COUPLING METHOD
The invention relates to a new method of determining the presence, absence or characteristics of an analyte. The analyte is coupled to a membrane. The invention also relates to nucleic acid sequencing.
1 .- 25 . (canceled)
26 . A method for determining the presence or characteristics of a target analyte, comprising:
(a) coupling the target analyte to an amphiphilic membrane containing a transmembrane pore, wherein the target analyte coupled to the membrane diffuses in a two-dimensional manner across the surface of the membrane to the nanopore; and
(b) interacting the target analyte coupled to the membrane with the transmembrane pore and thereby determining the presence or characteristics of the target analyte.
27 . The method of claim 26 , wherein the target analyte is coupled to the membrane via a linker.
28 . The method of claim 26 , wherein the target analyte comprises a linker, which couples the target analyte to the membrane.
29 . The method of claim 26 , wherein the amphiphilic membrane comprises a linker, which couples the target analyte to the membrane.
30 . The method of claim 26 , wherein the target analyte comprises two or more linkers and wherein the analyte is coupled to the membrane via the two or more linkers.
31 . The method of claim 30 , wherein the two or more linker is a polynucleotide.
32 . The method of claim 1 , wherein the transmembrane pore is a transmembrane protein pore.
33 . The method of claim 32 , wherein the protein pore is a α-helix bundle pore or a β-barrel pore.
34 . The method of claim 26 , wherein the target analyte is a polynucleotide.
35 . The method of claim 26 , wherein the target analyte is an amino acid, peptide, polypeptide, or protein.
36 . The method of claim 34 , wherein the polynucleotide analyte is coupled to the membrane at its 5′ end, its 3′ end or at one or more intermediate points along the polynucleotide strand.
37 . The method of claim 26 , wherein the coupling to the membrane is transient.
38 . The method of claim 26 , wherein the membrane comprises a first polynucleotide coupled to the membrane, and the coupling comprises modifying the target analyte by addition of a reactive group comprising a second polynucleotide that is complementary to the first polynucleotide, and wherein the target analyte is coupled to the membrane by hybridization between the first polynucleotide and the second polynucleotide.
39 . The method of claim 38 , wherein the target analyte is a single or double stranded polynucleotide analyte, and the reactive group is ligated to the polynucleotide analyte.
40 . The method of claim 34 , wherein the polynucleotide analyte is bound to a polynucleotide adaptor comprising a leader sequence, and wherein the lead sequence threads the polynucleotide analyte into the transmembrane pore.
41 . The method of claim 40 , wherein the method further comprises hybridizing the adaptor to a linker comprising a binding group.
42 . The method of claim 40 , wherein the method further comprises hybridizing or ligating the adapter to the polynucleotide analyte.
43 . The method of claim 26 , wherein the target analyte is a single stranded DNA (ssDNA) analyte comprising a leader sequence at one end that is adjacent to a region hybridized to a ssDNA tether, and wherein the ssDNA tether couples the polynucleotide analyte to the membrane to achieve a concentration enhancement at the membrane.
44 . The method of claim 41 , wherein the binding group or linker is mixed with the target analyte before contacting the membrane.
45 . The method of claim 41 , wherein the binding group or linker is contacted with the membrane prior to contacting the target analyte.
46 . The method of claim 26 , further comprising determining the presence or characteristics of the target analyte comprises measuring the current passing through the pore during the interaction.
47 . The method of claim 46 , wherein the target analyte is a polynucleotide, and wherein determining the presence or characteristics of the target analyte comprises estimating the sequence of the polynucleotide.
48 . The method of claim 47 , wherein a polynucleotide binding protein controls the movement of the target polynucleotide through the pore.
49 . The method of claim 48 , wherein the polynucleotide binding protein is a helicase.
50 . A method of sequencing an analyte which is a target polynucleotide, comprising:
(a) coupling the target polynucleotide to a membrane comprising a transmembrane pore;
(b) interacting the coupled target polynucleotide with the transmembrane pore and a polynucleotide binding protein, such that the polynucleotide binding protein controls the movement of the target polynucleotide through the transmembrane pore and nucleotides in the target polynucleotide interact with the transmembrane pore; and
(c) measuring the current passing through the transmembrane pore as the target polynucleotide moves with respect to the pore and thereby determining the sequence of the target polynucleotide.
51 . A complex comprising:
(a) a membrane comprising a transmembrane pore; and
(b) target polynucleotides, wherein the target polynucleotides are coupled to the membrane by one or more reactive group, wherein the coupling results in enhanced concentration of the target polynucleotides on the surface of the membrane in proximity to the transmembrane pore;
wherein the target polynucleotides are bound to respective polynucleotide binding proteins.
52 . The complex of claim 51 , wherein the respective polynucleotide binding proteins are configured to control the movement of the respective bound polynucleotides through the transmembrane pores.
53 . The complex of claim 51 , wherein the target polynucleotides each comprise a leader sequence.
54 . The complex of claim 53 , wherein the leader sequence is configured to thread the polynucleotide through the transmembrane pore.
55 . A kit for sequencing a target polynucleotide, comprising:
(a) a nanopore sequencing device comprising a membrane that comprises a transmembrane pore;
(b) a reactive group for coupling the target polynucleotide to the membrane;
(c) a leader sequence configured to be attached to the target polynucleotide for threading the polynucleotide through the transmembrane pore; and
(e) a polynucleotide binding protein that binds to the target polynucleotide for controlling the rate of interaction between the transmembrane pore and the target polynucleotide.