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. A method for detecting polynucleotides, comprising:
(a) providing a membrane in which is present a nanopore that provides a channel through the membrane;
(b) contacting the membrane, in an ionic solution, with polynucleotides, wherein following contact with the membrane the polynucleotides are tethered to the membrane via hydrophobic anchors, wherein the polynucleotides and the hydrophobic anchors form complexes, and wherein the complexes do not comprise streptavidin; and
(c) applying a potential difference across the membrane and detecting the polynucleotides using the nanopore, from among the polynucleotides tethered to the membrane.
2. The method according to claim 1 , wherein the membrane is an amphiphilic layer, a lipid bilayer, or a solid state layer.
3. The method according to claim 1 , wherein each hydrophobic anchor is a lipid, a fatty acid, a sterol, a carbon nanotube, or an amino acid.
4. The method according to claim 1 , wherein each hydrophobic anchor is capable of embedding in the membrane.
5. The method according to claim 1 , wherein the polynucleotides are tethered transiently to the membrane.
6. The method according to claim 1 , wherein the polynucleotides are detected based on ion flow through the nanopore that is measured via an electrical means.
7. The method according to claim 1 , wherein the nanopore is a protein nanopore, optionally wherein the protein nanopore is derived from Msp or α-hemolysin (α-HL).
8. The method according to claim 1 , wherein the nanopore comprises a molecular adaptor that mediates interaction of the polynucleotide with the nanopore.
9. The method according to claim 1 , wherein the nanopore is coupled to a polynucleotide binding protein, which is optionally an exonuclease or a polymerase.
10. The method according to claim 1 , wherein the polynucleotides are detected based on ion flow through the nanopore that is measured by measuring a current passing through the nanopore.
11. The method according to claim 1 , wherein each polynucleotide comprises a target polynucleotide.
12. The method according to claim 11 , wherein the method comprises digesting the target polynucleotide to provide a fragment and the fragment sequence is determined.
13. The method according to claim 1 , wherein the polynucleotides are present in the solution of (b) at a concentration of about 0.001 pM to about 1 nM.
14. The method according to claim 1 , wherein the rate of interaction of the polynucleotides with the nanopore is increased as compared to the rate of interaction of the polynucleotides with the nanopore in the absence of the hydrophobic anchors.
15. The method according to claim 1 , wherein an effective concentration of the polynucleotides at the nanopore is increased as compared to the concentration of the polynucleotides in the solution of (b).
16. The method according to claim 1 , wherein a single nanopore providing an ion channel through the membrane is present in the membrane.