Oxford Nanopore Technologies Limited

Daniel John Turner

James White

Andrew John Heron

Clive Gavin Brown

Daniel Ryan Garalde

James Anthony Clarke

Stuart William Reid

Daniel George Fordham

Lakmal Nishantha Jayasinghe

Christopher James Wright

Rebecca Victoria Bowen

Gavin Harper

Graham Hall

Marion Louise Crawford

Christopher Peter Youd

David Alves

Joseph Hargreaves Lloyd

Roger Charles Gill

Nicholas Antony Smith

David Jackson Stoddart

Andy Heron

David Antoni Alves

Rebecca Bowen

Stuart Reid

Nicholas Smith

The invention relates to a new method of characterising 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.

Method for nanopore RNA characterization

The invention relates to new methods of attaching one or more polynucleotide binding proteins to a target polynucleotide. The invention also related to new methods of characterising target polynucleotides.

Method for attaching one or more polynucleotide binding proteins to a target polynucleotide

The invention relates to a new method of determining in a sample the presence or absence of one or more analyte members of a group of two or more analytes. The invention therefore relates to a multiplex assay for determining the presence or absence of each analyte in a group of multiple analytes. The assay uses aptamers and transmembrane pores.

Aptamer method

The invention relates to a new method of delivering an analyte to a transmembrane pore in a membrane. The method involves the use of microparticles.

Methods for delivering an analyte to transmembrane pores

The invention relates to new methods of moving helicases past spacers on polynucleotides and controlling the loading of helicases on polynucleotides. The invention also relates to new methods of characterising target polynucleotides using helicases.

Enzyme stalling method

A biochemical analysis system analyses polymers by taking measurements of a polymer from a sensor element comprising a nanopore during translocation of the polymer through the nanopore. When a polymer has partially translocated, the series of measurements is analysed using reference data derived from a reference sequence to provide a measure of similarity. Responsive to the measure of similarity, the sensor element may be selectively operated to eject the polymer and thereby make the nanopore available to receive a further polymer. Where the biochemical analysis system comprises an array of sensor elements and is takes measurements from sensor elements selected in a multiplexed manner, responsive to the measure of similarity, the biochemical analysis system ceases taking measurements from the currently selected sensor element and to starts taking measurements from a newly selected sensor element.

Analysis of a polymer

A method for determining the presence, absence or amount of two or more target polynucleotides in a sample comprising additional components, the method comprising: (i) contacting the sample with a panel of two or more probes under conditions suitable for hybridisation of the target polynucleotides to the probes, wherein: (a) each probe comprises a non-hybridisation region and a hybridisation region that specifically hybridises to one of the target polynucleotides to form a hybridised probe; and (b) the hybridisation region of a probe of the panel comprises one or more non-natural nucleotides; (ii) contacting the sample prepared in step (i) with a transmembrane pore through which a single stranded polynucleotide but not a double stranded polynucleotide can pass and applying a potential difference to the transmembrane pore such that the hybridised probes in the sample interact with the pore; (iii) measuring current blockades having a duration within a defined window, wherein: (a) the one or more non-natural nucleotides present in the hybridisation region of the probe increase or decrease the duration of the current blockade due to the probe hybridised to its target polynucleotide such that the proportion of current blockades that occur within the window due to the interaction of the hybridised probes with the pore is increased compared to when the corresponding one or more natural nucleotides are present in the hybridisation region; and (b) each hybridised probe gives rise to a current blockade indicative of that probe; and (iv) correlating the measured current blockades with the probes, thereby determining the presence, absence or amount of the two or more target polynucleotides in the sample.

Method

The invention relates to a new method of characterising a target ribonucleic acid (RNA) involving forming a complementary polynucleotide. The method uses a transmembrane pore.

Method of characterizing a target ribonucleic acid (RNA) comprising forming a complementary polynucleotide which moves through a transmembrane pore

Method for nanopore RNA characterisation

Analysis Of A Polymer A biochemical analysis system analyses polymers by taking measurements of a polymer from a sensor element comprising a nanopore during translocation of the polymer through the nanopore. When a polymer has partially translocated, the series of measurements is analysed using reference data derived from a reference sequence to provide a measure of similarity. Responsive to the measure of similarity, the sensor element may be selectively operated to eject the polymer and thereby make the nanopore available to receive a further polymer. Where the biochemical analysis system comprises an array of sensor elements and is takes measurements from sensor elements selected in a multiplexed manner, responsive to the measure of similarity, the biochemical analysis system ceases taking measurements from the currently selected sensor element and to starts taking measurements from a newly selected sensor element.

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.

The invention relates to new methods of attaching one or more polynucleotide binding proteins to a target polynucleotide. The invention also relates to new methods of characterizing target polynucleotides.

The invention relates to new methods of moving helicases past spacers on polynucleotides and controlling the loading of helicases on polynucleotides. The invention also relates to new methods of characterizing target polynucleotides using helicases.

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Oxford, United Kingdom

Daniel John Turner