Enzyme method
AssigneeOxford Nanopore Technologies PLC
InventorsRuth Moysey, Andrew John Heron
Life of this patent
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Abstract
The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.
Claims21
1-43. (canceled)
A method of controlling the movement of a polynucleotide through a transmembrane pore, comprising:
- (a) contacting the polynucleotide with a transmembrane pore under an applied voltage field such that a portion of the polynucleotide is captured by the transmembrane pore, wherein the pore is present in a membrane;
- (b) contacting the polynucleotide with a Hel308 helicase on the cis side of the membrane such that the helicase binds to the polynucleotide, thereby controlling the movement of the polynucleotide through the transmembrane pore against the applied voltage field from the trans side of the membrane to the cis side of the membrane;
- and
- (c) measuring an ionic current as the polynucleotide moves through the transmembrane pore.
A method of controlling the movement of a polynucleotide through a transmembrane pore, comprising:
- (a) providing in an aqueous solution the transmembrane pore and a membrane, wherein the transmembrane pore is present in the membrane, and wherein the aqueous solution comprises a salt at a concentration in a range of 0.3 M to 3 M;
- (b) combining, in the aqueous solution of step (a), the polynucleotide and a Hel308 helicase, wherein the helicase binds to the polynucleotide to form a polynucleotide-helicase complex, thereby controlling the movement of the polynucleotide through the transmembrane pore;
- and
- (c) measuring, during an application of a potential across the transmembrane pore, an ionic current as the polynucleotide moves through the transmembrane pore.
A method of controlling the movement of a polynucleotide through a transmembrane pore, comprising:
- (a) contacting a polynucleotide with a transmembrane pore, wherein a first helicase is bound to the polynucleotide such that the first helicase controls the movement of the polynucleotide through the pore;
- (b) further contacting the polynucleotide with a second helicase as the first helicase controls the movement of the polynucleotide through the transmembrane pore, wherein the second helicase binds to an internal polynucleotide of the polynucleotide at a location on the polynucleotide different than where the first helicase is bound without concurrent binding to a terminal nucleotide of the polynucleotide, and wherein the second helicase controls movement of the polynucleotide through the pore after the first helicase disengages from the polynucleotide;
- and
- (c) measuring an ionic current as the polynucleotide moves through the transmembrane pore.
Dependencies are inferred from the claim text.