Abstract: The present invention relates to novel protein pores and their uses in analyte detection and characterisation. The invention particularly relates to an isolated pore complex formed by a CsgG-like pore and a modified CsgF peptide, or a homologue or mutant thereof, thereby incorporating an additional channel constriction or reader head in the nanopore. The invention further relates to a transmembrane pore complex and methods for production of the pore complex and for use in molecular sensing and nucleic acid sequencing applications.

Abstract: Methods of characterizing an analyte using a nanopore. One aspect features methods for characterizing a double-stranded polynucleotide using a nanopore, e.g., without using a hairpin connecting a template and a complement of the double-stranded polynucleotide. Another aspect features methods for characterizing an analyte using a tag-modified nanopore with increased sensitivity and/or higher throughput. Compositions and systems including, e.g., adaptors for attachment to double-stranded polynucleotides and tag-modified nanopores, which can be used in the methods are also provided.

Abstract: The invention relates to mutant forms of CsgG. The invention also relates to analyte detection and characterisation using CsgG.

Abstract: A microfluidic device comprises: a sensor provided in a sensing chamber; a liquid inlet and liquid outlet connecting to the sensor chamber for respectively passing liquid into and out of the sensing chamber and; a sample input port in fluid communication with the liquid inlet; a liquid collection channel downstream of the sensing chamber outlet; a flow path interruption between the liquid outlet and the liquid collection channel, preventing liquid from flowing into the liquid collection channel from upstream; a buffer liquid filling from the sample input port to the sensing chamber, and filling the sensing chamber and filing from the liquid outlet to the flow path interruption; an activation system operable to complete the flow path between the liquid outlet and the liquid collection channel such that the sensor remains unexposed to gas or a gas/liquid interface.

Abstract: The invention relates to adaptors for sequencing nucleic acids. The adaptors may be used to generate single stranded constructs of nucleic acid for sequencing purposes. Such constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) template. The invention also relates to the constructs generated using the adaptors, methods of making the adaptors and constructs, as well as methods of sequencing double stranded nucleic acids.

Abstract: The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterisation using nanopore sequencing. The method produces from the template a plurality of modified double stranded polynucleotides. These modified polynucleotides can then be characterised.

Abstract: Provided herein is a method of concentrating a tethering complex in a region of an amphiphilic layer, such as a lipid membrane. Also provided herein are methods of assembling a tethering complex; methods of concentrating an analyte in the region of a detector; amphiphilic layers; and arrays and devices for use in the disclosed methods.

Abstract: The present invention relates to novel protein pores and their uses in analyte detection and characterisation. The invention particularly relates to an isolated pore complex formed by a CsgG-like pore and a modified CsgF peptide, or a homologue or mutant thereof, thereby incorporating an additional channel constriction or reader head in the nanopore. The invention further relates to a transmembrane pore complex and methods for production of the pore complex and for use in molecular sensing and nucleic acid sequencing applications.

Abstract: Provided herein are methods of characterising a target polypeptide as it moves with respect to a nanopore. Also provided are related kits, systems and apparatuses for carrying out such methods.

Abstract: 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.

Abstract: An apparatus for supporting an array of layers of amphiphilic molecules, the apparatus comprising: a body, formed in a surface of the body, an array of sensor wells capable of supporting a layer of amphiphilic molecules across the sensor wells, the sensor wells each containing an electrode for connection to an electrical circuit, and formed in the surface of the body between the sensor wells, flow control wells capable of smoothing the flow of a fluid across the surface.

Abstract: The invention relates to an improved method for characterising a template polynucleotide. The method involves using a polymerase to prepare a modified polynucleotide which makes it easier to characterise than the template polynucleotide.

Abstract: Droplet interfaces are formed between droplets in an electro-wetting device comprising an array of actuation electrodes. Actuation signals are applied to selected actuation electrodes to place the droplets into an energised state in which the shape of the droplets is modified compared to a shape of the droplets in a lower energy state and to bring the two droplets into proximity. The actuation signals are then changed to lower the energy of the droplets into the lower energy state so that the droplets relax into the gap and the two droplets contact each other thereby forming a droplet interface. The use of sensing electrodes in the device permit electrical current measurements across the droplet interface. The sensing electrodes can be used for either (i) applying a reference signal during droplet actuation or (ii) recording electrical current measurements.

Abstract: A method of preparing a nucleic acid construct for single molecule characterisation, comprising contacting a target polynucleotide with: a polynucleotide-guided effector protein, a guide polynucleotide; a transposase; and a transposable element comprising a modified polynucleotide, wherein the polynucleotide-guided effector protein directs said transposase to a region of interest within the target polynucleotide and the transposase inserts the transposable element into the polynucleotide, thereby producing a nucleic acid construct for single molecule characterisation.

Abstract: The invention relates to a method of forming a sensing device for supporting a plurality of nanopores upon an array of wells. The method involves providing a substrate, said substrate having a surface having an array of electrodes located thereon for connecting to or for configuring upon an electronic circuit. Separately, a well array structure is provided, which has an array of walls defining through-holes for defining wells. The substrate and well array structures are aligning said array of electrodes define, at least in part, a portion of the bases of respective wells at the bottom of the through holes. The resulting sensing device overcomes problems with known sensing devices by employing a substrate and/or well array structure, or hybrid thereof, that employs alternative materials or manufacturing processes.

Abstract: The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.

Abstract: The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Dda helicase. The helicase controls the movement of the target polynucleotide through the pore. The invention also relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.

Abstract: The invention relates to modified helicases with reduced unbinding from polynucleotides. The helicases can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.

Abstract: Provided herein is a method of sequencing a target double stranded nucleic acid. The method comprises contacting the double stranded nucleic acid with a reagent as described herein to form a construct and sequencing the construct using a single-molecule sequencing technique as described herein. Associated products and kits are further provided.

Abstract: The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or a molecular 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.