Compositions, devices, systems, for using a Nanopore
First Claim
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.
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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.
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Citations
28 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
- 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;
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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−
1·
s−
1 (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. - View Dependent Claims (19, 20, 21, 22)
- (b) an enzyme having a binding affinity for a polynucleotide of at least 106 M−
- 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.
Specification