EP-4737389-A2 - COUPLING METHOD

Abstract

The invention relates to a new method of determining the presence, absence or characteristics of an analyte. The analyte is coupled to a membrane. The invention also relates to nucleic acid sequencing.

Inventors

• CLARKE, JAMES
• WHITE, JAMES
• MILTON, JOHN
• BROWN, Clive

Assignees

• Oxford Nanopore Technologies PLC

Dates

Publication Date

20260506

Application Date

20120525

Claims (17)

1. A method for determining the presence, absence or characteristics of an analyte, comprising: (a) coupling the analyte to a membrane and (b) allowing the analyte to interact with a nanopore present in the membrane, thereby determining the presence, absence or characteristics of the analyte, wherein the analyte is a peptide, a polypeptide or a protein, and wherein the coupling comprises functionalising the analyte for recognition by a specific binding group by binding the analyte to an adaptor which comprises a leader sequence designed to preferentially thread into the nanopore, and binding the functionalised analyte to a specific binding group coupled to the membrane.
2. A method according to claim 1, wherein the specific binding group is a DNA binding protein.
3. A method according to claim 3, wherein the DNA binding protein is coupled to the membrane using one or more linkers, e.g., a polynucleotide linker.
4. A method according to claim 1, wherein the analyte is functionalised with a peptide ligand for binding to the specific binding group.
5. A method according to any preceding claim, wherein the binding group is mixed with the analyte before contacting with the membrane.
6. A method according to any one of claims 1 to 4, wherein the binding group is contacted with the membrane and subsequence contacted with the analyte.
7. A method according to any preceding claim, wherein before it is contacted with the nanopore, the protein analyte is unfolded to form a polypeptide chain.
8. A method according to any preceding claim, wherein the membrane is an amphiphilic layer.
9. A method according to any preceding claim, wherein the membrane is a lipid bilayer.
10. A method according to any preceding claim, wherein the analyte is coupled transiently or permanently to the membrane.
11. A method according to any preceding claim, wherein the nanopore pore is a transmembrane protein nanopore.
12. A method according to claim 11, wherein the transmembrane protein nanopore comprises a β-barrel or channel or a transmembrane α-helix bundle or channel.
13. A method according to claim 11, wherein the transmembrane protein nanopore is derived from Msp or α-hemolysin (α-HL).
14. A method according any one of claims 11 to 13, wherein the transmembrane protein nanopore is an MspA nanopore embedded in a tri-block co-polymer.
15. A method according to any one of the preceding claims, wherein the method comprises: (a) allowing the analyte to interact with the nanopore; and (b) measuring the current passing through the nanopore during the interaction and thereby determining the presence, absence or characteristics of the analyte.
16. A method according to any one of the preceding claims, wherein the method is for identifying theanalyte.
17. A method of sequencing an analyte which is a target polypeptide, comprising: (a) coupling the target polypeptide to a membrane; (b) allowing the target polypeptide to interact with a nanopore in the membrane, such that the target polypeptide moves through the pore; and (c) measuring the current passing through the nanopore as the target polypeptide moves with respect to the pore and thereby determining the sequence of the target polypeptide.

Description

Field of the invention The invention relates to a new method of determining the presence, absence or characteristics of an analyte. The analyte is coupled to a membrane. The invention also relates to nucleic acid sequencing. Background of the invention There is currently a need for rapid and cheap nucleic acid (e.g. DNA or RNA) sequencing technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of nucleic acid and require a high quantity of specialist fluorescent chemicals for signal detection. Nanopores have great potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology. Two methods for DNA sequencing have been proposed; 'Exonuclease Sequencing', where bases are processively cleaved from the polynucleotide by an exonuclease and are then individually identified by the nanopore and also 'Strand Sequencing', where a single DNA strand is passed through the pore and nucleotides are directly identified. Strand Sequencing may involve the use of a DNA handling enzyme to control the movement of the polynucleotide through the nanopore. When a potential is applied across a nanopore, there is a drop in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nanopore detection of the analyte gives a current blockade of known signature and duration. The concentration of an analyte can then be determined by the number of blockade events per unit time to a single pore. For nanopore applications, such as DNA Sequencing, efficient capture of analyte from solution is required. For instance, in order to give the DNA handling enzyme used in DNA Sequencing a sufficiently high duty cycle to obtain efficient sequencing, the number of interactions between enzyme and polynucleotide needs to be maximal, so that a new polynucleotide is bound as soon as the present one is finished. Therefore, in DNA Sequencing, it is preferred to have the polynucleotide at as high a concentration as is possible so that, as soon as an enzyme finishes processing one, the next is readily available to be bound. This becomes a particular problem as the concentration of polynucleotide, such as DNA, becomes limiting, e.g. DNA from cancer cell samples for epigenetics. The more dilute the sample then the longer between sequencing runs, up to the point where binding the first polynucleotide is so limiting that it is unfeasible. The limits of nanopore detection have been estimated for various analytes. Capture of a 92-nucleotide synthetic piece of single strand DNA (ssDNA) by a protein nanopore (hemolysin) was determined to be at a frequency of 3.0±0.2 s-1 uM-1 (Maglia, Restrepo et al. 2008, Proc Natl Acad Sci U S A 105(50): 19720-5). Capture could be increased ~10 fold by the addition of a ring of positive charges at the entrance to the hemolysin barrel (23.0±2 s-1 uM-1). To put this into context, 1 uM of 92 nucleotide ssDNA is equivalent to 31 ug of DNA required per single channel recording, assuming a cis chamber volume of 1 ml. The market leading genomic DNA purification kit from human blood (Qiagen's PAXgene Blood DNA Kit) currently gives expected yields of between 150 - 500 ug of genomic from 8.5 ml of human whole blood. Therefore, this disclosed increase in analyte detection is still well short of the step change required for ultra-sensitive detection and delivery. Summary of the invention The inventors have surprisingly demonstrated ultra low concentration analyte delivery by coupling the analyte to a membrane in which the relevant detector is present. This lowers by several orders of magnitude the amount of analyte required in order to be detected. The extent to which the amount of analyte needed is reduced could not have been predicted. In particular, the inventors surprisingly report an increase in the capture of single stranded DNA by ~4 orders of magnitude over that previously reported. As both the detector and analyte are now on the same plane, then ~103 M s-1 more interactions occur per second, as diffusion of both molecules is in two dimensions rather than three dimensions. This has dramatic implications on the sample preparation requirements that are of key concern for diagnostic devices such as next-generation sequencing systems. In addition, coupling the analyte to a membrane has added advantages for various nanopore-enzyme sequencing applications. In Exonuclease Sequencing, when the DNA analyte is introduced the pore may become blocked permanently or temporarily, preventing the detection of individual nucleotides. When one end of the DNA analyte is localised away from the pore, for example by coupling or tethering to the membrane, surprisingly it was found that this temporary or permanent blocking is no longer observed. By occupying one e