Membrane-spanning nanopores

Patent metadata

Field	Value
Publication number	US-11485995-B2
Application number	US-201716317085-A
Country	US
Kind code	B2
Filing date	Jul 14, 2017
Priority date	Jul 14, 2016
Publication date	Nov 1, 2022
Grant date	Nov 1, 2022

Abstract

A membrane-spanning nanopore is provided that comprises: i. at least one scaffold polynucleotide strand; ii. a plurality of staple polynucleotide strands; and iii. 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. Membranes comprising the membrane-spanning nanopore and applications of those membranes are also provided.

First claim

The invention claimed is:

1. A membrane-spanning nanopore, comprising:
i. at least one scaffold polynucleotide strand;
ii. a plurality of staple polynucleotide strands; and
iii. 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 comprising a membrane spanning region, wherein the membrane spanning region has a wall thickness of more than one DNA duplex, and wherein the membrane spanning region defines a central channel with a minimum internal width of at least about 5 nm.

2. The membrane-spanning nanopore of claim 1 , wherein the or each scaffold polynucleotide strand, each of the plurality of staple polynucleotide strands, and the or each hydrophobically-modified polynucleotide strand comprises DNA.

3. The membrane-spanning nanopore of claim 1 , wherein the nanopore is assembled via DNA origami techniques.

4. The membrane-spanning nanopore of claim 1 , wherein the minimum internal width of the central channel of the nanopore is from about 5 nm to about 20 nm.

5. The membrane-spanning nanopore of claim 1 , wherein the nanopore comprises at least one cap region,
wherein the membrane-spanning region is arranged to abut the at least one cap region, and
wherein one cap region is present and the membrane-spanning region is located at one end of the nanopore.

6. The membrane-spanning nanopore of claim 5 , wherein the membrane-spanning region has a dimension co-axial with the channel of about 1 nm to about 7 nm.

7. The membrane-spanning nanopore of claim 5 , wherein the cap region has a dimension co-axial with the channel of about 20 nm to about 70 nm.

8. The membrane-spanning nanopore of claim 1 , wherein the nanopore further comprises one or more adaptor polynucleotide strands,
wherein the at least one hydrophobically-modified polynucleotide strand is hybridised to the 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.

9. The membrane-spanning nanopore of claim 8 , wherein the polynucleotide in the adaptor polynucleotide strands comprises DNA.

10. The membrane-spanning nanopore of claim 1 , 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.

11. The membrane-spanning nanopore of claim 10 , wherein:
the sterols are selected from the group consisting of: cholesterol; derivatives of cholesterol; phytosterol; ergosterol; and bile acid;
the alkylated phenols are selected from the group consisting of: methylated phenols; and
tocopherols;
the flavones are selected from the group consisting of: flavanone containing compounds;
and 6-hydroxyflavone;
the saturated and unsaturated fatty acids are selected from the group consisting of:
derivatives of lauric acid; oleic acid; linoleic acid; and palmitic acids; and
the synthetic lipid molecule is dodecyl-beta-D-glucoside.

12. The membrane-spanning nanopore of claim 1 , wherein the nanopore comprises at least one polynucleotide sequence selected from the group consisting of:
a scaffold strand polynucleotide sequence comprising the DNA sequence of m13mp18 DNA (SEQ ID NO. 1); and
one or more staple strand polynucleotide sequences selected from the group comprising SEQ ID Nos. 2 to 218.

13. The membrane-spanning nanopore of claim 8 , wherein the nanopore comprises at least one polynucleotide sequence selected from the group consisting of:
an adaptor strand polynucleotide sequence selected from the group comprising SEQ ID Nos. 219 to 241; and
a hydrophobically-modified polynucleotide sequence selected from the group comprising SEQ ID No. 242 or 243.

14. The membrane-spanning nanopore of claim 1 , wherein the nanopore has a cross-section perpendicular to a longitudinal axis of the channel that is quadrilateral in shape.

15. The membrane-spanning nanopore of claim 14 , wherein the quadrilateral is a square.

16. The membrane-spanning nanopore of claim 1 , wherein the membrane-spanning nanopore is modified, wherein the central channel comprises one or more constrictions.

17. A biological sensor, wherein the biological sensor comprises a membrane comprising a nanopore of claim 1 and an apparatus for measuring an ion flow through one or more membrane-spanning nanopores.

Assignees

• Ucl Business Ltd

Inventors

• Howorka Stefan
• Pugh Genevieve
• Burns Jonathan Richard

Classifications

• C12Q1/6825 Primary

  Nucleic acid detection involving sensors · CPC title

• C12Q2537/162

  Helper probe · CPC title

• C12Q2565/133

  conformational analysis · CPC title

• C12Q2563/157

  Nanotubes or nanorods · CPC title

• C12Q2525/113

  incorporating modified backbone · CPC title