Compositions, systems, and methods for sequencing polynucleotides using tethers anchored to polymerases adjacent to nanopores
First Claim
1. A composition including:
- a nanopore including a first side, a second side, and an aperture extending through the first and second sides;
a plurality of nucleotides, wherein each of the nucleotides comprises an elongated tag;
first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide;
a polymerase disposed adjacent to the first side of the nanopore, the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide;
a permanent tether including a head region, a tail region, and an elongated body disposed therebetween, the head region being anchored to the polymerase, wherein the elongated body is disposed in the aperture of the nanopore;
a first moiety disposed on the elongated body, wherein the first moiety is configured to bind to the elongated tag of a first nucleotide upon which the polymerase is acting; and
a reporter region disposed on the elongated body, wherein the reporter region is configured to indicate when the first nucleotide is complementary or is not complementary to a next nucleotide in the sequence of the second polynucleotide.
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Abstract
A composition includes a nanopore including first and second sides and an aperture, nucleotides each including an elongated tag, and a first polynucleotide that is complementary to a second polynucleotide. A polymerase can be disposed adjacent to the first side of the nanopore and configured to add nucleotides to the first polynucleotide based on a sequence of the second polynucleotide. A permanent tether can include a head region anchored to the polymerase, a tail region, and an elongated body disposed therebetween that occurs in the aperture of the nanopore. A first moiety can be disposed on the elongated body that binds to the elongated tag of a first nucleotide upon which the polymerase is acting. A reporter region can be disposed on the elongated body that indicates when the first nucleotide is complementary or is not complementary to a next nucleotide in the sequence of the second polynucleotide.
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Citations
49 Claims
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1. A composition including:
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a nanopore including a first side, a second side, and an aperture extending through the first and second sides; a plurality of nucleotides, wherein each of the nucleotides comprises an elongated tag; first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide; a polymerase disposed adjacent to the first side of the nanopore, the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide; a permanent tether including a head region, a tail region, and an elongated body disposed therebetween, the head region being anchored to the polymerase, wherein the elongated body is disposed in the aperture of the nanopore; a first moiety disposed on the elongated body, wherein the first moiety is configured to bind to the elongated tag of a first nucleotide upon which the polymerase is acting; and a reporter region disposed on the elongated body, wherein the reporter region is configured to indicate when the first nucleotide is complementary or is not complementary to a next nucleotide in the sequence of the second polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. A method including:
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(a) providing a nanopore including a first side, a second side, and an aperture extending through the first and second sides; (b) providing a plurality of nucleotides, wherein each of the nucleotides comprises an elongated tag; (c) providing first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide; (d) providing a polymerase disposed adjacent to the first side of the nanopore, the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide, wherein the polymerase is anchored to a permanent tether including a head region, a tail region, and an elongated body disposed therebetween, the elongated body is disposed in the aperture of the nanopore; (e) determining that a first nucleotide is being acted upon by the polymerase based on binding of the elongated tag to a first moiety disposed on the elongated body; and (f) with a reporter region disposed on the elongated body, indicating when the first nucleotide is complementary or is not complementary to a next nucleotide in the sequence of the second polynucleotide. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)
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Specification