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US 10,480,026 B2Granted
Method for nanopore RNA characterisation
AssigneeOxford Nanopore Technologies PLC
InventorsDaniel Ryan Garalde, Andrew John Heron, Lakmal Jayasinghe, Daniel John Turner, James White
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Abstract
The invention relates to a new method of characterizing a target RNA polynucleotide by taking one or more measurements as the target RNA polynucleotide moves with respect to a transmembrane pore. The movement is controlled by a DNA helicase. The invention also relates to a modified RNA construct wherein the RNA polynucleotide has been modified to increase DNA helicase binding thereto.
Claims32
A method of characterising a target RNA polynucleotide comprising:
- a) providing (i) an RNA polynucleotide wherein the RNA polynucleotide is modified to comprise a non-RNA polynucleotide and (ii) a DNA helicase enzyme;
- wherein the DNA helicase comprises a modification to reduce the size of an opening in the polynucleotide binding domain through which in at least one conformational state the RNA polynucleotide can unbind from the helicase
- b) contacting the RNA polynucleotide and DNA helicase enzyme provided in a) with a transmembrane pore such that the DNA helicase controls the movement of the RNA polynucleotide through the transmembrane pore;
- c) taking one or more measurements as the RNA polynucleotide moves with respect to the transmembrane pore, wherein the measurements are indicative of one or more characteristics of the RNA polynucleotide, and thereby characterising the target RNA polynucleotide.
1 more element
A method of characterising an RNA polynucleotide comprising:
- a) providing (i) an RNA polynucleotide wherein the RNA polynucleotide is modified to comprise a non-RNA polynucleotide, (ii) a DNA helicase enzyme, and (iii) one or more molecular brakes that are derived from helicases and are modified such that they bind the polynucleotide but do not function as a helicase;
- b) contacting the RNA polynucleotide and DNA helicase enzyme provided in a) with a transmembrane pore such that the DNA helicase controls the movement of the RNA polynucleotide through the transmembrane pore;
- c) taking one or more measurements as the RNA polynucleotide moves with respect to the transmembrane pore, wherein the measurements are indicative of one or more characteristics of the RNA polynucleotide, and thereby characterising the RNA polynucleotide.
Dependencies are inferred from the claim text.