METHOD USING FLUORINATED AMPHIPHILES
The invention relates to a method of inhibiting the insertion of one or more membrane proteins into a lipid bilayer. The invention also relates to a method of inserting a pre-determined number of membrane proteins into a lipid bilayer and lipid bilayers having a pre-determined number of membrane proteins inserted therein. The lipid bilayers of the invention are useful as sensor arrays, particularly for sequencing nucleic acids.
1 . A method for inhibiting the insertion of one or more membrane proteins into a membrane, comprising (a) contacting the proteins and membrane with a fluorinated amphiphile (F-amphiphile) under conditions that in the absence of the F-amphiphile allow the insertion of the proteins into the membrane and (b) thereby inhibiting the insertion of the proteins into the membrane.
2 . A method according to claim 1 , wherein the one or more membrane proteins are not derived from the mechanosensitive channel of large conductance, MscL, of Escherichia coli.
3 . A method according to claim 1 , wherein the one or more membrane proteins are derived from a-hemolysin (a-HL), derived from MspA from Mycobacterium smegmatis or derived from Kcv of chlorella virus PBCV-1.
4 . A method according to claim 3 , wherein the one or more membrane proteins each comprise (a) the sequence shown SEQ ID NO: 2 or a variant thereof, (b) the sequence shown in SEQ ID NO: 14 or a variant thereof or (c) the sequence shown in SEQ ID NO: 16 or a variant thereof.
5 - 6 . (canceled)
7 . A method according to claim 1 , wherein the F-amphiphile (a) comprises (i) a polar head group and (ii) a hydrophobic tail comprising a fluorinated chain or (b) is F-fos-choline (F 6 FC) with a zwitterionic head group, F-octyl maltoside (F 6 OM) with a non-ionic disaccharide head group and C 6 F 13 C 2 H 4 —S-poly[tris(hydroxymethyl)aminomethane] (F 6 TAC) with a non-ionic polymeric head group.
8 . (canceled)
9 . A method according to claim 1 , wherein the F-amphiphile is contacted with the proteins and membrane on the cis side.
10 . A method according to claim 1 , wherein the F-amphiphile is contacted with the proteins and membrane at a concentration greater than the critical micelle concentration (CMC) or at a concentration at least five times greater than the CMC.
11 . (canceled)
12 . A method according to claim 1 , wherein the membrane is a lipid membrane and the lipid-to-protein ratio is lower than 40:1 (w/w) or is 1:1 (w/w).
13 . (canceled)
14 . Use of a F-amphiphile for inhibiting the insertion of one or more membrane proteins into a membrane.
15 . A method for inserting a pre-determined number of membrane proteins into a membrane, comprising (a) contacting more than the pre-determined number of the proteins with the membrane under conditions that allow the insertion of the proteins into the membrane and (b) once the pre-determined number of membrane proteins have inserted in the membrane, contacting the proteins and membrane with a F-amphiphile and thereby inhibiting further insertion of the proteins into the membrane.
16 . A method according to claim 15 , wherein the method is for inserting a single pore into the membrane.
17 . A method according to claim 15 , wherein the membrane proteins are derived from a-hemolysin (a-HL), derived from MspA from Mycobacterium smegmatis or derived from Kcv of chlorella virus PBCV-1.
18 . A membrane having a predetermined number of membrane proteins inserted therein produced using a method according to claim 15 .
19 . A method of determining the presence or absence of an analyte, comprising:
(a) contacting the analyte with a membrane according to claim 18 , which comprises a transmembrane pore or an ion channel, so that the analyte interacts with the pore or channel; and
(b) measuring the current passing through the pore or channel during the interaction and thereby determining the presence or absence of the analyte.
20 . A method according to claim 19 , wherein the analyte is an individual nucleotide or is a nucleic acid sequence.
21 - 22 . (canceled)
23 . A method of estimating the sequence of a target nucleic acid sequence, comprising:
(a) contacting the target sequence with a membrane according to claim 18 , which comprises at least one transmembrane pore so that the target sequence translocates through the pore and a proportion of the nucleotides in the target sequence interacts with the pore; and
(b) measuring the current passing through the pore during each interaction and thereby determining the sequence of the target sequence.
24 . A method according to claim 23 , wherein the transmembrane pore has a molecular adaptor attached thereto.
25 . A method according to claim 23 , wherein the transmembrane pore has a nucleic handling enzyme covalently attached thereto and the enzyme controls the translocation of the target sequence through the pore.
26 . A kit for inserting a pre-determined number of membrane proteins into a membrane comprising (a) one or more membrane proteins and (b) a fluorinated amphiphile, wherein the membrane proteins are derived from α-hemolysin (α-HL), MspA from Mycobacterium smegmatis or Kcv of chlorella virus PBCV-1.