Nanodiscs as a delivery vehicle for insertion of nanopores into sensor membranes

Abstract

Compositions comprised of nanodiscs and methods for their manufacture are provided. The methods comprise contacting lipid or synthetic lipid analogues with one or more nanopores in aqueous solution, thereby forming a membrane vesicle that encompasses the one or more nanopores embedded within the membrane vesicle; contacting the one or more membrane vesicles with a nanodisc forming polymer; and generating a nanodisc composition wherein the nanodisc forming polymers solubilise the membrane vesicle and encircle the one or more nanopores embedded within the membrane vesicle so as to form a plurality of nanodiscs. The nanodiscs may be used to facilitate incorporation of protein or nucleic acid nanopores into flow cell or membrane array nanopore sensor devices.

Description

[0001] NANODISCS AS A DELIVERY VEHICLE FOR INSERTION OF NANOPORES INTO SENSOR MEMBRANES

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to novel nanodisc compositions and methods of making the compositions. In particular, it relates to methods and uses of nanodiscs as delivery vehicles for insertion of one or more membrane-spanning nanopores into a target semi-fluid or solid state membrane.

[0004] BACKGROUND OF THE INVENTION

[0005] Nanopores are nanoscale structures that mediate molecular transport across both solid state and semifluid membranes. In biological systems, nanopores are embedded into the membrane and function as selective gatekeepers, allowing the passage of ions, nutrients, and genetic material to support cellular processes.

[0006] Nanopores have been used in sensors for nucleic acid sequencing (Quick, J. et al, 2016), wherein an atomically engineered inner lumen of the nanopore helps discern the DNA sequences from individual translocating strands. The nanopores used in nucleic acid sequencing can be comprised of proteins, i.e. protein or polypeptide nanopores, or can be rationally designed from nucleic acids to generate required dimensions and sensitivity using DNA origami (Lanphere et al., 2020). Nanopore based sensing and sequencing has further enabled rapid and accurate identification of nucleic acids and proteins (Howorka, 2017). Beyond nucleic acid sequencing, nanopore technology has been applied to protein analysis, allowing the detection of protein translocation events alongside real-time kinetic analysis of thousands of translocation processes simultaneously (Diederichs et al., 2019). Further, analysis at the post-translation modifications level within single polypeptides has been performed (Martin-Baniandres et al., 2023).

[0007] Nucleic acid nanopore design can be precisely tailored for specific functions (Burns et al., 2013), enabling their use in a wide range of applications. Some examples of the applications include model systems for studying the physics of fluid transport (Schoch et al., 2008) and smart biosensing devices that measure intracellular pH levels (Bhatia et al., 2011). Furthermore, nucleic acid nanopores have been incorporated into DNA nanocages for the targeted delivery of bioactive cargo and used as DNA scaffolds to accurately position proteins and other biomolecular components for research purposes in biophysics and molecular biology (Walsh et al., 2011).

[0008] Further, nanopores have been used in therapeutic applications. For instance, the design of customizable lids for nanopores can equip a nanopore to regulate the release of therapeutic proteins from drug delivery vesicles (Lanphere et al., 2020). Moreover, these resealable nanopores can be engineered to facilitate membrane translocation or rupture in response to specific stimuli, such as light or the acidic microenvironment characteristic of tumour cells (Offenbartl-Stiegert et al., 2022). The targeting efficiency of nanopores can be further enhanced by their ability to selectively interact with cells expressing specific markers, such as cholesterol lipid anchors found on immune cell membranes (Arulkumaran et al., 2021). Additionally, custom-designed DNA nanopores can be engineered to achieve enhanced structural stability, thereby providing resistance to enzymatic degradation (Burns et al., 2018).

[0009] However, despite the considerable promise, the practical application of nanopores is limited by the considerable difficulty of insertion into the robust target membranes. Some nanopores, specifically DNA nanopores, are net negatively charged, hindering efficient insertion into hydrophobic, lipid target membranes. Similarly, insertion of nanopores into solid state target membrane apertures may be hindered due to differences in hydrophobicity and membrane structure, posing a significant energy barrier for insertion. This challenge is further complicated by inserting nanopores into high-throughput flow cells, or membrane arrays containing thousands of membranes comprised of synthetic or artificial lipid analogues. Low insertion frequency of nanopores into a membrane array compromises the throughput level of the sensor, and prevents high accuracy, real-time readout that can be obtained from parallel sample processing.

[0010] There have been efforts to facilitate insertion of membrane proteins into a target bilayer membrane, such as application of an electrical potential across the target membrane (Vreeker et al., 2024). It is thought that this potential difference stretches and thins the membrane, allowing easier insertion of membrane proteins, channels and nanopores. Voltage assisted insertion combined with simultaneous detection of current flow through the membrane may also be used to control the number of membrane channels inserted into a membrane. There have been further methods devised to automatically control the membrane potential applicable to a large membrane array (US12042790B2).

[0011] Recent studies have shown that ion channel proteins reconstituted in nanodiscs can be subsequently inserted into planar bilayer membranes. Nanodiscs that are used to accommodate protein ion channels comprise of a central core of lipids, combined with a few scaffold proteins. Nanodiscs with ion channel proteins, once directly added to bilayers, results in spontaneous insertion of the channel proteins the final target membrane (Winterstein et al., 2018).

[0012] However, current conventional approaches are limited to membrane proteins inserted into lipid bilayers (Vreeker et al, 2024). Therefore, there is a need to further develop methods that can efficiently insert various types of nanopores, including highly negatively charged nucleic acid nanopores, into diverse range of target membranes, such as triblock copolymer, artificial lipid analogue or artificial amphiphilic layer compatible with high-throughput flow cells. The present invention addresses the deficiencies in the art and provides an improved solution of enhancing the insertion frequency of both protein and nucleic acid nanopores into target membranes compatible with high-throughput flow cells. These and other uses, features and advantages of the invention should be apparent to those skilled in the art from the teachings provided herein.

[0013] WO 2017 / 044899 B2 describes that synthetic apolipoproteins based on native / naturally occurring homolog proteins can be prepared using solid-phase peptide synthesis approaches combined with native chemical ligation methods to create analogs of full length apolipoproteins for nanolipoprotein particle formation.

[0014] WO 2016 / 019030 A relates to a detection apparatus comprising a solid support with an array of solid-state nanopores and a plurality of lipid nanodiscs with protein nanopores, wherein the lipid nanodiscs are disposed on the solid support such that they form seals at the solid- state nanopores. Further described is a detection apparatus comprising one or more nanopores that are surrounded by a membrane, wherein each nanopore is tethered to an electrode.

[0015] WO 2024 / 200192 A1 describes lipid binding molecules and / or combinations of the lipid binding protein with a lipid component (i.e. , a mispid) that are used to modify the interaction of a target molecule with a lipid membrane. This includes use of the lipid binding molecules and / or mispids, for example, to improve sequencing efficiency and throughput of nanoporebased sequencing systems.

[0016] GB 2608837 A describes a zwitterionic copolymer comprising optionally at least partially substituted styrene monomer and maleic anhydride derivative monomer wherein the maleic anhydride derivative monomer includes a zwitterionic carboxybetaine moiety. Also described are a lipid nanodisc comprising a lipid bilayer encircled by the zwitterionic copolymer and a method of synthesising the zwitterionic copolymer; and a method of solubilising lipid bilayers to form lipid nanodiscs and isolate membrane proteins comprising use of the zwitterionic copolymer.

[0017] US 2021 / 0171673 A1 describes compositions, methods, and methods of making and using a polymer-encased nanodisc comprising: one or more integral membrane proteins in a lipid layer; and a polymer comprising zwitterionic styrene-maleic acid derivative repeating units that carry zero or nearly zero negative charge, and the polymer-encased nanodiscs.

[0018] WO 2024 / 085693 A1 describes a nanodisc comprising a membrane scaffold protein or an amphipathic polymer, and phosphatidylethanolamine, which is a phospholipid, and to an antiviral use thereof.

[0019] SUMMARY OF THE INVENTION

[0020] In a first aspect, the invention provides a method of making a nanodisc composition, comprising contacting a composition comprised of lipid or synthetic lipid analogues with one or more nanopores in aqueous solution, thereby forming a membrane vesicle that encompasses the one or more nanopores embedded within the membrane vesicle, and then contacting the one or more membrane vesicles with a nanodisc forming polymer, and thereby generating a nanodisc composition wherein the nanodisc forming polymer solubilises the membrane vesicle and encircles the one or more nanopores embedded within the membrane vesicle so as to form a plurality of nanodiscs that comprise one or more nanopores embedded within.

[0021] A second aspect of the invention provides a nanodisc composition comprising a semi-fluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the nanodisc composition further comprises one or more nanopores embedded within the semifluid lipid membrane.

[0022] A third aspect of the invention provides a nanodisc comprising a semi-fluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the one or more NFPs comprise an amphipathic polymer and / or copolymer; wherein the semi-fluid lipid membrane comprises first and second opposing hydrophilic faces and a hydrophobic edge defined between the first and second hydrophilic faces, and the NFPs encircle the hydrophobic edge of the lipid membrane; and wherein the nanodisc further comprises one or more nucleic acid nanopores embedded within the semi-fluid lipid membrane.

[0023] A fourth aspect of the invention provides a use of a nanodisc as defined herein, as a delivery vehicle for insertion of one or more nanopores into a target membrane.

[0024] A fifth aspect of the invention provides a method of increasing frequency of insertion of a plurality of nanopores into a target membrane array, comprising using a nanodisc composition as defined herein as a delivery vehicle to increase the said frequency.

[0025] A sixth aspect of the invention provides a method for regenerating a high-throughput flow cell or membrane array configured with a plurality of nanopore sensors, the method comprising contacting a high-throughput flow cell or membrane array with an aqueous repair solution comprising a composition of repair nanodiscs, wherein the repair nanodiscs comprise a semifluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the one or more NFPs comprise an amphipathic polymer and / or copolymer, and wherein the semi-fluid lipid membrane comprises first and second opposing hydrophilic faces and a hydrophobic edge defined between the first and second hydrophilic faces, and the NFPs encircle the hydrophobic edge of the lipid membrane. Optionally, the composition of repair nanodiscs may comprise compositions of the second or third aspects.

[0026] A seventh aspect of the invention provides a method of preparing a repair formulation for regenerating a high-throughput flow cell or a membrane array comprising a plurality of nanopore sensors, the method comprising, contacting lipid or synthetic lipid analogues with an aqueous solution thereby forming one or more membrane vesicles; contacting the one or more membrane vesicles with a nanodisc forming polymer; and generating a repair nanodisc composition wherein the nanodisc forming polymers solubilise the membrane vesicle and encircle the lipid or synthetic lipid analogues comprising the membrane vesicle so as to form a plurality of nanodiscs in suspension.

[0027] Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible. DESCRIPTION OF THE DRAWINGS

[0028] One or more embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings.

[0029] Figure 1 shows a schematic of Nanodisc Mediated Membrane Insertion of Nanopores (A) Schematic representation and dimensions of archetypical DNA nanopore. (B) Addition of lipid to nanopore in aqueous solution results in formation of membrane vesicle around the nanopore, circumventing energetically expensive membrane remodelling that occurs when trying to insert nanopores into pre-formed membranes. (C) Addition of Nanodisc Forming Polymer (NFP) such as styrene-maleic acid or diisobutylene-maleic acid results in solubilisation of membrane vesicle into polymer encircled membrane nanodiscs, with transmembrane nanopores. (D) Purification of the nanodiscs acts to remove excess NFP which would otherwise solubilise membranes in downstream applications. (E) The nanodisc then acts to as delivery vehicle to insert the nanopore into pre-formed membranes, such as the sensing array in the minlON, a portable nanopore analysis device. (F) Inset showing individual transmembrane nanopore in a minlON array. (G) Addition of an analyte, such as a bio-polymer like DNA or a peptide, can then translocate through the nanopore while under voltage, producing unique current blockages for analyte identification. (H) Schematic demonstration that nanopores of varying dimensions can be assembled into nanodiscs to enable high frequency membrane insertion.

[0030] Figure 2 shows Assembly, Purification and Characterisation of Nanodiscs

[0031] (A) Schematic representation and (B) Dynamic Light Scattering (DLS) analysis formation of lipid nanodisc from POPC vesicles by addition of increasing amount of nanodisc forming polymer (NFP), in this instance it is the NFP diisobutylene maleic acid (DIBMA). The DLS traces show a POPC vesicle, a 0.5:1 molar ratio of DIBMA to POPC, and a 2:1 ratio. (C) Size Exclusion Chromatograph showing purification of 2:1 DIBMA: POPC vesicle, generated with 0.1 mol % PE-Cy5. Right-hand axis shows fluorescence measurement of fractions, demonstrating co-localisation of lipid and polymer. (D) DLS analysis of fraction eluting at 6 mL, demonstrating removal of excess polymer. (E) Confocal fluorescent microscopy showing binding of purified fluorescence nanodiscs to giant uni-lamellar POPC vesicles. An external fluorescence dye has been added to demonstrate the maintained integrity of the vesicles. Scale bar is 50 pm. (F) Fluorescent Spectrogram showing release of encapsulated fluorescence dye (sulfurhodamine B) from DPhPC LUVs upon addition of osmotically balanced buffer (grey trace) or purified nanodiscs (pink trace). Each trace is an average of three technical repeats and the coloured ribbon shows the standard deviation. (G) DLS analysis of polymer vesicle (polymersome) composed of a PEO-b-PBO block copolymer. (H) Addition of 0.5:1 molar ratio of DIBMAto PEO-b-PBO polymersome, demonstrating formation of lipid-free nanodiscs.

[0032] Figure 3 shows Assembly and Characterisation of DNA Nanopores

[0033] (A) Schematic diagram and dimensions of Six Helical Bundle (6HB) DNA Nanopore. (B) 10% TBE-PAGE analysis of 6HB DNA nanopore generated with 0,1 or 3 cholesterol moieties (C) Schematic representation and dimensions of Ring style DNA Nanopore. (D) 10% PAGE gel showing assembly of Ring style DNA nanopore. (E) Size Exclusion Chromatogram showing analytical purification of 6HB-0C. (F) DLS analysis showing volume and intensity distributions of 6HB-0C. (G) Schematic diagram describing the dye efflux experiment. Sulfarhoamine B (inset) is encapsulated within a vesicle (depicted as a grey membrane) at a self-quenching concentration. Addition of a nanopore (pictured, blue) allows the release of the dye into the bulk solution, lowering the effective concentration and causing the dye to fluoresce. (H) Fluorescent spectrogram showing release of encapsulated fluorescence dye from DPhPC LUVs upon addition of osmotically balanced buffer (grey trace), 200 nM of 6HB-3C (blue) or 100 nM of protein nanopore alpha-heamolysin (red, purchased from Sigma). Each trace is an average of three technical repeats and the coloured ribbon shows the standard error.

[0034] Figure 4 shows Generation and Characterisation of Lipid Nanodiscs with Transmembrane DNA Nanopores

[0035] (A) Representative chromatograms showing the SEC purification of the 6HB nanopore inside nanodiscs formed of a 2:1 ratio of the nanodisc forming polymer Styrene-Malic acid (SMA) and POPC. (B) DLS traces showing the purified products from the major peaks (retention time = 8.52 minutes) from (A). (C) UV spectra of the purified products from (A). In all plots POPC:SMA(2:1)-6HB is depicted in a solid lilac line. For comparison, the data received when purifying and characterising POPC:SMA(2:1) in the absence of 6HB is plotted below in a dashed purple line. (D) Normalised fluorescent spectrograms showing the release of encapsulated dye (SRB) from DPhPC lipid vesicles upon addition of 6HB (200 nM, blue), SEC purified POPC:SMA(2:1) (5 pM wrt polymer concentration, purple), SEC purified POPC:SMA(2:1)-6HB (5 pM wrt polymer concentration, 77 nM wrt DNA concentration, lilac, or equivalent volumes of buffer (grey). (E) Normalised fluorescence spectrograms showing the equilibration of pH across the membrane of HPTS filled block copolymer vesicles upon addition of 6HB, SEC purified POPC:SMA(2:1), SEC purified POPC:SMA(2:1)-6HB or equivalent volumes of buffer (grey). All fluorescent traces are an average of three technical repeats and the coloured ribbon shows the standard deviation. (F) Representative chromatograms showing the SEC purification of DIBMA:POPC(1 :1)-Ring (dark cyan). For comparison, the chromatograms received when purifying the Ring in solution in the absence of complementary cholesterol modified oligonucleotides (solid grey line) were also included. (G) DLS traces showing the purified products from the major peak (retention time = 8.14 minutes) from (F). (H) UV spectra of the purified products from (F). (I) Normalised fluorescent spectrograms showing the release of encapsulated dye (SRB) from DPhPC lipid vesicles upon addition of Ring (8 nM, dark cyan), SEC purified DIBMA:POPC(1 :1) (5 pM wrt polymer concentration, gold, denoted “Disc”), SEC purified DIBMA:POPC(1 :1)-Ring (5 pM wrt polymer concentration, 8.02 nM wrt DNA concentration, green, denoted “Ring-Disc”) or equivalent volumes of buffer (grey). (J) Normalised fluorescence spectrograms showing the equilibration of pH across the membrane of HPTS filled block copolymer vesicles upon addition of Ring, SEC purified DIBMA:POPC(1 :1), SEC purified DIBMA:POPC(1 : 1 )-Ring or equivalent volumes of buffer. As with previous, each trace is an average of three technical repeats and the coloured ribbon shows the standard deviation.

[0036] Figure 5 shows use of Nanodiscs to insert DNA Nanopores into a DNA sequencer array.

[0037] (A) Schematic representation of nanodisc mediated insertion of the 6HB nanopore into a membrane array (the minlON from Oxford Nanopore Technologies (ONT)). (B) Time- averaged current- voltage curve from nanodisc inserted 6HB nanopore in a minlON. The trace is an average of 10 traces and the coloured ribbon shows the standard deviation. (C) Current- Voltage (IV) analysis showing average current received through the 6HB nanopore when increasing voltage in 5 mV increments from -50 to 50 mV. Schematic representation of nanodisc mediated insertion of the 6HB nanopore into the membrane array in the minlON from Oxford Nanopore Technologies (ONT). Histogram of conductance at -25 mV. The data plotted in grey represents multiple 6HB’s inserted in a single membrane and was omitted from (C). (E) Comparison of insertion frequency when adding the 6HB directly to the minlON, or when added using nanodiscs. (F) Schematic representation of nanodisc mediated insertion of the Ring nanopore into the membrane array in the minlON ONT. (G) Representative current trace from the small-state of the Ring nanopore in a minlON membrane. (H) Representative current trace for the large-state of the Ring nanopore in a minlON membrane. (I) IV analysis showing average current received through the large and small states of the Ring nanopore when increasing voltage in 5 mV increments from -50 to 50 mV. (J) Histogram of conductance at -25 mV. (K) Comparison of insertion frequency when adding the Ring nanopore directly to the minlON, or when added using nanodiscs.

[0038] Figure 6 shows Analyte Translocation Through DNA Nanopores Inserted into Semi- Fluid Membranes Using Nanodiscs

[0039] (A) Schematic diagram and (B) representative current trace of nanodisc inserted 6HB nanopore in minlON membrane, recorded at 75 mV, (C) histogram showing the frequency of translocation events, and (D) analysis of the duration, or dwell time, of the translocation events recorded in the absence (top row) and presence (bottom row) of 500 nM of the Thrombin Binding Aptamer DNA oligonucleotide. (E) Schematic Diagram and (F) Representative current trace of nanodisc inserted Ring nanopore in minlON membrane, (G) histogram showing the frequency of translocation events, and (H) analysis of the dwell time of translocation events recorded in the absence (top row), presence of 2.6 pM (middle row) and presence of 14.8 pM of the protease Trypsin. Representative current traces have been filtered with a low pass Bessel filter at 1 kHz.

[0040] Figure 7 shows Healing of damaged minlON membranes with 6HB containing POPC- DIBMA nanodiscs.

[0041] Bar plot showing increase in usable membranes within a used sensor flow cell upon addition of 6HB-containing nanodiscs. The Y-axis shows the number of membranes within the flow cell array classified as "Good" or "Large" (which are the optimal categories for nanopore insertion) by an Oxford Nanopore Technologies (“ONT”) membrane QC script, which is an assessment of membrane capacitance. The X-axis shows the concentration of affinity purified 6HB-containing POPC-DIBMA nanodiscs that were added to the membranes within the sensor array, followed by addition of a stop solution. It shows that a number of highly damaged membranes ("pre-addition") are significantly regenerated by addition of diluted nanodiscs (1 / 1000 mil and 1 / 10 mil).

[0042] Figure 8 shows Nanodisc healing of a defective flow cell array: minlON™ chip.

[0043] Bar plots showing the distribution of different classifications from the ONT automatic membrane classification tool, MembraneQC. (A) shows the defective chip before addition of a regenerating nanodisc composition, while (B) shows after addition.

[0044] Figure 9 shows nanodisc mediated reduction of aggregation of nucleic acid nanopores in solution.

[0045] Aggregation was measured by centrifuging fluorescently labelled nanopores overnight. Aggregated material would pellet and be removed from solution. Percentage aggregation is determined as the fluorescence of the supernatant (non-pelleted material) as a percentage of the fluorescence of the sample pre-centrifugation. Each experiment was performed in triplicate, and the error bars show the standard deviation.

[0046] Figure 10 shows that nanodiscs can stabilise nucleic acid nanopores for long-term storage.

[0047] (A). Unitary conductance histogram of nanopores inserted into minlON™ membranes after long-term storage. (B). Schematic representation of the nanopore-nanodisc construct used in the experiment. (C) Dynamic Light Scattering traces showing minimal changes in the size of the nanopore before and after two years of storage.

[0048] Figure 11 shows nanodisc lipids only disperse when inserted into membranes of similar properties.

[0049] Fluorescent chromatogram showing the dispersal of nanodisc lipids upon insertion into different target membranes. Nanodiscs formed of POPC lipids containing a FRET pair were added to vesicles formed of the same lipid type (POPC), a synthetic membrane of a similar size (PEO-PBO), or a much larger synthetic membrane, comparable to that used in a high- throughput flow cell such as the minlON™ (PMOXA-PDMS-PMOXA). The dispersal of the nanodisc lipids once inserted into the target membrane was measured by the reduction in FRET. Percentage dispersal was determined by solubilising the nanodiscs using TritonX- 100. Experiments were performed in triplicate, each trace shows the average, and the grey ribbon shows the standard deviation.

[0050] Figure 12 shows nanodisc insertion of protein nanopores aHL into minlON™ membranes.

[0051] (A). Schematic representation of the protein nanopore used, alpha heamolysin (aHL) inserted into a lipid nanodisc. The protein pore nanodisc with POPC lipids and the SMA nanodiscforming polymer. (B) Current-Voltage curve of the inserted nanopores showing rectification characteristic of aHL. Each point is an average of all inserted nanopores and the error bars show the s.e.m. (C) Unitary conductance histogram showing over 200 aHL nanopores inserted into minlON™ membranes using lipid nanodiscs.

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] Prior to setting forth the invention, a number of definitions are provided that will assist in the understanding of the invention.

[0054] Unless otherwise indicated, the practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, and chemical methods, which are within the capabilities of a person of ordinary skill in the art. Such techniques are also explained in the literature, for example, M.R. Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, Fourth Edition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F. M. et al. (Current Protocols in Molecular Biology, John Wiley & Sons, Online ISSN:1934-3647); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridisation: Principles and Practice, Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Synthetic Biology, Part A, Methods in Enzymology, Edited by Chris Voigt, Volume 497, pages 2-662 (2011); Synthetic Biology, Part B, Computer Aided Design and DNA Assembly, Methods in Enzymology, Edited by Christopher Voigt, Volume 498, Pages 2-500 (2011). Each of these general texts is herein incorporated by reference.

[0055] As used herein, the term ‘comprising’ means any of the recited elements are necessarily included and other elements may optionally be included as well. ‘Consisting essentially of’ means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. ‘Consisting of’ means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

[0056] The term ‘nucleic acid’ refers to a sequence of nucleotides that may be single or doublestranded, with the 3' and 5' ends of each nucleotide connected by phosphodiester bonds. These polynucleotides may consist of either deoxyribonucleotide or ribonucleotide bases. Nucleic acids include DNA and RNA, which are typically produced synthetically but can also be extracted from natural sources. Additionally, nucleic acids may involve modified forms of DNA or RNA, such as those that have been methylated or chemically altered, for example, 5’-capping with 7-methylguanosine or its analogues, 3’-processing like cleavage and polyadenylation, splicing, or labelling with fluorophores or other substances. Nucleic acids may also include synthetic nucleic acids (XNA) or nucleic acid analogues, such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA) and peptide nucleic acid (PNA). Hence, where the terms ‘DNA’ and ‘RNA’ are used herein it should be understood that these terms are not limited to only include naturally occurring nucleotides. Sizes of nucleic acids, also referred to herein as ‘polynucleotides’ are typically expressed as the number of base pairs (bp) for double stranded polynucleotides, or in the case of single stranded polynucleotides as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides of less than around 100 nucleotides in length are typically called ‘oligonucleotides’.

[0057] Polynucleotides or oligonucleotides may be functionalized by, for example, using a modified phosphoramidite in the strand synthesis reaction. Enzymic modification using a terminal transferase can also be used to incorporate an oligonucleotide, which incorporates a modification such as an anchor, to the 3’ of a single stranded nucleic acid (e.g. ssDNA). The polynucleotides or oligonucleotides of the disclosure may be modified to have phosphorothioate (PS) linkage in the backbone to increase resistance to nuclease degradation, or to have 2'-O-Methyl (2'-OMe) and 2'-0-Methoxyethyl (2 -MOE) modifications on the 2' position of the ribose sugar in RNA to improve stability against ribonucleases (enzymes that degrade RNA).

[0058] As used herein, the term ‘nanostructure’ refers to a geometrically predefined or ‘predesigned’ two or three dimensional molecular structure typically comprised from a biopolymer, suitably a naturally or non-naturally occurring nucleic acid or a polypeptide, which structure has at least one dimension or an aspect of its geometry that is within the nanoscale (i.e. 10'9metres). Nanoscale structures suitably have dimensions or geometry of less than around 100 nm, typically less than around 50 nm, and most suitably around 20 nm. Nanoscale structures suitably possess dimensions or geometry greater than around 0.1 nm, typically greater than around 1 nm, and optionally greater than around 2 nm.

[0059] Assembly of nucleic acid-based nanostructures may occur spontaneously in solution, such as by heating and cooling a mixture of DNA strands of preselected sequences, or may require presence of additional co-factors including, but not limited to, nucleic acid scaffolds, nucleic acid aptamers, nucleic acid staples, co-enzymes, and molecular chaperones. Where desired nanostructures result from one or more predesigned spontaneously self-folding nucleic acid molecules, such as DNA or RNA, this is typically referred to as nucleic acid ‘origami’. Rational design and folding of DNA to create two dimensional or three-dimensional nanoscale structures and shapes is known in the art (e.g. Rothemund (2006) Nature 440, 297-302). An embodiment of nucleic acid rational design may be DNA origami, wherein single-stranded DNA molecule is self-assembled into specific shapes by the complementary binding of multiple short "staple" strands.

[0060] The nucleic acid sequences that form the nucleic acid nanostructures will typically be manufactured synthetically, although they may also be obtained by conventional recombinant nucleic acid techniques. DNA constructs comprising the required sequences may be comprised within vectors grown within a microbial host organism (such as E. coli). This would allow for large quantities of DNA or RNA to be prepared within a bioreactor and then harvested using conventional techniques. The vectors may be isolated, purified to remove extraneous material, with the desired DNA sequences excised by restriction endonucleases and isolated, such as by using chromatographic or electrophoretic separation. A ‘polypeptide’ is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or in vitro by synthetic means. Polypeptide of less than around 12 amino acid residues in length is typically referred to as a “peptide”. The term “polypeptide” as used herein denotes the product of a naturally occurring polypeptide, precursor form or proprotein. Polypeptides also undergo maturation or post-translational modification processes that may include, but are not limited to: glycosylation, proteolytic cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, and such like. A “protein” is a macromolecule comprising one or more polypeptide chains.

[0061] As used herein the term ‘hydrophobic’ refers to a molecule having apolar character including organic molecules and polymers. Examples are saturated or unsaturated hydrocarbons. The molecule may have amphipathic properties. As used herein, the term ‘amphipathic’ describes a molecule with both hydrophobic and hydrophilic regions.

[0062] The term ‘membrane’ in the context of this application refers to a thin layer or barrier that separates two environments and selectively prevents the movement of molecular substances in solution, such as ions, small molecules, peptides, sugars, proteins and nucleic acids, between them. The membrane may comprise biological materials, synthetic materials, or a combination of these. A membrane may comprise a plurality of molecules that are charged and hydrophilic at one end, and hydrophobic and non-polar at the other end - also described as being amphipathic. In embodiments of the invention, the plurality of molecules of a membrane are all substantially amphipathic. Biological material suitable for a membrane composition may typically include lipids, specifically phospholipids. A membrane may be comprised of lipids, typically phospholipids and cholesterol. Examples of phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) or phosphatidylglycerol (PG). Examples of PC may be 1 ,2- Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or 1 ,2- diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). An example of phosphatidylglycerol may be 1 ,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG). An example of phosphatidylinositol may be phosphatidylinositol 4,5-bisphosphate (PIP2). Biological membrane material may also typically comprise cholesterol. A membrane may additionally comprise proteins suitable to be embedded in a membrane.

[0063] The membrane may also, or alternatively, comprise synthetic materials such as a synthetic polymer. In embodiments, the synthetic polymers may be synthetic lipid analogues. The synthetic polymer membrane may include a block copolymer, a di-block copolymer, a triblock copolymer, a terpolymer, an alternating copolymer, or a combination thereof. In some embodiments, the polymer membrane is made of tri-block polymers comprising poly 2- methyl-2-oxazoline (PMOXA) or polydimethylsiloxane (PDMS). In embodiments, the tri-block polymer comprising PMOXA and PDMS can be organised into alternating layers of hydrophilic PMOXA and hydrophobic PDMS, for example, PMOXA-PDMS-PMOXA. Hydrophilic PMOXA layers can interact with water, while the hydrophobic PDMS layers provide stability between the two PMOXA layers. In some embodiments, each block of polymers may be longer than a single unit. For example, membranes used can include PMOXA6-PDMS35-PMOXA6; PMOXA6-PDMS65-PMOXA6 or PMOXA11-PDMS65- PMOXA11 . In a different embodiment, the polymer membrane is made of di-block polymers comprising poly(1 ,2-butadiene)-b-polyethylene oxide (PBD-PEO), with hydrophobic PBD and hydrophilic PEO components. The membrane may be a hybrid membrane comprising both synthetic polymers and lipids. In some embodiments, a hybrid membrane may comprise 1 :1 PBD-PEO and DPhPC. In some embodiments, a hybrid giant unilamellar vesicle comprising both lipid and polymer components may be generated. There may be more than one type of lipid and polymer respectively. A polymer membrane may comprise synthetic lipid analogues. In an embodiment, the polymer membrane is made of poly (ethylene oxide)- block-poly (butylene oxide) (PEO-b-PBO) block copolymers. PEO-b-PBO is an amphipathic molecule with hydrophilic PEO and hydrophobic PBO.

[0064] A membrane may be in a form of a variety of macromolecular structures, such as micelles, inverted micelles, monolayers, bilayers, polymersomes, or other lamellar structures. Lamellar structures may include a giant unilamellar vesicle and a large unilamellar vesicle. In a micelle, the polar head groups are positioned on the outer surface, while the hydrocarbon chains are oriented towards the interior. Typically, the terminal methyl groups of the fatty acyl chains are located at the centre of the hydrophobic core, which can take the form of a sphere, ellipsoid, or cylinder. A lipid bilayer is characterised by orientation of the polar head groups facing the exterior surfaces in an aqueous environment, and the hydrophobic hydrocarbon chains in the interior of the bilayer.

[0065] Lipid bilayers tend to close in on themselves, preventing the exposure of hydrophobic aliphatic chains to water, which leads to the formation of a compartment enclosed by lipids. A lamellar structure refers to a layered arrangement of lipid, copolymer or synthetic lipid analogues. In an embodiment, a lamellar structure may include a unilamellar structure with a single layer of lipid, copolymer of synthetic lipid analogues. In an embodiment, a unilamellar structure may include vesicle or liposome referring to a structure made up of lipid bilayers enclosing an aqueous compartment. In an embodiment, a lamellar structure may be a multilamellar structure comprising multiple layers of lipid, copolymer or synthetic lipid analogues. In an embodiment, a vesicle-like structure made from amphipathic block copolymers are referred to polymersomes. Polymersomes typically comprise a bilayer membrane that encloses an aqueous core.

[0066] The term ‘fluidity’ in the context of membrane is defined as the degree in which individual molecules of the lipid membrane are free to rotate and move in lateral directions. The fluidity of a lipid membrane can be determined by several factors including composition of the membrane such as the ratio of saturated to unsaturated fatty acids. High proportions of phospholipids with unsaturated fatty acids typically increases the fluidity of the membrane by preventing tight packing of the fatty acids using its kinked tailed. Cholesterol may impact the fluidity of the membrane by restricting the movement of phospholipid fatty acid chains. The lipid membrane comprising both cholesterol and phospholipids may exhibit a ‘semi-fluidity’, as the mosaic structure of the lipid components maintains structural organisation while at least some lipid molecules, typically phospholipids, can move laterally.

[0067] A ‘semi-fluid membrane’ as used herein means a membrane that exhibits a semi-fluidity. A semi-fluid membrane typically comprises lipid and / or synthetic lipid analogues. In some embodiments, a membrane vesicle, a nanodisc or a nanodisc composition can comprise semi-fluid membrane. In embodiments, a lipid composition of a semi-fluid membrane of a nanodisc may not be identical to that of a membrane vesicle. In some embodiments, the lipid composition of a semi-fluid membrane of a nanodisc may be a selectively enriched version of the lipid composition of a membrane vesicle.

[0068] As used herein, the term ‘polymer’ comprises repeating structural units or monomers that are chemically bonded typically in a chain structure. The term ‘copolymer’ refers to a polymer comprising two or more different monomers that are bonded in the same polymer chain. Copolymers can be classified into various types, including a ‘random copolymer’ wherein the monomers are distributed randomly along the chain, an ‘alternating copolymer’ wherein the different monomers alternate in a regular pattern, and a ‘block copolymer’ wherein two or more chemically distinct polymer segments, or ‘blocks’ are covalently bonded. The term ‘diblock polymer’ is a type of block copolymer that consists of two distinct polymer segments, or blocks. The term ‘terpolymer’ refers to a polymer having three units that are different from each other.

[0069] As used herein, the term ‘membrane vesicle’ refers to a substantially spherical membrane structure that encloses a fluid-filled interior compartment, primarily composed of biological components such as phospholipids and cholesterol. The term ‘nanopore’ refers to a nanostructure that is embedded within a membrane and allows passing of molecular substances or analytes across the membrane via a central channel, or lumen. The diameter of the central channel of the nanopores delimits the size of the substance or analyte that is able to pass through the pore. Nanopores can be engineered to enhance cellular export of specific molecules, or to perform DNA sequencing and biosensing, wherein individual molecules pass through membrane-embedded nanopores to cause detectable changes in the ionic currents passing through the pore and / or tunnelling currents laterally across the pore. Ionic currents are produced when ions move through a nanopore. When a constant potential difference is applied across the electrodes around the vertical ends of the nanopore, a steady-state ion current is established due to the flow of ions across the nanopore within the membrane. As individual analyte molecules pass through or interact with the pore, short-lived obstructions of the pore create detectable modulations in the ionic current. The amplitude, duration, and frequency of these transient current modulations depend on characteristics of the analyte molecule such as size, shape, and charge as well as interactions that take place between the molecule and pore. However, accuracy of the ionic currents may be limited due to the rapid speed of DNA movement and the low signal-to-noise ratio. Tunneling current may be measured by incorporating electrodes, sensors or nanowires typically at the pore entrance in a direction transverse to the membrane and, thereby, monitoring the change in the electron tunnelling current in a transverse direction to the membrane. Analytes do not always need to pass through the entirety of the nanopore, in certain embodiments mere obstruction of the central channel by an analyte to create a current blockade may be sufficient for a sensing application.

[0070] The term ‘solid state nanopore’ refers to a pore formed in solid state layer of material or membrane. These types of membrane are typically 10 to 100 nm in thickness. Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as silicon nitride (SiNx) typically in the form of Si3N4, aluminium oxide typically in the form of AI2O3, and silicon oxide typically in the form of SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and various glasses. A solid state nanopore may be generated by applying focused ion beam or electron beam to the solid state layer, applying high voltage across the solid state layer, or chemical etching using a chemical solution or reactive gas that dissolves or reacts with the solid state layer. A solid state nanopore may also be a hybrid nanopore, wherein a nucleic acid or polypeptide nanopore is provided in an aperture of a solid support such as those described above.

[0071] The analyte can be any one of nucleic acids, amino acids, peptides, proteins, polymers, polysaccharides, and chemical small molecules. The three-dimensional configuration of an assembled nanopore may define at least one nanoscale pore that spans the membrane located substantially centrally in the pore structure. The nanoscale pores may comprise a channel having a lumen that has a minimum diameter of at least about 1 nm, suitably at least about 5 nm, typically at least about 50 nm, and the maximum opening of the channel is at most about 200 nm, about 100 nm, about 50 nm or about 10 nm.

[0072] The term ‘DNA nanopore’ refers to a nanostructure that comprises a nucleic acid. In one embodiment, a nucleic acid may be formed into a bundle, such as 6-helix-bundle (6HB), 4- helix-bundle (4HB), or 2-helix-bundle (2HB), or a series of modules comprised of bundles, that cooperate to define the desired geometry of nanostructure. The geometry may comprise a combination of secondary structural motifs and more open unstructured regions. The geometry may change from pre-assembly to post-assembly. In an embodiment, DNA nanopore may comprise one or more polynucleotide strands that provide a functional scaffold component, wherein the polynucleotide strands comprised within the scaffold component include a polynucleotide backbone; and a plurality of polynucleotide strands that provide a plurality of functional staple components. The scaffold strand(s) cooperate with and hybridise to themselves or the plurality of staple polynucleotide strands - e.g. via appropriate Watson- Crick base pairing hybridisation - in order to form a three-dimensional configuration of the nanostructure. In an embodiment, to enable the insertion of the negatively charged DNA nanopore to the lipid bilayer, hydrophobic cholesterol tags may be incorporated at selected positions in the structure. According to another embodiment of the present invention a nucleic acid nanostructure is provided that comprises a plurality of component modules. Suitably, each module comprises a nucleic acid sequence (e.g. DNA) and at least one attached membrane anchor molecule. The nucleic acid sequence comprises at least a portion / region of double helix that serves to define a secondary structural element having a defined length as well as a level of structural rigidity. The secondary structure may comprise one or more nucleic acid duplex bundles. In a specific embodiment of the invention, the duplex bundles may be oriented 5’ to 3’ substantially perpendicularly to the planar axis of the semifluid membrane. Alternatively, these duplex bundles may be oriented 5’ to 3’ substantially coaxially to the planar axis of the semifluid membrane. In some nanostructures there may be a combination of modules comprising both orientations in combination depending on the requirements of the resultant nanostructure. DNA nanopores that are suitable for use in the methods and compositions of the present invention are described in International Patent applications published as WO2018 / 011603, W02020 / 025974, WO2022 / 263670; and WO2022 / 263669, which are hereby incorporated by reference.

[0073] As used herein, the term ‘protein nanopore’ refers to a polypeptide subunit and multimers of subunits that can create an aperture through a membrane when an appropriate higher-order structure is formed. Nanopore protein may refer to a single polypeptide subunit of a multimeric nanopore protein or different oligomeric forms of single polypeptide subunits. A “mixture of nanopore proteins” refers to a solution that may contain a heterogenous combination of single and / or oligomeric forms of a nanopore protein. Exemplary naturally occurring nanopore proteins may include a-hemolysin (aHL), Mycobacterium smegmatis porin A (MspA), aerolysin, phi29, gramicidin A, maltoporin, OmpG, OmpF, OmpC, Vibrio cholerae cytolysin, PhoE, Tsx, fragaceatoxin C (FraC), cytolysin A (ClyA) F-pilus, and E. coli curli transport channel CsgG.

[0074] As used herein, the term ‘nanodisc’ refers to a membrane system comprising a semi-fluid membrane, typically a lipid membrane, surrounded by amphipathic molecules, including proteins, peptides, and / or synthetic polymers. The semi-fluid membrane component of a nanodisc may be stabilised by the encircling amphipathic molecules. Nanodiscs provide a stable environment that mimics a larger semi-fluid lipid membrane, while maintaining solubility in aqueous environments. Nanodiscs have been used to reconstitute membrane proteins or nanopores following extraction from their native membranes. Nanodiscs can be engineered to a range of specific sizes, typically ranging from about 10 to up to around 100 nanometers in diameter. The diameter of a given nanodisc may exceed that of any nanopore that is embedded within by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100% or more.

[0075] A ‘nanodisc forming polymer’ refers to a polymer that surrounds and stabilises the lipid membrane of a nanodisc. A nanodisc forming polymer typically comprises a synthetic copolymer as described herein. In an embodiment, the nanodisc forming polymer may comprise amphiphilic polymers, di-isobutylene-maleic acid (DIBMA), styrene-maleic acid (SMA), polyacrylic acid-co-styrene (AASTY) or a combination thereof. Di-isobutylene-maleic acid (DIBMA) is a copolymer formed by the polymerization of di-isobutylene and maleic acid in an alternating order. DIBMA is an amphiphilic molecule with hydrophilic maleic acid and hydrophobic di-isobutylene components. Styrene-maleic acid (SMA) is a type of copolymer made from the polymerization of styrene and maleic acid in an alternating order. The styrene portion of the molecule is hydrophobic due to its aromatic hydrocarbon structure, and the maleic acid portion is hydrophilic due to carboxyl groups. In embodiments, the ratio of styrene and maleic acid may be at least 1 :1 , 2:1 or 3:1. Polyacrylic acid-co-styrene (AASTY) is a highly-alternating copolymer comprising of acrylic acid and styrene monomer units.

[0076] A ’nanopore device’ refers to a device that makes use of nanopores for sensing interactions with molecular entities, such as target analytes (e.g. nucleic acids, small molecules or proteins / peptides in solution). A nanopore device typically comprises a membrane array that may embed a plurality of nanopores, typically a single nanopore, within each membrane comprised within the array. A nanopore device may typically employ an electrical signal across a nanopore channel to generate a measurement signal that is interpreted to sense and / or characterise molecular entities as they interact with the nanopore. In use, an electrical signal is detected as a change in potential difference (voltage) or current across the array of nanopore channels that provides a meaningful measurement to be interpreted. The measurement can include, for example, changes in one or more of ionic current flow, electrical resistance, electrical impedance, voltage, or capacitance. Optionally nanopores of the nanopore device are referred to as ‘sensors’. A membrane array, by comprising an array of nanopore sensing elements, increases data collection by allowing plural nanopores to sense interactions in parallel. The membrane array is comprised within a sensor device, wherein the sensor device is a nanopore based sensor device. The sensor devices may further comprise special purpose hardware and systems (e.g., circuitry, processors, memory, GUIs etc.) that perform the specified functions or acts, or combinations of special purpose hardware and computer instructions, in order to render a functioning sensor device capable of providing a meaningful readout to a user.

[0077] The disclosure relates to a nanodisc composition and a method of making the nanodisc composition by contacting a membrane vesicle with one or more nanopores in aqueous solution, thereby forming a membrane vesicle that encompass the one or more nanopores embedded within the membrane vesicle, contacting the one or more membrane vesicles with a nanodisc forming polymer, generating a nanodisc composition wherein the nanodisc forming polymers solubilise the membrane vesicle and encircle the one or more nanopores embedded within the membrane vesicle so as to form a plurality of nanodiscs.

[0078] Further, the disclosure relates to a use of a nanodisc composition as a delivery vehicle for insertion of one or more nanopores into a target membrane, and a method of increasing the frequency of insertion of nanopores into a membrane array, using a nanodisc composition of the present invention as a delivery vehicle.

[0079] Method of making a nanodisc composition

[0080] Provided herein is a method of making a nanodisc composition, a nanodisc composition comprising a semi-fluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the nanodisc composition further comprises one or more nanopores embedded within the semi-fluid lipid membrane. Exemplary methods of making a nanodisc composition include stabilising a lipid bilayer by encircling it with an amphipathic copolymer. According to the disclosure, a method comprises first contacting a nanopore with lipid in an aqueous solution, resulting in formation of a membrane vesicle around the nanopore that may be a protein or nucleic acid nanopore. The diameter of the membrane vesicle may be, for example, in a range of about 100 to 1000nm, about 200 to 900nm, about 300 to 800nm, about 400 to 700nm, or about 500 to 600nm. Suitably the membrane vesicle is at least 100, 200, 300, 400, or 500 nm in diameter; and / or at most 500, 600, 700, 800, 900 or 1000 nm in diameter.

[0081] In some embodiments, a semi-fluid membrane may be a semi-fluid lipid membrane. Alternatively, a semi-fluid membrane may comprise synthetic polymers or synthetic lipid analogues. Suitable lipids for forming the semi-fluid membrane include phosphatidylethanolamines (PE), phosphatidylcholines (PC), phosphatidylglycerols (PG), phosphatidylserines (PS), cholesterols, sphingomyelin, gangliosides, lipopolysaccharides, phosphatidylinositols (PI), and derivatives of the foregoing. In some embodiments, the lipid is a phospholipid. In some embodiments, the phospholipid includes a phosphatidylcholine. In an embodiment, a phosphatidylcholine includes 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC).

[0082] In some embodiments, the semi-fluid membrane may comprise synthetic materials such as synthetic polymer. The polymer membrane may include a block copolymer, a di-block copolymer, a tri-block copolymer, a terpolymer, an alternating copolymer, or a combination thereof. In some embodiments, the polymer membrane comprises a tri-block polymer comprising poly 2-methyl-2-oxazoline (PMOXA) or polydimethylsiloxane (PDMS). In embodiments, the tri-block polymer comprising PMOXA and PDMS can be organised into alternating layers of hydrophilic PMOXA and hydrophobic PDMS, for example, PMOXA- PDMS-PMOXA. Hydrophilic PMOXA layers can interact with water, while the hydrophobic PDMS layers provide stability between the two PMOXA layer. In some embodiments, each block of polymers may be longer than a single unit. For example, membranes used can include PMOXA6-PDMS35-PMOXA6; PMOXA6-PDMS65-PMOXA6 or PMOXA11- PDMS65-PMOXA11 . In a different embodiment, the polymer membrane is made of di-block polymers comprising poly(1 ,2-butadiene)-b-polyethylene oxide (PBD-PEO), with hydrophobic PBD and hydrophilic PEO components. The membrane may be a hybrid membrane comprising both synthetic polymers and lipids. Use of lipid-polymer hybrid membrane in nanodisc advantageously enhances stability, resistance to physical, osmotic, or electrical perturbations, and / or prolonged stability in aqueous environments over extended time periods, compared to non-hybrid membranes. Hybrid membranes therefore increases the capacity to stably accommodate nanopores. Furthermore, the membranes may exhibit reduced signal noise during nanopore analysis, may possess advantageous properties that are unattainable with membranes composed solely of lipids or solely of copolymers. Such properties are typically associated with enhanced stability, resistance to physical, osmotic, or electrical perturbations, or prolonged stability in aqueous environments over extended time periods. Additionally, these membranes may exhibit particular characteristics such as membrane thickness, which correlates with overall stability, and the capacity to accommodate nanopores. Furthermore, the membranes may exhibit reduced signal noise during nanopore analysis. In some embodiments, a hybrid membrane may comprise 1 :1 PBD-PEO and DPhPC. In some embodiments, a hybrid giant unilamellar vesicle comprising both lipid and polymer components may be generated. There may be more than one type of lipid and polymer respectively. A polymer membrane may comprise synthetic lipid analogues. In an embodiment, the polymer membrane is made of poly (ethylene oxide)-block-poly (butylene oxide) (PEO-b-PBO) block copolymers. PEO-b-PBO is an amphipathic molecule with hydrophilic PEO and hydrophobic PBO. In other embodiments, the semi-fluid lipid membrane may be a hybrid membrane comprising both synthetic polymers and lipids. In some embodiments, a hybrid membrane may comprise 1 :1 PBD-PEO and DPhPC.

[0083] Hybrid membranes can have desirable properties that are not achievable with pure lipid or pure copolymer membranes. These properties are usually related to stability, resistance to physical, osmotic or electrical shock, or stable over long timer periods in aqueous media; size, the height, or thickness, of the membrane which relates to its stability and; ability to accommodate nanopores; as well as noise imparted to the signal generated during nanopore analysis.

[0084] The semi-fluid membrane may additionally comprise a membrane protein such that the resulting lipid nanodisc includes a membrane protein spanning across at least one half of the lipid bilayer from one hydrophilic face to the centre of the hydrophobic edge. In some embodiments, the lipid includes a membrane protein such that the resulting lipid nanodisc includes a membrane protein spanning across the entire lipid bilayer from one hydrophilic face to the second hydrophilic face at least once. In some embodiments, the membrane protein spans across the entire lipid bilayer from the first hydrophilic face to the second hydrophilic face more than once.

[0085] According to the disclosure, the nanopore comprises a protein nanopore or nucleic acid nanopore, wherein the protein nanopore comprises amino acids, and the nucleic acid nanopore comprises DNA or RNA. Typically, a nanopore comprises and defines a membrane-spanning central channel, wherein the central channel has a minimum internal width of at least 0.5 nm, optionally at least 0.8 nm, suitably up to or around 2 nm or more. In an embodiment, a nucleic acid nanopore may be a computationally designed synthetic nanopore comprising polynucleotides with hydrophobic modifications. The central channel may extend along a central axis of the nanopore and optionally defining at least one aperture. When the nanopore is embedded within a membrane the nanopore may permit a measurable flow of electrically charged ions to pass through the lumen of the central channel, generating a measurable electrical current that pass across the membrane via the nanopore. Examples of nanopores may include biological protein nanopores including but not limited to any of those described herein, or particularly alpha-hemolysin (aHL), MspA derived from bacterium Mycobacterium smegmatis, FraC or the E. coli curli transport channel CsgG. In an embodiment, the nanopore may comprise a plurality of component modules comprising a nucleic acid sequence, and wherein the plurality of component modules is capable of undertaking a controlled assembly in response to an external stimulus to form the nanostructure; and wherein the nanostructure is configured to penetrate a semifluid membrane upon or following the controlled assembly. In another embodiment, the nucleic acid nanopore may comprise a scaffold polynucleotide strand and a plurality of staple polynucleotide strands 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.

[0086] The first contacting step of membrane vesicle to nanopore may involve addition of lipid or synthetic lipid analogues to nanopores in aqueous solution. In an embodiment, prior to the first contacting step, an aqueous nanopore solution may be prepared. In an embodiment, lipids I synthetic lipid analogues may be prepared in a ‘dry’ form by being stored under chloroform. In an embodiment, lipids I synthetic lipid analogues are dried to a thin film using a rotary evaporator or a thin stream of argon. In the first contacting step, dry lipids I synthetic lipid analogues in a film form are brought into contact with nanopores by re-suspending the lipid in an aqueous nanopore solution.

[0087] Following the first contacting step, the nanopores are resuspended in the lipid I synthetic lipid analogues following an incubation with agitation until the solution is clarified. In some embodiments, the resuspended synthetic lipid analogue-nanopore solution is incubated with slow agitation over a time period of hours, days or even up to one week.

[0088] The solutions and suspensions described in the disclosure are largely free of detergents. Here, “substantially free” refers to the solution or suspension containing no significant amount of intentionally added detergent. However, trace amounts of detergents (e.g., less than about 100 ppb) may be present and still fall within the scope of the present disclosure. The contacting step can optionally further include providing a buffer to regulate the pH of the solution. Without intending to be bound by theory it is believed that in some embodiments, the pH of the solution can affect the charge of the nanodisc forming polymer, thus ultimately affecting solubility of the polymer and stability of the resulting nanodiscs. The contacting step can be carried out at any suitable pH in which the polymer is stable and soluble, for example, in a range of about 3 to about 11 , about 4 to about 10, about 5 to about 9.5, or about 6 to about 9, or about 6.5 to 9..

[0089] Following the initial contact of the nanopores with the lipid, the hydrophobic regions / anchors of the nanopore interact with the hydrophobic core of the lipid component, stabilising the nanopore-lipid assembly. With this hydrophobic effect, a non-polar portion of the nanopore- lipid assembly aggregates when immersed in the aqueous environment, and minimises exposure of hydrophobic tails to its environment, forming a nanopore embedded membrane vesicle structure. In an embodiment, the membrane vesicle comprises first and second opposing hydrophilic faces and a hydrophobic edge defined between the first and second hydrophilic faces. In an embodiment, the membrane vesicle may be a micelle or a liposome.

[0090] Nanodisc forming polymers (NFPs) are put in contact with the nanopore embedded membrane vesicles. In an embodiment, NFPs are provided at a membrane vesicle solubilising concentration. The solubilising concentration in terms of molar ratio of NFPs to membrane vesicle may be at least 0.2:1 , 0.5:1 , or 1 :1 . The solubilising concentration may be at most 1 :1 , 2:1 , or 5:1 . In an embodiment, the NFPs comprise amphipathic copolymers. In this embodiment, the method of making a nanodisc involves contacting a lipid to nanodiscforming amphipathic polymers comprising hydrophobic and hydrophilic groups. At least one of hydrophobic and hydrophilic groups may be pendant hydrophobic groups and hydrophilic groups. In an embodiment, the hydrophobic and hydrophilic groups may be distributed within the NFPs. In some embodiments, the NFPs may comprise maleic acid copolymers. In further embodiments, the maleic acid copolymers are selected from copolymers comprising: styrene-maleic acid (SMA) and / or di-isobutylene-maleic acid (DIBMA). In an embodiment, a nanodisc comprises 0.5:1 or 2:1 molar ratio of DIMBA to 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC). In a different embodiment, a nanodisc comprises 0.5:1 molar ratio of DIBMA to poly (ethylene oxide)-block-poly(butylene oxide) (PEO-b-PBO) polymersome. Optionally, the nanodisc forming polymer comprises scaffold protein or non-structural protein family (Nsp).

[0091] Upon contact of NFPs with nanopore embedded membrane vesicles, hydrophobic groups of the NFPs insert themselves into the lipids, leading to fracture of the membrane vesicles. NFPs are in a cluster form prior to contact with the membrane vesicles, and following the contact, disaggregate within the membrane to further separate and deform the lipid membrane of the membrane vesicle. In embodiments where the NFP comprises SMA, the nanopore embedded membrane vesicles are eventually solubilised by both styrene-induced disruptions of the membrane vesicle lipid tail packing and repulsions between the maleic acid groups, leading to encircling of the hydrophobic edge of the lipid membrane by nanodisc forming polymers. A plurality of nanodiscs is formed wherein semi-fluid lipid membrane is enclosed by one or more NFPs, wherein the nanodisc composition further comprises one or more nanopores embedded within the semi-fluid lipid membrane. Optionally, following the formation of nanodiscs, excess NFPs may precipitate upon addition of divalent cations. In embodiments, divalent cations may include at least one of calcium (Ca2+), magnesium (Mg2+), iron (Fe2+), copper (Cu2+), zinc (Zn2+), strontium (Sr2+), manganese (Mn2+), barium (Ba2+), lead (Pb2+), or mercury (Hg2+).

[0092] Advantageously, the nanodisc-forming method of the present disclosure allows circumvention of energetically intensive membrane remodelling processes during nanopore insertion into target lipid membranes by formation of intermediary lipid vesicles. Additionally, by directly solubilising vesicle membranes, the method removes the need for use of conventional detergent mediated lipid membrane solubilisation, which can lead to disruption of nanopore protein structure and / or function. Further, the nanodisc forming method produces nanodiscs that may comprise synthetic lipid analogues. The disclosure provides a method that is compatible with a high throughput flow cell, by reproducibly generating nanodiscs with a single nanopore per disc. Importantly, the nanodiscs of the present disclosure serve as delivery vehicles for facilitating improved uptake of nanostructures and nanopores into target synthetic membranes comprised within a membrane array, typically within a nanopore sequencing device. In addition, the nanodiscs may facilitate insertion of complex nanostructures and nanopores under conditions that would otherwise be energetically unfavourable into target membranes, by acting and an intermediary shuttle.

[0093] The formation of the nanodiscs of the disclosure can be confirmed and characterized using static light scattering (SLS), dynamic light scattering (DLS), size-exclusion chromatography (SEC), Fourier-transform infrared spectroscopy (FT-IR), solid-state nuclear magnetic resonance (ssNMR), and transmission electron microscopy (TEM). Advantageously, when the nanodiscs are less than or equal to about 40 nm in diameter, the structure of the nanodiscs can be determined based on solution NMR techniques and when the nanodiscs are greater than about 40 nm, the nanodiscs can be magnetically aligned which is advantageous for solid-state NMR studies. Furthermore, as the NFPs of the disclosure can be free of styrene and / or aromatic groups, the nanodiscs can be characterized using biophysical techniques such as circular dichroism (CD), ultraviolet-visible spectroscopy (UV / Vis), and fluorescence spectroscopy.

[0094] Nanodiscs and nanodisc compositions

[0095] The present disclosure provides, in various aspects and embodiments, a nanodisc comprising a semi-fluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the one or more NFPs comprise an amphipathic polymer and / or copolymer. The nanodisc also comprises the semi-fluid lipid membrane which comprises first and second opposing hydrophilic faces and a hydrophobic edge defined between the first and second hydrophilic faces. In aspects and embodiments, the NFPs of the nanopore encircle the hydrophobic edge of the lipid membrane. In embodiments of the disclosure the the nanodisc further comprises one or more nanopores embedded within the semi-fluid lipid membrane. The one or more nanopores may be comprised of nucleic acid nanopores, or alternatively polypeptide / protein based nanopores. In alternative embodiments of the disclosure, wherein the nanodisc is intended for use in the regeneration of a damaged high-throughput flow cell or membrane sensor array, the nanodisc need not comprise an embedded nanopore.

[0096] Further, the present disclosure relates to a nanodisc composition comprising a semi-fluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the nanodisc composition further comprises one or more nanopores embedded within the semifluid lipid membrane. The nanodiscs disclosed herein further include a semi-fluid lipid membrane comprises lipids or synthetic lipid analogue. Optionally, a composition may comprise a plurality of nanodiscs according to the present disclosure in liquid suspension, suitably in aqueous suspension (e.g. in an emulsion).

[0097] The lipid of the nanodisc forms a lipid bilayer including two opposing hydrophilic faces, and a hydrophobic edge between the hydrophilic faces. The hydrophobic edge is made up of the hydrophobic tails from both layers of the lipid bilayer. The centre of the hydrophobic edge is the point at which the hydrophobic tail from one layer of the bilayer meets the hydrophobic tail from the second layer of the bilayer. The nanodisc further includes a copolymer of the disclosure encircling and enclosing the hydrophobic edge of the lipid bilayer. In some embodiments, the semi-fluid lipid membrane retains, or enriches, components of membrane vesicles.

[0098] In some embodiments, the semi-fluid lipid membrane of the nanodisc is POPC, NFPs are styrene-maleic acid copolymers, and a nanopore is a DNA nanopore comprised of scaffold and staple strands. In an embodiment, the molar ratio of the first monomeric unit to the second monomeric unit of the NFPs can be in a range of about 1 :1 to about 3:1 , about 1 :1 to about 2:1 , about 1.1 :1 to about 1.8:1 , about 1.1 :1 to about 1.5:1 , for example, about 1.1 :1 , about 1 .2:1 , about 1.3:1 , about 1.4:1 , or about 1 .5:1 . In some cases, the copolymer includes a styrene / modified maleic anhydride copolymer having a styrene to maleic anhydride molar ratio of about 1 .3:1 . Without intending to be bound by theory, it is believed that the ability of a polymer to form a lipid nanodisc depends on the ratio of hydrophobic:hydrophilic monomer units, for a given polymer molecular weight.

[0099] The lipid nanodiscs of the disclosure can have a diameter in a range of about 6 nm to about 100 nm, for example, about 6 nm to about 100 nm, about 10 nm to about 90 nm, about 20 nm to about 90 nm, about 30 nm to about 80 nm, about 40 nm to about 80 nm, about 50 nm to about 70 nm, or about 55 nm to about 65 nm. In some cases, the nanodisc has a diameter less than or equal to 40 nm, for example, in a range of about 6 nm to 40 nm, about 10 nm to about 35 nm, about 20 nm to about 35 nm, or about 25 nm to about 30 nm. In some cases, the nanodisc has a diameter greater than 40 nm, for example, 41 nm to about 100 nm, about 45 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, or about 60 nm.

[0100] Nanodiscs as a delivery vehicle for inserting nanopores into a target membrane

[0101] The nanodiscs and nanodisc compositions defined in the present disclosure can be used as a novel delivery vehicle for insertion of one or more nanopores into a target membrane, suitably a synthetic polymer-containing membrane within a sensor array. Insertion of one or more nanopores into a target membrane involves reconstitution of nanopores into a target membrane. The reconstitution typically involves fusion of the nanodisc membrane with the target membrane, allowing the nanopore integration into the target membrane. Previous uses of nanodiscs include transfer of membrane proteins such as potassium channels from nanoparticles to lipid bilayers (Banerjee and Nimigean, 2011), and transfer of a membrane protein from nanoparticles to mammalian cell membranes (Umbach et al., 2022). However, these are limited to the methods of insertion of membrane proteins into a target biological lipid membrane of similar composition to the initial vesicle. Therefore, these methods do not address the significant technical problem of overcoming the energy barrier of inserting nanopores, especially highly negatively charged nucleic acid nanopores, into a synthetic lipid analogue membrane, i.e. a target membrane, of the type typically comprised within a high- throughput flow cell or membrane sensor array.

[0102] Prior to insertion, the nanodisc of the present invention is brought close to a target membrane. During the insertion process, hydrophobic component of semi-fluid membrane of the nanodisc may be reconstituted into the target membrane by fusing with the target membrane, in an embodiment, the NFP mediates the contact of nanodisc composition to the target membrane.

[0103] Following the nanodisc insertion to the target membrane, the nanodisc forming polymers may dissociate from the nanodisc periphery, and precipitate, optionally in the presence of divalent cations (e.g. Ca2+or Mg2+). Following the removal of the nanodisc forming polymers, the semi fluid lipid membrane of nanodisc merges with the target membrane of the membrane, generating a nanodisc-target hybrid membrane. Finally, the semi-fluid membrane components of the nanodisc dissipate across the nanodisc-target hybrid membrane. In an embodiment, the relative area of the nanodisc semi fluid membrane to the target membrane is at least about 1 :50, 1 :100, or 1 :200.

[0104] A target membrane may be comprised of a polymer membrane, a lipid membrane, a hybrid membrane, or a solid-state membrane / layer. Preferably, a target membrane may comprise synthetic polymers or synthetic lipid analogues. In an embodiment, a target membrane may be in a planar or vesicular form. In an embodiment, a target membrane may be in a form of hybrid giant unilamellar vesicles, which comprises both lipid and polymer components. There may be more than one type of lipid and polymer respectively. A polymer membrane may comprise synthetic lipid analogues or synthetic polymers.

[0105] In another aspect of the invention, nanodiscs of the present disclosure are configured to be inserted into an aperture formed within a solid state membrane or the lumen of a solid state nanopore. Nanodiscs of the present invention may improve functionality of nanopores embedded in the nanodisc by optimally accommodating the nanodisc to the solid state membrane / nanopores. Nanodiscs of the present invention further provide a resilient seal that minimises any gap between the periphery of the nanodisc and the surface of a solid state nanopore. Efficient sealing between the periphery of a membrane embedded nanopore and the surrounding aperture within the solid-state material reduces current leakage across the membrane. In an embodiment, solid state nanopore accommodates the nanodisc via hydrophobic interaction between hydrophobic components of the NFPs and hydrophobic surface of the solid state nanopore. In an embodiment, the solid-state membrane is designed to have an orifice / aperture that modulates the diameter of the nanopore across the solid- state membrane. In an embodiment, the solid-state orifice / aperture varies in diameter across the thickness of the membrane material assuming a conical, frustoconical or tapered configuration that facilitates placement of the nanodisc in situ. In an embodiment, the lumen of the solid-state material orifice / aperture is wetted or coated with hydrophilic or hydrophobic materials that facilitate electrostatic interactions with the NFP of the nanodisc and improve placement. In an embodiment, the hydrophobic material may comprise a silicone, a silane, a siloxane or a hydrophobic polymeric material.

[0106] In an embodiment of the invention, nanodiscs containing at least one nanopore are selected prior to insertion into a target membrane. In an embodiment, the nanopore containing nanodiscs are purified using affinity purification. In further embodiments, the affinity purification involves purification of a nanopore bound tag against an affinity column. In embodiments, the nanopore containing nanodiscs are purified using a combination of purification methods such as size-exclusion chromatography (SEC), affinity purification, or centrifugation.

[0107] In an embodiment of the invention, target membrane is comprised within a membrane array.

[0108] A membrane array is an organised structure of membranes. Membrane array is comprised within a sensor device, wherein the sensor device is a nanopore based sensor device.

[0109] Advantageously, the current disclosure provides an improved method in multiple aspects: first, the method lowers the energy barrier required for insertion of the nanopores. Furthermore, using nanodiscs as a delivery vehicle prevents the nanopore structure from becoming a limiting factor in membrane insertion. The current method allows the insertion of nanopores with structural properties which may be very desirable in an analyte sensor but are not well suited to membrane insertion. For example, according to the embodiments of the invention, highly charged DNA nanopores are efficiently reconstituted in a target synthetic semi-fluid membrane. Further, the nanodisc of the present invention ensures optimal sealing with the solid-state membranes or nanopores, thereby preventing damage, deformation, or loss of functionality in the inserted nanopore. The nanodisc composition of the present invention eliminates the need for external forces or electric fields to position the nanodisc, preserving its integrity and functionality.

[0110] Additionally, the method demonstrates a stable insertion and consistent functioning of a highly charged nanopores to a semi-fluid target membrane environment. Therefore, the current invention discloses a novel use of nanodiscs as a delivery vehicle that is capable of inserting highly charged nanopores into a variety of semi-fluid target membranes, including synthetic polymer membranes or hybrid membranes.

[0111] Method of increasing frequency of nanopore insertion into a target membrane Nanopore sensors have been designed to detect a variety of species, including individual molecules such as peptide and nucleic acid analytes. One example of such a device is the minlON™, produced and distributed by Oxford Nanopore Technologies Ltd. This nanoporebased detection system works by measuring the ionic current passing through a biological nanopore embedded within a highly resistive amphiphilic membrane. The minlON™ contains an array of nanopore sensors (see Figure 1 E-G).

[0112] Devices comprising nanopore sensors are suitably incorporated into nucleic acid sequencing systems. Such devices and systems typically comprise a high-throughput flow cell or membrane array configured with a plurality of nanopore sensors arranged in a spatially addressable format. In one embodiment, the flow cell comprises a polymer membrane (e.g., a lipid bilayer or synthetic polymer membrane) suspended across an array of electrodecontaining wells or microfluidic channels, wherein each well supports one or more biological or solid-state nanopores. The array may comprise between around 512 and around 4,000 or more individual sensing channels, each channel being independently addressable via integrated circuitry disposed beneath or adjacent to the membrane structure. In a particular implementation analogous to the minlON™ platform mentioned above, the flow cell integrates an application-specific integrated circuit (ASIC) chip containing microelectrode arrays, wherein each electrode is capacitively or resistively coupled to a nanopore to detect ionic current modulations as nucleic acid polymers translocate through the nanopore under an applied voltage differential (typically 140-200 mV). The flow cell further comprises fluidic inlet and outlet ports in fluid communication with upper and lower chambers separated by the membrane, allowing continuous perfusion of electrolyte buffer and sample delivery while maintaining stable nanopore insertion and electrical contact throughout the sequencing run.

[0113] In one embodiment, the disclosure provides a method of increasing the frequency of insertion of a nanopore into a target amphipathic membrane comprised within a membrane array of the type described. In an embodiment, a membrane array may comprise at least 512, 1024, 2048 or 4096 individual membrane units, and each membrane unit of the membrane array has a single nanopore inserted within it. In a different embodiment, each membrane unit of the membrane array has more than one nanopore inserted. The membrane array may be comprised within a sensor device, wherein the sensor device is a nanopore based sensor device. A nanopore device may typically employ an electrical signal detection configuration across each nanopore channel (i.e. within each membrane unit) to generate a measurable signal that is interpreted to sense and / or characterise molecular entities as they interact with, and optionally pass through, the nanopore. A membrane array, by comprising an array of nanopore sensing elements, increases data collection by allowing multiplexed testing of multiple analytes. In an embodiment, the frequency of nanopore insertion into a target membrane array (or a given unit within the array) is increased by at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, or at least 1000 fold relative to direct insertion of nanopores in the absence of a nanodisc delivery vehicle.

[0114] Advantageously, use of a nanodisc as a delivery vehicle increases nanopore insertion efficiency for each membrane unit of the membrane array, increasing throughput and scalability in nanopore mediated applications. Increasing insertion frequency of highly charged nucleic acid nanopores significantly increases the potential of nucleic acid nanopores as therapeutic, prognostic and diagnostic tools. While nucleic acid nanopores have been known to respond to specific stimuli, such as the acidic micro-environment of tumour cells, and regulate membrane flux, their therapeutic applications have been limited by the low insertion frequency of DNA nanopores into sensor membranes. The present invention addresses this limitation by introducing a method to increase nanopore insertion frequency into target membranes comprised within a membrane array of a sensor device. The advancement allows fast and efficient integration of nucleic acid nanopores in a high- throughput, multiplex system. The invention overall enables exploration of novel therapeutic, prognostic and diagnostic uses of synthetic nanopores, by providing comprehensive insights through reliable, high-throughput data generation.

[0115] Membrane regeneration using nanodiscs

[0116] In certain embodiments, nanodiscs of the type described elsewhere herein but devoid of transmembrane nanopore proteins may be employed to regenerate, repair, reconstitute, or otherwise restore compromised membrane structures within a flow cell sensor device. The compromised membrane structures may include ruptured membranes, perforated membranes, mechanically damaged membranes, electrically damaged membranes, or membranes exhibiting loss of bilayer integrity in one or more sensing channels of a nanopore sequencing system, such as a flow cell cartridge compatible with MinlON™ or related sequencing platforms (Oxford Nanopore Technologies, Oxford, UK).

[0117] The membrane repair process comprises introducing a population of nanodiscs lacking incorporated nanopores into a fluidic chamber of the flow cell, wherein the nanodiscs spontaneously associate with and integrate into the damaged membrane regions within the array. This integration effects a transformation of the respective membranes from a nonfunctional state (characterized by breaches, multilayer defects, or insufficient electrical seal resistance) to a functionally restored state comprising intact, unilamellar lipid bilayer structures capable of supporting subsequent further insertion of nanodiscs comprising nanopores for nucleic acid sensing operations (if required).

[0118] The efficacy of membrane regeneration and the transition from a damaged membrane state to a sensing-competent membrane state may be quantitatively assessed by electrical impedance measurements, particularly by determining the electrical capacitance of the membrane structure within the sensor array (see Examples below). In one embodiment, membrane capacitance is measured across individual sensing channels using integrated electrode arrays within the application-specific integrated circuit (ASIC) underlying the membrane sensor array. A successful repair event is indicated by capacitance values characteristic of intact lipid bilayers (e.g. designated as ‘good’), as opposed to the anomalous capacitance signatures associated with membrane defects, such as elevated capacitance due to thinned regions or reduced capacitance due to multilayer formation or complete membrane rupture.

[0119] Accordingly, the invention further provides for sensors and sensor devices comprising the membrane arrays modified by nanodisc insertion as described by any of the methods herein.

[0120] The invention is further exemplified by reference to the following non-limiting examples.

[0121] Examples

[0122] Example 1. Assembly, Purification and Characterisation of Nanodiscs without nanopores

[0123] All un-modified and fluorophore modified DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT) at a 100 nmol scale, pre-resuspended to 100 pM in IDT- TE buffer. Cholesterol or fluorophore modified oligonucleotides were purchased from either IDT or ATD-Bio with HPLC purification and were resuspended to 100 pM in deionised water or TE buffer respectively and stored in aliquots at -20 °C. All lipids were purchased from Avanti Lipids and resuspended to 10 mg / mL in anhydrous chloroform. Nanodisc Forming Polymer (NFP) Styrene maleic acid (3:1) was purchased Pre-hydrolysed from Avanti Lipids, while Diisobutylene-maleic acid was purchased powdered with Tris, pH 7.5 from Cube Biotech. NFPs were resuspended in deionised water at 5 and 10 mM respectively, and stored sub-aliquoted at -20 °C. The triblock copolymer used to generate vesicles in Fig. 4E,J was purchased from PolymerSource, resuspended to 10 mg / mL in anhydrous chloroform and stored at -20 °C. All enzymes, DNA markers and gel loading dyes were purchased from New England Biolabs. All other materials were purchased from Merck (UK), unless otherwise stated. Nanodisc Generation

[0124] Nanodisc Forming Polymers (NFP) such as Styrene-Malic Acid (SMA) diisobutylene maleic acid (DIBMA) were added to a lipid vesicle suspension at 2:1 or 1 :1 polymedipid for SMA and DIBMA respectively and allowed to incubate at 25 °C with 800 rpm agitation for 30 minutes or until the solution clarified.

[0125] Dynamic Light Scattering (DLS)

[0126] DLS was performed to confirm complete assembly, purification and sizing of the assembled nanodiscs. All DLS measurements were performed on a Punk DLS Analyser (Unchained Labs) using a 15 pL quartz cuvette with a path length of 1 mm (Hellma Analytics). Each DLS trace shown in this work is an average of between 8 and 20 technical repeats, each of which is average of 10 individual scans. Samples were allowed to thermally equilibrate to 25 °C for 1-5 minutes prior to scanning.

[0127] Dynamic Light Scattering (DLS) analysis formation of lipid nanodisc from POPC vesicles by addition of increasing amount of nanodisc forming polymer (NFP), in this instance (Figure 2B) it is the NFP diisobutylene maleic acid (DIBMA). The DLS traces show a POPC vesicle, a 0.5:1 molar ratio of DIBMA to POPC, and a 2:1 ratio. DLS analysis also characterises polymer vesicle (polymersome) composed of a PEO-b-PBO block copolymer (Figure 2G). Addition of 0.5:1 molar ratio of DIBMA to PEO-b-PBO polymersome, demonstrating formation of lipid-free nanodiscs (Figure 2H).

[0128] Size exclusion chromatography (SEC)

[0129] SEC purification was performed to separate assembled nanodiscs from excess NFP in solution. Chromatography was performed on an AKTA Purified 100 FPLC system with a UV- 900 detector. A Superdex 75 30 / 100 (Cytivia) column with a bed volume of 24 mL and a fractionation range from 3 - 70 kDa was pre-equilibrated with a degassed solution buffer of 50 mM Tris. Cl, pH 7.5, supplemented with 500 mM KCI. For purification, samples were injected using a super-loop and flow was set to 0.7 mL / min. Data was monitored at 260 nm.

[0130] Size Exclusion Chromatograph showing purification of 2:1 DIBMA:POPC vesicle, generated with 0.1 mol % PE-Cy5. Right-hand axis shows fluorescence measurement of fractions, demonstrating co-localisation of lipid and polymer (Figure 2C).

[0131] Giant Unilamellar Vesicle (GUV) Generation

[0132] GUVs were generated from POPC to demonstrate the continued integrity of vesicle membranes after nanodisc fusion via light microscopy. To generate GUVs, 5 pL of 10 mM POPC in chloroform was evaporated on the surface of an indium tin-oxide (ITO) glass slide. This slide was inserted into a Vesicle PrepPro (Nanion Technologies). A rubber O-ring was placed around the lipid film, 300 uL of a 1 M Sucrose solution was added and another ITO glass slide added to seal the chamber. A 3 V electric field was applied for 120 minutes alternating at 5 Hz. The GUV suspension was collected via pipette and used that day.

[0133] Large Unilamellar Vesicle (LUV) Generation

[0134] LUVs were generated from POPC to study the interactions of the DNA nanopores with and without encircling nanodiscs with lipid membranes. 1 nmol of lipid in chloroform was gently evaporated under a stream of argon. Once a thin film had formed the sample was further evaporated using a rotary evaporator (Buchi) for 1 hour under high vacuum. The lipid film was resuspended by vortexing with 1 mL aqueous buffer. Lipid vesicles were then extruded 31x to the desired size using Mini Extruder (Avanti Lipids) and polycarbonate membrane (Whatman) of the requisite size. Where mentioned, 1 mol % of PE-Cy5 or PE-Rhodamine (Avanti Lipids) was added for visualisation. Polymeric vesicles were generated in a similar manner, however instead of vortexing and sonicating the samples, thin polymer films were allowed to resuspend slowly over 2-4 hours with 150 rpm agitation prior to extrusion.

[0135] Confocal Laser Scanning Microscopy (CLSM)

[0136] CLSM was performed using a Leica TCS SPE high-resolution Spectral Confocal microscope (Leica). 100 pL of SEC purified SMA-mediated nanodiscs generated using a fluorescent tag on the lipid was added to a fluorodish (World Precision Instruments). 1 pL of 100 pM Atto633 (Sigma) in deionised water was added, followed by 10 uL of GUV suspension. Sample was incubated for ~10 minutes to allow the GUVs to sink. A 96x optical zoom was used to locate the GUVs in brightfield and images were taken using 570 and 670 nm laser lines. Data analysis was performed using Imaged.

[0137] Confocal fluorescent microscopy showing binding of purified fluorescence nanodiscs to giant uni-lamellar POPC vesicles. An external fluorescence dye has been added to demonstrate the maintained integrity of the vesicles. Scale bar is 50 pm.

[0138] Fluorescent Spectrogram

[0139] Fluorescent spectrogram are used to monitor the release of encapsulated fluorescent dyes from nanoparticles, liposomes, or nanodiscs. Fluorescent spectrogram demonstrates release of encapsulated fluorescence dye (sulfurhodamine B) from DPhPC LUVs upon addition of osmotically balanced buffer (grey trace) or purified nanodiscs (pink trace). Each trace is an average of three technical repeats and the coloured ribbon shows the standard deviation. Example 2. Generation and Characterisation of Lipid Nanodiscs with Transmembrane DNA Nanopores

[0140] This example relates to generation and characterisation of 6 Helical Bundle (6HB) DNA Nanopore.

[0141] DNA Nanopore Assembly

[0142] DNA oligonucleotides were diluted to 1 pM in a solution of 50 mM Tris. Cl, pH 7.5, supplemented with 500 mM KCI, to a final volume of 100 pL. Assembly of DNA nanopores was performed on a T100 Thermocycler (BioRad). Samples were heated to 95 °C for 10 minutes, followed by cooling to 65 °C over 5 minutes, then slowly cooled to 25 °C at a rate of 0.05 °C a minute, then to 10 °C at a rate of 0.2 °C per minute. Overall run time was under 15 hours.

[0143] Gel Electrophoresis

[0144] Gel Electrophoresis was used to confirm the correct assembly of the DNA Nanopores. Agarose gels were cast from 150 mL 1 .5 or 2% solution of UltraPure Agarose (ThermoFisher) in 1x UltraPure TAE buffer (ThermoFisher) with 0.00003% v / v Ethidium Bromide. 2 pmol of DNA was applied to the gel from a 100 nM solution in Purple SDS-free loading Dye (NEB) with 2.5 uL of 100 bp or 1 kbp DNA Marker (NEB) used as a reference. Gels were electrophoresed for 60 minutes at 60 volts at 4 °C unless otherwise specified. For PAGE analysis 10% Mini-PROTEAN TGX pre-cast PAGE gels (BioRad) were pre-equilibrated in 1x TBE buffer (Thermo Fisher) for 30 minutes, 120 volts, 4 °C. 10 pL of 100 nM DNA pre-diluted in 1x Purple loading dye (NEB), was applied to the gel, followed by electrophoresis for 60 minutes at 60 volts. After electrophoresis, gel was first washed for 5 minutes in 150 mL deionised water, then stained in 150 mL 0.00003% Ethidium Bromide for 20 minutes with gentle agitation. Excess stain was removed with a final 1-hour wash in deionised water. PAGE gels were imaged the same way as agarose.

[0145] 10% TBE-PAGE analysis of6HB DNA nanopore generated with 0,1 or 3 cholesterol moieties (Figure 3B). Schematic representation and dimensions of Ring style DNA Nanopore (Figure 3C). 10% PAGE gel showing assembly of Ring style DNA nanopore (Figure 3D). Size Exclusion Chromatogram showing analytical purification of 6HB-0C (Figure 3E). DLS analysis showing volume and intensity distributions of 6HB-0C (Figure 3F).

[0146] Ultra-Violet (UV) Spectrophotometry UV analysis was performed to quantify the DNA and polymer contents of the purified nanopore-nanodisc construct. UV analysis was performed on a Varian Cary 300 Bio-UV-Vis spectrophotometer (Agilent) using a in quartz cuvette with a 10 mm path length (Hellma Analytics). Prior to sample analysis the spectrophotometer was first zero’d using a blank buffer solution.

[0147] Dye Efflux from Liposomes

[0148] Dye efflux experiments were used to assess the relative insertion frequencies and subsequent cargo translocation abilities of nanopores with and without encircling nanodiscs into spherical lipid membranes. POPC vesicles were prepared as described previously, with the lipid thin film being re-suspended in the DNA pore folding buffer supplemented with 50 mM sulfarhodamine B dye. After extrusion, the vesicles were purified from any external dye using a NAP-25 column (Cytivia). To perform the assay, 10 pL of the purified vesicles were added to 110 pL of 50 mM Tris. Cl, pH 7.5, supplemented with 500 mM KCI. Fluorescence was monitored at 586 nm and excited at 565 nm. Once the baseline had stabilised (~5 min), 30 pL of sample or control solution was added. To determine maximal fluorescence for normalisation, Triton-X100 was added to a final concentration of 0.1% to lyse the LU s. This assay was performed on an Eclipse fluorescence spectrometer (Agilent).

[0149] Schematic diagram describing the dye efflux experiment. Sulfarhoamine B (inset) is encapsulated within a vesicle (depicted as a grey membrane) at a self-quenching concentration. Addition of a nanopore (pictured, blue) allows the release of the dye into the bulk solution, lowering the effective concentration and causing the dye to fluoresce (Figure 3G). Fluorescent spectrogram showing release of encapsulated fluorescence dye from DPhPC LUVs upon addition of osmotically balanced buffer (grey trace), 200 nM of 6HB-3C (blue) or 100 nM of protein nanopore alpha-heamolysin (red, purchased from Sigma) (Figure 3H). Each trace is an average of three technical repeats and the coloured ribbon shows the standard error.

[0150] Representative chromatograms showing the SEC purification of the 6HB nanopore inside nanodiscs formed of a 2:1 ratio of the nanodisc forming polymer Styrene-Malic acid (SMA) and POPC (Figure 4A). DLS traces showing the purified products from the major peaks (retention time = 8.52 minutes) from (A) (Figure 4B). UV spectra of the purified products from (A). In all plots POPC:SMA(2:1)-6HB is depicted in a solid lilac line. For comparison, the data received when purifying and characterising POPC:SMA(2:1) in the absence of 6HB is plotted below in a dashed purple line (Figure 4C). Normalised fluorescent spectrograms showing the release of encapsulated dye (SRB) from DPhPC lipid vesicles upon addition of 6HB (200 nM, blue), SEC purified POPC:SMA(2:1) (5 pM wrt polymer concentration, purple), SEC purified POPC:SMA(2:1)-6HB (5 pM wrt polymer concentration, 77 nM wrt DNA concentration, lilac, or equivalent volumes of buffer (grey). (Figure 4D) Normalised fluorescence spectrograms showing the equilibration of pH across the membrane of H PTS filled block copolymer vesicles upon addition of 6HB, SEC purified POPC:SMA(2:1), SEC purified POPC:SMA(2:1)-6HB or equivalent volumes of buffer (grey). All fluorescent traces are an average of three technical repeats and the coloured ribbon shows the standard deviation (Figure 4E). Representative chromatograms showing the SEC purification of DIBMA:POPC(1 :1)-Ring (dark cyan). For comparison, the chromatograms received when purifying the Ring in solution in the absence of complementary cholesterol modified oligonucleotides (solid grey line) were also included (Figure 4F). DLS traces showing the purified products from the major peak (retention time = 8.14 minutes) from (F). (H) UV spectra of the purified products from (F) (Figure 4G). Normalised fluorescent spectrograms showing the release of encapsulated dye (SRB) from DPhPC lipid vesicles upon addition of Ring (8nM, dark cyan), SEC purified DIBMA:POPC(1 :1) (5 pM wrt polymer concentration, gold, denoted “Disc”), SEC purified DIBMA:POPC(1 :1)-Ring (5 pM wrt polymer concentration, 8.02 nM wrt DNA concentration, green, denoted “Ring-Disc”) or equivalent volumes of buffer (grey) (Figure 4I). Normalised fluorescence spectrograms showing the equilibration of pH across the membrane of HPTS filled block copolymer vesicles upon addition of Ring, SEC purified DIBMA:POPC(1 :1), SEC purified DIBMA:POPC(1 :1)-Ring or equivalent volumes of buffer. As with previous, each trace is an average of three technical repeats and the coloured ribbon shows the standard deviation (Figure 4J).

[0151] Example 3. Using Nanodiscs to insert DNA Nanopores into the minlON™, DNA sequencer from Oxford Nanopore Technologies.

[0152] Ion Influx to Polymersomes

[0153] Ion Influx experiments were used to assess the relative insertion frequencies and subsequent cargo translocation abilities of nanopores with and without encircling nanodiscs into spherical polymeric membranes. Polymer vesicles were prepared by re-suspending the dry copolymer film with a solution of 20 mM Phosphate buffer pH 6, 250 pM HPTS, 500 mM KCI. After extrusion, vesicles were freeze-thawed 10x before purification into a solution of the same buffer without HPTS using a NAP-25 column (Cytivia). The assay was performed using a Synergy2 plate reader (BioTek). 130 pL reactions were prepared in a flat bottomed black 96 well microplate (Corning) containing nanopore samples in solution of 1x TAE, pH 8.3 (Sigma), supplemented with 490 mM KCI Samples were first scanned at using an excitation filter of 400 / 30 and an emission filter of 485 / 20, then 20 pL of the LUV suspension was injected into each well using the reagent dispenser and the plate shaken rigorously for 1 second. Fluorescence was monitored using the above settings with the mirror set to top 50% and gain at 35. After 2.5 hours, the reagent dispenser was used to dispense 20 pL 10% Triton-X100 into each well, and the plate was shaken and rescanned to determine maximum fluorescence. minlON™ Current Recordings

[0154] The minlON™ was used to assess the ability of nanodisc-nanopore constructs to insert into planar polymer membranes, and asses their ability to interrogate analytes in a high- throughput manner such as might be used in a commercial device. minlON™ chips without pre-inserted nanopores were kindly provided by Oxford Nanopore Technologies (ONT). Samples were diluted in the provided minlON™ running buffer up to a maximum concentration of 1 / 60, which is 5 nM with regard to nanopore content. 300 pL was then applied to the flow-cell with a P1000 pipette via the sample entry port. After brief (sub 1 minute) incubation, voltage was applied run using in-house scripts. Data extraction and analysis was performed using R.

[0155] Schematic representation of nanodisc mediated insertion of the 6HB nanopore into the membrane array in the minlON™ from Oxford Nanopore Technologies (ONT) (Figure 5A). Time-averaged current- voltage curve from nanodisc inserted 6HB nanopore in a minlON™. The trace is an average of 10 traces and the coloured ribbon shows the standard deviation (Figure 5B). Current-Voltage (IV) analysis showing average current received through the 6HB nanopore when increasing voltage in 5 mV increments from -50 to 50 mV (Figure 5C). Schematic representation of nanodisc mediated insertion of the 6HB nanopore into the membrane array in the minlON™ from Oxford Nanopore Technologies (ONT) (Figure 5D). Histogram of conductance at -25 mV. The data plotted in grey represents multiple 6HB’s inserted in a single membrane and was omitted from (C). Comparison of insertion frequency when adding the 6HB directly to the minlON™, or when added using nanodiscs (Figure 5E). Schematic representation of nanodisc mediated insertion of the Ring nanopore into the membrane array in the minlON™ ONT (Figure 5F). Representative current trace from the small-state of the Ring nanopore in a minlON™ membrane (Figure 5G). Representative current trace for the large-state of the Ring nanopore in a minlON™ membrane (Figure 5H). IV analysis showing average current received through the large and small states of the Ring nanopore when increasing voltage in 5 mV increments from -50 to 50 mV (Figure 5I). Histogram of conductance at -25 mV. (K) Comparison of insertion frequency when adding the Ring nanopore directly to the minlON™, or when added using nanodiscs (Figure 5J).

[0156] Example 4. Analyte Translocation Through DNA Nanopores Inserted into minlON™ Membranes Using Nanodiscs Schematic diagram and representative current trace of nanodisc inserted 6HB nanopore in minlON™ membrane, recorded at 75 mV (Figures 6A, 6B), Histogram showing the frequency of translocation events, and analysis of the duration, ordwell time, of the translocation events recorded in the absence (top row) and presence (bottom row) of 500 nM of the Thrombin Binding Aptamer DNA oligonucleotide (Figures 6C, 6D). Schematic Diagram and Representative current trace of nanodisc inserted Ring nanopore in minlON™ membrane (Figures 6E, 6F), Histogram showing the frequency of translocation events, and analysis of the dwell time of translocation events recorded in the absence (top row), presence of 2.6 pM (middle row) and presence of 14.8 pM of the protease Trypsin. Representative current traces have been filtered with a low pass Bessel filter at 1 kHz (Figures 6G, 6H).

[0157] Example 5. Nanodisc Membrane Regeneration

[0158] Nanodiscs formed without a transmembrane nanopore can be used to regenerate burst or damaged membranes in a high-throughput flow cell or membrane array, such asin minlON™ chips from Oxford Nanopore Technologies (ONT). The induced change in membrane properties, from damaged to unilamellar membranes able to accommodate nanopores for sensing, is assessed by measuring the electrical membrane capacitance (Vreeker et al., 2025). Accordingly, membranes in the minlON™ chips designated as competent to accommodate nanopores are classified as “Good” or “Large”. The membrane-healing properties of nanodiscs is shown in Figure 7 when the addition of dilute solutions of a mixed preparation containing nanodiscs with and without transmembrane nanopores resulted in a two-fold increase in the number of membranes classified as “Good” or “Large”.

[0159] This experiment was repeated with a defective chip that appeared to have no “Good” membranes, with ~2000 membranes being designated as “oil seal” by the automatic classifier. After addition of the nanodisc solution, over 1000 “Good” membranes were available (Figure. 8).

[0160] Example 6. Using nanodisc to improve nanopore stability

[0161] The observed stabilising effects of nanodiscs on nanopores are threefold:

[0162] (i) Nanodiscs can prevent aggregation of nanopores in solution.

[0163] Nanopores typically have exposed hydrophobic regions, such as anchor moieties (e.g. cholesterol), required to insert into hydrophobic membranes. In aqueous solution, this can cause the nanopores to aggregate via the hydrophobic interaction between the regions. Aggregated nanopores cannot insert easily into flow cell sensor array chips. By contrast, nanodiscs provide a shielded environment around the hydrophobic perimeter of the pore, thereby reducing aggregation of nanopores (see Figure 9). The use of nanodiscs was observed to reduce aggregation of six helix barrel (6HB) nucleic acid nanopores in aqueous suspension by over 50%.

[0164] (ii) Nanodiscs can stabilise assembled nanopores for long term storage.

[0165] Pre-assembled nucleic acid based nanopores are at risk of limited stability given the oxidation of membrane anchors composed of cholesterol. However, unexpectedly it it has been shown that when incorporated into nanodiscs nanopores have been demonstrated to be stable for over 2 years at -80 °C, and could be inserted into flow cell sensor array membranes in a minlON™ device when thawed (see Figure 10). Without wishing to be bound by theory, it is postulated that within the nanodiscs, the cholesterol anchors are embedded within the shielding environment of a lipid membrane thereby reducing exposure to an oxidising environment.

[0166] (iii) Nanodiscs can stabilise nanopores within flow cell sensor array membranes by providing a highly localized native environment.

[0167] Commercial sequencing devices, such as the minlON™, use synthetic lipids within the membrane array for enhanced stability as well as improved mechanical and electrical properties. However, this can result in a non-native membrane environment that impacts the conductance properties of inserted biological nanopores. Inserting lipid nanodiscs into a membrane formed of larger synthetic membrane polymers, similar to those used in the minlON™, results in lipid islands which stay clustered together inside the target membrane (see Figure 11). Without being bound by theory, this is believed to be due to the difference in line tension between the different membrane components, as the nanodisc lipids will disperse if inserted into a synthetic membrane of similar line tension (Figure 11).

[0168] Example 8. Nanodisc Insertion of Biological Protein Nanopores

[0169] In the same manner as nanodiscs have been shown to insert synthetic DNA nanopores into flow cell sensor array membranes, they can also be used to insertion biological protein nanopores (Figure 12). In this example, alpha haemolysin (aHL) protein nanopore was inserted into lipid nanodiscs. The protein pore containing nanodisc was formulated with POPC lipids and the SMA nanodisc-forming polymer. The aHL nanopore-nanodiscs were prepared as per DNA nanopores (see Example 2) and purified by size exclusion chromatography. Figure 12B shows a Current-Voltage curve of the inserted nanopores showing rectification characteristic of aHL. The results indicated that over 200 aHL nanopores were successfully inserted into minlON™ membranes using lipid nanodiscs (see Figure 12C).

[0170] Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the appended claims, which follow. For example, the choice of starting material is believed to be a routine matter for the person of skill in the art with knowledge of the presently described embodiments. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

[0171] Reference List:

[0172] Quick, J., Loman, N.J., Duraffour, S., Simpson, J.T., Severi, E., et al. (2016) Real-time, portable genome sequencing for Ebola surveillance. Nature. 530 (7589), 228-232.

[0173] Lanphere, C. et al. (2021) ‘Design, assembly, and characterization of membrane-spanning DNA nanopores’, Nature Protocols, 16(1), pp. 86-130.

[0174] Howorka, S. (2017) ‘Building membrane nanopores’, Nature Nanotechnology, 12(7), pp. 619-630.

[0175] Diederichs, T., Pugh, G., Dorey, A., Xing, Y., Burns, J.R., Hung Nguyen, Q., Tornow, M., Tampe, R. & Howorka, S. (2019) Synthetic protein-conductive membrane nanopores built with DNA. Nature Communications. 10 (1), 5018

[0176] Martin-Baniandres, P., Lan, W.-H., Board, S., Romero-Ruiz, M., Garcia-Manyes, S., Qing, Y. & Bayley, H. (2023) Enzyme-less nanopore detection of post-translational modifications within long polypeptides. Nature Nanotechnology. 18 (11), 1335-1340

[0177] Burns, J.R., Stulz, E. and Howorka, S. (2013) Self-assembled dna nanopores that span lipid bilayers, Nano Letters, 13(6), pp. 2351-2356.

[0178] Bhatia, D., Surana, S., Chakraborty, S., Koushika, S.P. & Krishnan, Y. (2011) A synthetic icosahedral DNA-based host-cargo complex for functional in vivo imaging. Nature Communications. 2 (1), 339

[0179] Walsh, A. S., Yin, H., Erben, C. M., Wood, M. J., Turberfield, A. J. (2011) DNA cage delivery to mammalian cells. ACS Nano 5, 5427-5432.

[0180] Offenbartl-Stiegert, D., Rottensteiner, A., Dorey, A. & Howorka, S. (2022) A Light-Triggered Synthetic Nanopore for Controlling Molecular Transport Across Biological Membranes. Angewandte Chemie. 134 (52), e202210886. Arulkumaran, N., Lanphere, C., Gaupp, C., Burns, J.R., Singer, M. & Howorka, S. (2021) DNA Nanodevices with Selective Immune Cell Interaction and Function. ACS Nano. 15 (3), 4394-4404.

[0181] Burns, J. R., Howorka, S. (2018) Defined bilayer interactions of DNA nanopores revealed with a nuclease-based nanoprobe strategy. ACS Nano, 12, 3263-3271.

[0182] Winterstein, L.-M., Kukovetz, K., Rauh, O., Turman, D.L., Braun, C., Moroni, A., Schroeder, I. & Thiel, G. (2018) Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers. Journal of General Physiology. 150 (4), 637-646.

[0183] Vreeker, E., Griinewald, F., Heide, N.J. van der, Marrink, S.-J., Tych, K. (Kasia) & Maglia, G. (2024) Hybrid lipid-block copolymer membranes enable stable reconstitution of a wide range of nanopores and robust sampling of serum.

[0184] Banerjee, S. & Nimigean, C.M. (2011) Non-vesicular transfer of membrane proteins from nanoparticles to lipid bilayers. Journal of General Physiology. 137 (2), 217-223.

[0185] Umbach, S., Levin, R., Neumann, S., Steinmetzer, T., Dbtsch, V. & Bernhard, F. (2022) Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells. Frontiers in Bioengineering and Biotechnology. 10, 906295.

[0186] Vreeker E, Griinewald F, van der Heide NJ, Bonini A, Marrink SJ, Tych K, Maglia G. Nanopore-Functionalized Hybrid Lipid-Block Copolymer Membranes Allow Efficient SingleMolecule Sampling and Stable Sensing of Human Serum. Advanced Materials. 2025 Apr;37(15):2418462.

Claims

Claims1 . A method of making a nanodisc composition, comprising: a. contacting lipid or synthetic lipid analogues with one or more nanopores in aqueous solution, thereby forming a membrane vesicle that encompasses the one or more nanopores embedded within the membrane vesicle; b. contacting the one or more membrane vesicles with a nanodisc forming polymer; c. generating a nanodisc composition wherein the nanodisc forming polymers solubilise the membrane vesicle and encircle the one or more nanopores embedded within the membrane vesicle so as to form a plurality of nanodiscs.

2. The method of claim 1 , wherein the vesicle comprises first and second opposing hydrophilic faces and a hydrophobic edge defined between the first and second hydrophilic faces.

3. The method of claim 1 , wherein the vesicle comprises a semi-fluid lipid membrane.

4. The method of claim 3, wherein the semi-fluid lipid membrane comprises natural or synthetic lipids.

5. The method of claim 3, wherein the semi-fluid lipid membrane comprises lipid-like polymers.

6. The method of claim 3, wherein the semi-fluid lipid membrane comprises at least one lipid bilayer.

7. The method of claim 6, wherein the semi-fluid lipid membrane comprises a phospholipid, optionally selected from a phosphatidylcholine (POPC) or 1 ,2-diphytanoyl-glycero-3- phosphocholine.

8. The method of claim 1 , wherein the nanodisc forming polymers comprise amphipathic copolymers.

9. The method of claim 8, wherein the amphipathic copolymers comprise both hydrophilic and hydrophobic component groups.

10. The method of claim 9, wherein the hydrophobic and hydrophilic groups are distributed within the nanodisc forming polymers.

11. The method of claim 1 , wherein the nanodisc forming polymers comprise maleic acid copolymers.

12. The method of claim 11 , wherein the maleic acid copolymers are selected from copolymers comprising: styrene-maleic acid (SMA) and / or di-isobutylene-maleic acid (DIBMA).

13. The method of claim 1 , wherein the nanodisc forming polymer comprises scaffold protein.

14. The method of claim 13, wherein the scaffold protein comprises non-structural protein family (Nsp).

15. The method of claim 1 , wherein the nanopores comprise a protein nanopore, a nucleic acid nanopore, or a hybrid nanostructure that is embedded within a membrane and allows passing of molecular substances or analytes across the membrane via a central channel.

16. The method of claim 15, wherein the protein nanopore comprises a protein channel or protein engineered variants thereof.

17. The method of claim 15, wherein the nucleic acid nanopore comprises DNA and / or RNA.

18. The method of claim 17, wherein the nucleic acid nanopore is a computationally designed synthetic nanopore comprising polynucleotides with hydrophobic modifications, optionally wherein the hydrophobic modifications comprise one or more hydrophobic anchor molecules.

19. The method of claim 1 , wherein the nanopore comprises a membrane-spanning nanopore that defines a central channel.

20. The method of claim 19, wherein the central channel has a minimum internal width of at least 0.5nm, optionally at least 0.8 nm.21 . The method of claim 1 , wherein the nanodisc forming polymers solubilise the one or more membrane vesicles by simultaneously contacting hydrophobic components within hydrophobic membrane vesicles, and hydrophilic components within aqueous solution.

22. The method of claim 1 , wherein the nanodisc is not in contact with the membrane vesicle.

23. A nanodisc composition comprising a semi-fluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the nanodisc composition further comprises one or more nanopores embedded within the semi-fluid lipid membrane.

24. The nanodisc composition of claim 23, wherein the semi-fluid lipid membrane comprises lipids or synthetic lipid analogues.

25. The nanodisc composition of any preceding claim, wherein the semi-fluid lipid membrane retains components of membrane vesicles.

26. The nanodisc composition of any preceding claim, wherein the semi-fluid lipid membrane enriches some components of membrane vesicles.

27. The nanodisc composition of any preceding claim, wherein the nanodisc forming polymers encircle a hydrophobic edge of the lipid membrane.

28. The nanodisc composition of any preceding claim, wherein the nanodisc has a diameter of at least 10nm, at least 25nm, at least 50nm, or at least 10Onm.

29. The nanodisc composition of claims 23 to 28, wherein the nanopore 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 portion of the at least one scaffold polynucleotide strand.

30. A nanodisc comprising a semi-fluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the one or more NFPs comprise an amphipathic polymer and / or copolymer; wherein the semi-fluid lipid membrane comprises first and second opposing hydrophilic faces and a hydrophobic edge defined between the first and second hydrophilic faces, and the NFPs encircle the hydrophobic edge of the lipid membrane; and wherein the nanodisc further comprises one or more nanopores embedded within the semifluid lipid membrane.31 . The nanodisc of claim 30, wherein one or more nanopores comprise a membrane spanning nanopore, optionally wherein the membrane spanning nanopore is comprised of a nucleic acid or a polypeptide.

32. A composition comprising a plurality of nanodiscs according to claims 30 or 31 in liquid suspension.

33. Use of a nanodisc as defined in claim 30, as a delivery vehicle for insertion of one or more nanopores into a target membrane.

34. The use of claim 33, wherein the nanopores comprises a membrane-spanning nanopore that defines a membrane-spanning channel.

35. The use of claim 33, wherein the nanodisc facilitates insertion of the nanopore into the target membrane by fusing nanodisc semi-fluid membrane with the target membrane.

36. The use of claim 33, wherein the nanodisc facilitates insertion of the nanopore into an aperture within a solid-state membrane.

37. The use of any of claims 33 to 36, wherein the target membrane is comprised within a membrane array.

38. The use of claim 37, wherein the target membrane is a synthetic polymer membrane.

39. The use of claim 33, wherein the membrane array is comprised within a sensor device.

40. The use of claim 39, wherein the sensor device is a nanopore-based sensor device.

41. A method of increasing the frequency of insertion of a nanopore into a target semi-fluid synthetic membrane, comprising using a nanodisc composition as defined in claim 30, as a delivery vehicle to increase the frequency of insertion of nanopores into a membrane array.

42. The method of claim 41 , wherein the method further comprises removal of nanodisc forming polymers from the nanopores in the process of insertion of nanopores into a membrane array.

43. The method of claim 41 , wherein the nanodisc forming polymers precipitate in the presence of divalent cations.

44. The method of claim 41 , wherein the nanodisc forming polymers precipitate without any detergent.

45. The method of claim 41 , wherein the concentration of Mg2+is at least 0.5mM, 1 mM, or 5mM.

46. The method of claim 41 , wherein a single nanopore is inserted into each membrane of the membrane array.

47. The method of claim 41 , wherein the semi-fluid membrane of the nanodisc merges with the membrane of the membrane array and generates a hybrid membrane.

48. The method of claim 47, wherein the hybrid membrane, the relative area of the nanodisc semi-fluid membrane to the membrane array is at least 1 :50, 1 :100, or 1 :200.

49. The method of claim 41 , wherein the membrane array comprises at least 1024, 2048, or 4096 membranes.

50. The method of claim 41 , wherein the membrane array comprises a synthetic lipid analogue.51 . The method of claim 41 , wherein the frequency of insertion is increased by at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold relative to direct insertion of nanopores in the absence of a nanodisc delivery vehicle.

52. A method of increasing the frequency of insertion of a nanopore into a target solid-state membrane, comprising using a nanodisc composition as defined in claim 30, as a delivery vehicle to increase the frequency of locating a plurality of nanopores into a solid-state membrane array.

53. A method for repairing a semi-fluid lipid membrane of the nanodisc composition of any one of claims 23 to 29 or the nanodisc of claims 30 or 31 , comprising: contacting a composition of any one of claims 23 to 29 or a nanodisc of claims 30 or 31 with a repair aqueous solution comprising a composition of repair nanodiscs, wherein the repair nanodiscs comprise a semi-fluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the one or more NFPs comprise an amphipathic polymer and / or copolymer, and wherein the semi-fluid lipid membrane comprises first and second opposing hydrophilic faces and a hydrophobic edge defined between the first and second hydrophilic faces, and the NFPs encircle the hydrophobic edge of the lipid membrane.

54. The method of claim 53, wherein the repair nanodiscs do not comprise a nanopore embedded within the semi-fluid lipid membrane.

55. A method for regenerating a high-throughput flow cell or membrane array configured with a plurality of nanopore sensors, the method comprising contacting a high-throughput flow cell or membrane array with an aqueous repair solution comprising a composition of repair nanodiscs, wherein the repair nanodiscs comprise a semi-fluid lipid membrane enclosed by one or more nanodisc forming polymers (NFPs), wherein the one or more NFPs comprise an amphipathic polymer and / or copolymer, and wherein the semi-fluid lipid membrane comprises first and second opposing hydrophilic faces and a hydrophobic edge defined between the first and second hydrophilic faces, and the NFPs encircle the hydrophobic edge of the lipid membrane.

56. A method of preparing a repair composition for regenerating a high-throughput flow cell or a membrane array comprising a plurality of nanopore sensors, the method comprising, contacting lipid or synthetic lipid analogues with an aqueous solution thereby forming one or more membrane vesicles; contacting the one or more membrane vesicles with a nanodisc forming polymer; and generating a repair composition wherein the nanodisc forming polymers solubilise the membrane vesicle and encircle the lipid or synthetic lipid analogues comprising the membrane vesicle so as to form a plurality of nanodiscs in suspension.

57. A repair nanodisc composition obtainable or obtained by the method of claim 56.

58. Use of the repair nanodisc composition of claim 57 for regenerating a high-throughput flow cell or a membrane array device comprising a plurality of nanopore sensors.

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