Membrane-spanning nanopores

Abstract

A membrane-spanning nanopore is provided that comprises:i. at least one scaffold polynucleotide strand;ii. a plurality of staple polynucleotide strands; andiii. at least one hydrophobically-modified polynucleotide strand, wherein the at least one hydrophobically-modified polynucleotide strand comprises a polynucleotide strand and a hydrophobic moiety; wherein each of the plurality of staple polynucleotide strands hybridises to the at least one scaffold polynucleotide strand to form the three-dimensional structure of the membrane-spanning nanopore, and wherein the at least one hydrophobically-modified polynucleotide strand hybridises to a portion of the at least one scaffold polynucleotide strand, the membrane-spanning nanopore defining a central channel with a minimum internal width of at least about 5 nm.

1 .- 46 . (canceled) 
     
     
         47 . A method for molecular sensing comprising:
 i) providing a semi-fluid or lipid membrane having a first side and a second side;   ii) providing a nucleic acid membrane-spanning nanopore located in the membrane, wherein the nucleic acid membrane-spanning nanopore defines a central channel that allows for fluid communication between the first side of the membrane and the second side of the membrane, wherein the minimum internal width of the central channel of the nanopore is from about 5 nm to about 20 nm; and   iii) measuring a change in electrical properties of the nucleic acid membrane-spanning nanopore in the presence of an analyte, wherein detection of a change in electrical properties is indicative of the presence of the analyte.   
     
     
         48 . The method of  claim 47 , wherein the semi-fluid or lipid membrane is selected from the group consisting of: a membrane comprising a lipid bilayer; and a membrane comprising a semi-fluid membrane formed of polymers. 
     
     
         49 . The method of  claim 48 , wherein the polymer forming the semi-fluid membrane is composed of amphiphilic synthetic block copolymers, suitably selected from hydrophilic copolymer blocks and hydrophobic copolymer blocks. 
     
     
         50 . The method of  claim 47 , wherein the change in electrical properties is a change in the flow of ions through the nucleic acid membrane-spanning nanopore. 
     
     
         51 . The method of  claim 50 , wherein the change in the flow of ions is from the first side of the membrane to the second side of the membrane. 
     
     
         52 . The method of  claim 47 , wherein the change in electrical properties is a change in electron flow across the nucleic acid membrane-spanning nanopore. 
     
     
         53 . The method of  claim 52 , wherein the change in electrical properties is measured across an aperture of the nucleic acid membrane-spanning nanopore, and the electrical property is selected from one or more of the group consisting of: tunnelling current; local potential; and capacitance. 
     
     
         54 . The method of  claim 53 , wherein the change in electron flow is measured by a field effect transistor (FET) device or a FET nanopore device. 
     
     
         55 . The method of  claim 47 , wherein the analyte is selected from one or more of the group consisting of: folded or unfolded proteins; DNA-protein constructs such as nucleosomes and polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); polysaccharides; and synthetic polymers. 
     
     
         56 . The method of  claim 47 , wherein the analyte comprises a nucleic acid. 
     
     
         57 . The method of  claim 56 , wherein the nucleic acid is selected from one or more of the group consisting of: DNA; RNA; a synthetic nucleic acid; and a modified nucleic acid. 
     
     
         58 . The method of  claim 57 , wherein the nucleic acid is comprised of one or more nucleotides and the sequence of the one or more nucleotides may be determined from characteristic disruptions in the measured electrical properties over time. 
     
     
         59 . The method of  claim 47 , wherein the nucleic acid membrane-spanning nanopore comprises:
 a) at least one scaffold polynucleotide strand;   b) a plurality of staple polynucleotide strands; and   c) at least one hydrophobically-modified polynucleotide strand, wherein the at least one hydrophobically-modified polynucleotide strand comprises a polynucleotide strand and a hydrophobic moiety;   
       wherein each of the plurality of staple polynucleotide strands hybridises to the at least one scaffold polynucleotide strand to form the three-dimensional structure of the nucleic acid membrane-spanning nanopore; and 
       wherein the at least one hydrophobically-modified polynucleotide strand hybridises to a portion of the at least one scaffold polynucleotide strand. 
     
     
         60 . The method of  claim 47 , wherein the nucleic acid membrane-spanning nanopore comprises a membrane spanning region, wherein the membrane spanning region has a wall thickness of more than one DNA duplex. 
     
     
         61 . The method of  claim 59 , wherein the polynucleotide strand of the or each at least one scaffold strand, each of the plurality of staple polynucleotide strands, and the or each hydrophobically-modified polynucleotide strand comprises DNA. 
     
     
         62 . The method of  claim 59 , wherein the assembly of the nucleic acid membrane-spanning nanopore is via DNA origami techniques. 
     
     
         63 . The method of  claim 59 , wherein the nucleic acid membrane-spanning nanopore further comprises one or more adaptor polynucleotide strands,
 wherein the at least one hydrophobically-modified polynucleotide strand is hybridised to the nucleic acid membrane-spanning nanopore via the one or more adaptor polynucleotide strands, the one of more adaptor polynucleotide strands each having a first end and a second end,   wherein the first end of the adaptor polynucleotide strand hybridises with the at least one scaffold polynucleotide strand, and the second end of the adaptor polynucleotide strand hybridises with the at least one hydrophobically-modified polynucleotide strand.   
     
     
         64 . The method of  claim 63 , wherein the polynucleotide in the adaptor polynucleotide strands comprises DNA. 
     
     
         65 . The method of  claim 59 , wherein the at least one hydrophobic moiety comprises a lipid, wherein the lipid is selected from the group consisting of: sterols; alkylated phenols; flavones; saturated and unsaturated fatty acids; and synthetic lipid molecules (including dodecyl-beta-D-glucoside). 
     
     
         66 . The method of  claim 47 , wherein the semi-fluid or lipid membrane is provided with a plurality of nucleic acid membrane-spanning nanopores.