Method of target molecule characterisation using a molecular pore
The invention relates to a new method of determining the presence, absence or one or more characteristics of multiple analytes. The invention concerns coupling a first analyte to a membrane containing a detector and investigating the first analyte using the detector. The invention also concerns coupling a second analyte to the membrane and investigating the second analyte. The first analyte is uncoupled from the membrane prior to investigating the second analyte. The invention also relates to polynucleotide sequencing.
1. A method for determining the presence, absence or one or more characteristics of two or more analytes in two or more samples, comprising:
(a) coupling a first analyte in a first sample to a membrane using one or more anchors;
(b) allowing the first analyte to interact with a nanopore present in the membrane and thereby determining the presence, absence or one or more characteristics of the first analyte;
(c) uncoupling the first analyte of the first sample from the membrane;
(d) after step (c), coupling a second analyte in a second sample to the membrane using one or more anchors, under conditions in which the first analyte of the first sample does not recouple to the membrane; and
(e) allowing the second analyte of the second sample to interact with a nanopore in the membrane and thereby determining the presence, absence or one or more characteristics of the second analyte.
2. A method according to claim 1 , wherein the one or more anchors comprise a polypeptide anchor and/or a hydrophobic anchor.
3. A method according to claim 2 , wherein the hydrophobic anchor comprises a lipid, fatty acid, sterol, carbon nanotube or amino acid.
4. A method according to claim 1 , wherein step (c) comprises uncoupling the first analyte of the first sample from the membrane by removing the one or more anchors from the membrane.
5. A method according to claim 4 , wherein step (c) comprises contacting the one or more anchors with an agent which has a higher affinity for the one or more anchors than the anchors have for the membrane.
6. A method according to claim 5 , wherein (i) the one or more anchors comprises cholesterol and the agent is a cyclodextrin or a derivative thereof or a lipid; (ii) the one or more anchors comprises streptavidin, biotin or desthiobiotin and the agent is biotin, desthiobiotin or streptavidin; or (iii) the one or more anchors comprises a protein and the agent is an antibody or fragment thereof which specifically binds to the protein.
7. A method according to claim 1 , wherein step (c) comprises contacting the one or more anchors with an agent which reduces their ability to couple to the membrane.
8. A method according to claim 7 , wherein (i) the one or more anchors comprises cholesterol and the agent is cholesterol dehydrogenase; (ii) the one or more anchors comprises a lipid and the agent is a phospholipase; or (iii) the one or more anchors comprises a protein and the agent is a proteinase or urea.
9. A method according to claim 1 , wherein step (c) comprises uncoupling the first analyte of the first sample from the membrane by separating the first analyte from the one or more anchors.
10. A method according to claim 1 , wherein step (c) comprises uncoupling the first analyte of the first sample from the membrane by contacting the first analyte and the one or more anchors with an agent which competes with the first analyte for binding to the one or more anchors.
11. A method according to claim 10 , wherein the agent is a polynucleotide which competes with the first analyte of the first sample for hybridisation to the one or more anchors.
12. A method according to claim 9 , wherein step (c) comprises (i) contacting the first analyte of the first sample and the one or more anchors with urea, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), streptavidin or biotin, UV light, an enzyme or a binding agent; (ii) heating the first analyte and one or more anchors; or (iii) altering the pH.
13. A method according to claim 9 , wherein step (d) comprises coupling the second analyte of the second sample to the membrane using the one or more anchors that were separated from the first analyte of the first sample.
14. A method according to claim 1 , wherein between steps (c) and (d) the method comprises removing at least some of the first sample from the membrane.
15. A method according to claim 1 , wherein the first analyte of the first sample and the second analyte of the second sample are polynucleotides and wherein the method is for identifying or estimating the sequence of the first polynucleotide of the first sample and/or the second polynucleotide of the second sample.