATP, NAD + , NADP + , and S-adenosylmethionine.
8. The method of claim 1 , wherein the medium is electrically conductive.
9. The method of claim 8 , wherein the medium is an aqueous solution.
10. The method of claim 5 wherein the at least one reagent is a deoxyribonucleotide.
11. The method of claim 1 which comprises a further step of characterizing the first or second polynucleotide.