METHODS AND COMPOSITIONS FOR ASSEMBLY OF BIOLOGICAL NANOPORES
Methods and compositions for the manufacture and use of a detection apparatus based on one or more native biological nanopores are provided. Uses include, but are not limited to, detection and sequencing of nucleic acids.
1 . A method of making a detection apparatus comprising one or more native nanopore proteins, comprising the steps of:
(a) forming an aqueous mixture comprising a nanopore protein, a membrane scaffold protein (MSP), and a first lipid to produce a sample of nanodisc-nanopore protein complexes, wherein a population of the nanodisc-nanopore protein complexes in the sample each comprise a native nanopore protein;
(b) providing a solid support comprising one or more apertures, wherein a membrane is formed over each of the apertures, wherein the membrane comprises a second lipid, and wherein the membrane separates a cis chamber from a trans chamber in the detection apparatus; and
(c) contacting the one or more membranes with the population of nanopore-nanodisc complexes comprising the native nanopore protein to assimilate a native nanopore protein into each of the membranes.
2 . The method of claim 1 , further comprising the step of purifying the population of nanopore-nanodisc complexes comprising the native nanopore protein from the aqueous mixture prior to the step of contacting the one or more membranes with the population of nanopore-nanodisc complexes comprising the native nanopore protein.
3 . The method of claim 2 , wherein the step of purifying the population of nanopore-nanodisc complexes comprising the native nanopore protein comprises one or both of size-exclusion chromatography and affinity chromatography.
4 . The method of claim 1 , wherein the aqueous mixture further comprises a detergent, wherein the final concentration of the detergent is from about 14 mM to about 40 mM.
5 . The method of claim 4 , wherein the first lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), the MSP is MSP1D1, or a variant thereof, the nanopore protein is α-hemolysin (α-HL) or a variant thereof, the detergent is cholate, and the second lipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE).
6 . The method of claim 5 , wherein the molar ratio of lipid to MSP to nanopore protein is about 101:6:1 or about 120:6:1.
7 . The method of claim 1 , wherein the solid support comprises a plurality of apertures, wherein a membrane is formed over each of the plurality of apertures, and wherein each of the membranes is contacted with the nanopore-nanodisc complex comprising the native nanopore protein.
8 . A method of sequencing a polymer comprising use of the detection system of claim 1 .
9 . The method of claim 8 , wherein the polymer is an Xpandomer.
10 . A method of forming a native nanopore protein in a membrane comprising the steps of:
(a) forming an aqueous mixture comprising a nanopore protein, a membrane scaffold protein (MSP), and a first lipid to produce a sample of nanodisc-nanopore protein complexes, wherein a population of the nanodisc-nanopore protein complexes each comprise a native nanopore protein;
(b) providing a membrane comprising a second lipid; and
(c) contacting the membrane with the population of nanopore-nanodisc complexes comprising the native nanopore protein to assimilate a native nanopore protein the membranes.
11 . The method of claim 10 , further comprising the step of purifying the population of nanopore-nanodisc complexes comprising the native nanopore protein from the aqueous mixture prior to the step of contacting the membrane with the population of nanopore-nanodisc complexes comprising the native nanopore protein.
12 . The method of claim 11 , wherein the step of purifying the population of nanopore-nanodisc complexes comprises one or both of size-exclusion chromatography and immobilized metal affinity chromatography.
13 . The method of claim 11 , wherein the aqueous mixture further comprises a detergent, wherein the final concentration of the detergent is from over 14 mM to 40 mM.
14 . The method of claim 13 , wherein the first lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), the MSP is MSP1D1, or a variant thereof, the nanopore protein is α-hemolysin (α-HL) or a variant thereof, the detergent is cholate, and the second lipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE).
15 . The method of claim 14 , wherein the molar ratio of lipid to MSP to nanopore protein is about 101:6:1 or about 120:6:1.