Methods and compositions for the assembly of biological nanopores

Abstract

The present invention provides methods and compositions for making and using detection devices based on one or more native biological nanopores. Use includes, but is not limited to, the detection and sequencing of nucleic acids. The present invention generally relates to new methods and compositions for preparing protein-based nanopore sensors, and more specifically, to methods for assembling native nanopore proteins in lipid nanodiscs, which serve as carriers to deliver the nanopores to the lipid membrane component of the sensor system, and to methods of using them, particularly in nanopore-based nucleic acid sequencing methods.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 928,207, filed Oct. 30, 2019, under 35 U.S.C. § 119(e); the entire disclosure of which is incorporated herein by reference for all purposes. Technical field

[0003] The present invention generally relates to new methods and compositions for preparing protein - based nanopore sensors, and more specifically, to methods for assembling native nanopore proteins in lipid nanodiscs, which serve as carriers to deliver the nanopores to the lipid - membrane component of the sensor system, and to methods of using them, particularly in nanopore - based nucleic acid sequencing methods. Background art

[0004] In the past two decades, nanopore sensors have emerged as a powerful tool and have had a huge impact on science and biotechnology. Nanopore technology is generally classified into biological nanopores and solid - state nanopores according to their materials. Conventionally, solid - state nanopores are fabricated by drilling nanoscale holes using semiconductor or microfluidic techniques, such as ion or electron beam lithography in silicon - or graphene - based membranes (such as Si, SiN or SiO 2 )). However, most nanopore applications, such as DNA sequencing, small - molecule sensing, drug screening, molecular sieving, and biomolecule analysis, require high - precision geometries, sensitivity, and reproducibility, which are not achievable with solid - state pores.

[0005] DNA sequencing is the most important and highly anticipated application of nanopores. However, the translocation speed of DNA through nanopores is very fast (multiple nucleotides pass through the nanopore in a few microseconds), and the main problems associated with this result in few data points per base, which hinders further analysis of the data. To address such problems, Stratos Genomics has developed a method called Sequencing by Expansion (“SBX”), which uses a biochemical process to transcribe DNA sequences onto a measurable polymer called an “Xpandomer” (see, for example, Kokoris et al., U.S. Pat. No. 7,939,259, “High Throughput Nucleic Acid Sequencing by Expansion”). The transcribed sequences are encoded in high signal-to-noise ratio reporters along the Xpandomer backbone, which are spaced approximately 10 nm apart and are designed for high signal-to-noise ratio, well-differentiated responses. These differences provide a significant performance enhancement in terms of the sequence read efficiency and accuracy of the Xpandomer relative to native DNA. The Xpandomer enables a variety of next-generation DNA sequencing detection techniques and is well-suited for nanopore sequencing.

[0006] α-Hemolysin (α-HL) is the most widely used biological nanopore for single molecule analysis, mainly due to its small inner diameter and structural reproducibility. α-HL is a monomeric polypeptide that self-assembles in a lipid bilayer membrane to form a transmembrane heptameric pore, which has a vestibule with a diameter of 2.6 nm and a constriction pore with a diameter of 1.5 nm (the narrowest point of the pore). The constriction pore of the α-HL nanopore allows linear molecules with dimensions on the same order of magnitude as single-stranded DNA to pass through or “translocate”; however, molecules with a diameter greater than approximately 2.0 nm, such as double-stranded DNA, are blocked from translocation. Although α-HL (and other oligomeric, transmembrane protein nanopores) have advantages in DNA sequencing, they still have inherent structural limitations, for example, due to the reduced stability of native oligomers in aqueous solution and the incomplete assembly of native proteins in lipid membranes, thus requiring improved methods and compositions for preparing biological nanopore sensors.

[0007] The present invention meets these needs and provides further related improved advantages as described below.

[0008] All topics discussed in the background section are not necessarily prior art and should not be assumed to be prior art merely because of their discussion in the background section. Along these lines, unless expressly stated to be prior art, any recognition of problems in the prior art discussed in the background section or related to such topics should not be regarded as prior art. Instead, the discussion of any topic in the background section should be considered part of the inventors' approach to solving a particular problem, which may itself be creative. Summary of the Invention

[0009] The details of one or more embodiments are set forth in the accompanying drawings and the following description. Other features, objects, and advantages will be apparent from the description, the drawings, and the claims.

[0010] Briefly, the present disclosure provides methods and compositions for improving the fabrication of nanopore-based sensors. In certain embodiments, the methods and compositions enable the fabrication of bio-nanopore-based sensors using improved assimilation of native nanopore structures.

[0011] In one aspect, the present invention provides a method of preparing a detection device comprising one or more native nanopore proteins, the method comprising the steps of: (a) forming an aqueous mixture comprising a nanopore protein, a membrane scaffold protein (MSP), and a first lipid to produce a sample of a nanodisc-nanopore protein complex, wherein the population of nanodisc-nanopore protein complexes in the sample each comprises a native nanopore protein; (b) providing a solid support comprising one or more orifices, wherein a membrane is formed over each of the orifices, wherein the membrane comprises a second lipid, and wherein the membrane separates a cis chamber from a trans chamber in the detection device; and (c) contacting one or more of the membranes with a population of nanodisc-nanopore complexes comprising a native nanopore protein to assimilate the native nanopore protein into each of the membranes. In one embodiment, the method further comprises the step of purifying the population of nanodisc-nanopore complexes comprising a native nanopore protein from the aqueous mixture prior to the step of contacting one or more of the membranes with the population of nanodisc-nanopore complexes comprising a native nanopore protein. In a further embodiment, the step of purifying the population of nanodisc-nanopore complexes comprising a native nanopore protein comprises one or both of size exclusion chromatography and affinity chromatography. In another embodiment, the aqueous mixture further comprises a detergent, wherein the final concentration of the detergent is from about 14 mM to about 40 mM. In a further embodiment, the first lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), the MSP is MSP1D1 or a variant thereof, the nanopore protein is α-hemolysin (α-HL) or a variant thereof, the detergent is cholate, and the second lipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE). In yet a further embodiment, the molar ratio of lipid to MSP to nanopore protein is about 101:6:1 or about 120:6:1. In another embodiment, the solid support comprises a plurality of orifices, wherein a membrane is formed over each of the plurality of orifices, and each of the membranes is contacted with a nanodisc-nanopore complex comprising a native nanopore protein.

[0012] In another aspect, the present invention provides a method for sequencing a polymer, the method comprising the use of any of the above-described detection devices. In certain embodiments, the polymer is Xpandomer.

[0013] In another aspect, the present invention provides a method for forming a native nanopore protein in a membrane, the method comprising the steps of: (a) forming an aqueous mixture comprising a nanopore protein, a membrane scaffold protein (MSP), and a first lipid to produce a sample of a nanodisc-nanopore protein complex, wherein the population of nanodisc-nanopore protein complexes each comprise a native nanopore protein; (b) providing a membrane comprising a second lipid; (c) contacting the membrane with the population of nanodisc-nanopore complexes comprising the native nanopore protein to assimilate the native nanopore protein into the membrane. In one embodiment, the method further comprises the step of purifying the population of nanodisc-nanopore complexes comprising the native nanopore protein from the aqueous mixture prior to the step of contacting the membrane with the population of nanodisc-nanopore complexes comprising the native nanopore protein. In certain embodiments, the step of purifying the population of nanodisc-nanopore complexes comprises one or both of size exclusion chromatography and immobilized metal affinity chromatography. In another embodiment, the aqueous mixture further comprises a detergent, wherein the final concentration of the detergent is from greater than 14 mM to 40 mM. In a further embodiment, the first lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), the MSP is MSP1D1 or a variant thereof, the nanopore protein is α-hemolysin (α-HL) or a variant thereof, the detergent is cholate, and the second lipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE). In yet another embodiment, the molar ratio of lipid to MSP to nanopore protein is about 101:6:1 or about 120:6:1.

[0014] In another aspect, the present invention provides a composition comprising a nanodisc-nanopore complex in an aqueous buffer, wherein the nanodisc-nanopore complex comprises a native nanopore protein, a membrane scaffold protein (MSP), and a lipid, and wherein the aqueous buffer comprises a detergent. In one embodiment, the native nanopore protein is α-hemolysin (α-HL) or a variant thereof, the MSP is MSP1D1 or a variant thereof, the lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), and the detergent is cholate. In a further embodiment, the molar ratio of lipid to MSP to nanopore protein is about 101:6:1 or about 120:6:1, and the concentration of cholate is from greater than 14 mM to 40 mM.

[0015] In another aspect, the present invention provides a composition comprising a lyophilized powder, the lyophilized powder comprising a nanopore-nanodisc complex, wherein the nanopore-nanodisc complex comprises a native nanopore protein, a membrane scaffold protein (MSP), and a lipid. In one embodiment, the native nanopore protein is α-hemolysin (α-HL) or a variant thereof, the MSP is MSP1D1 or a variant thereof, and the lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). In a further embodiment, the molar ratio of lipid to MSP to nanopore protein is about 101:6:1 or about 120:6:1. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a flowchart illustrating one embodiment of a method of preparing a biosensor nanopore-based detection system.

[0017] Figure 2A 、 2B Figures 2C and 2D are schematic diagrams illustrating the main features of a general XNTP and its use in extended sequencing (SBX).

[0018] Figure 3 is a SEC trace showing the elution of sample A 280 as a function of time.

[0019] Figure 4 is a gel showing protein samples taken at different stages of the nanopore purification process. DETAILED DESCRIPTION

[0020] The present invention can be more readily understood by reference to the following detailed description of the preferred embodiments of the invention and the examples included herein. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined.

[0021] Biological nanopore proteins for use in the field of nucleic acid sequencing have been found to include those based on native transmembrane proteins that form pores when individual polypeptide subunits self-assemble in a membrane and oligomerize into their native higher-order structures. Conventional biological nanopore sensors (e.g., αHL nanopores) are typically assembled by applying an aqueous solution of the dissolved protein to a micron-scale membrane assembly of the detector system. To form a functional nanopore, the soluble protein subunits must insert into the membrane and correctly self-assemble to form the native higher-order structure. Reconstituting native membrane proteins in a lipid bilayer presents several technical challenges due to, for example, low solubility and stability of the protein in aqueous solution and difficulty in efficiently and consistently assembling the appropriate native structure in the lipid substrate. The present disclosure addresses these challenges by providing methods and compositions for fabricating nanopore sensors, wherein the native oligomeric nanopore structure is assembled in lipid nanodiscs prior to assimilating the nanopore in the membrane. Nanodiscs incorporating the native nanopore protein can optionally be purified from the mixture (e.g., complexes having an appropriate size and / or incorporating a heterologous detection “tag”) to provide a more uniform sample of native nanopores. The purified nanopore-nanodisc complex can then be applied to a lipid bilayer membrane so that the native protein structure is assimilated into the membrane to form a functional nanopore sensor or detector. An additional advantage provided by the present invention is that the structure of the native nanopore protein is very stable when formed in nanodiscs, thus providing an improved composition for, e.g., the storage and transport of the native nanopore protein.

[0022] Nanodisc technology is well known in the art. In some embodiments, a nanodisc is a nanoscale discoidal phospholipid bilayer that is stabilized and made water-soluble by two surrounding amphipathic helical proteins “bands” called membrane scaffold proteins (MSPs). Nanodiscs can be used as mediators for incorporating a target membrane protein (MP) into the bilayer to maintain the MP structure and activity, and have traditionally been used for biophysical, enzymatic, or structural studies of MPs (reviewed, e.g., in Bayburt and Sligar, FEBS Lett.;584(9):1721-1727 (2010)). In this method, the target membrane protein and / or phospholipids are transiently solubilized using a detergent in the presence of the surrounding amphipathic helical MSP. When the detergent is removed by dialysis or adsorption to hydrophobic beads, the target MP simultaneously assembles with the phospholipids into a discoidal bilayer, the size of which is controlled by the length of the MSP. Thus, the resulting nanodiscs hold the membrane protein in solution, providing a native-like phospholipid bilayer environment that provides the stability and functional requirements for incorporation of the target, and also allows control of the oligomeric state of the target membrane protein. Nanodiscs are known to be robust and can be frozen or lyophilized with the incorporated MP. As further discussed herein, the inventors have found that nanodiscs offer several advantages as mediators for nanopore delivery and storage.

[0023] As used herein, the term "membrane scaffold protein" refers to a protein that can stabilize the phospholipid bilayer in a nanodisc by binding to the bilayer periphery. Generally, a membrane scaffold protein has a hydrophobic surface that can associate with the nonpolar interior of the phospholipid bilayer and a hydrophilic surface that can preferably interact with a polar solvent such as an aqueous buffer. The membrane scaffold protein sequence can be naturally occurring, or can be engineered or de novo constructed using recombinant techniques. Naturally occurring membrane scaffold proteins include apolipoproteins, which are components of lipoproteins. Known classes of apolipoproteins include: A (including, for example, apo A-I and apo A-II), B, C, D, E, and H. Non-naturally occurring membrane scaffold proteins include MSP1 and MSP2 described in U.S. Patent No. 7,691,414, the entire contents of which are incorporated herein by reference in their entirety. An exemplary commercially available non-naturally occurring MSP is MSP1D1 available from, for example, Sigma. The membrane scaffold protein can be a full-length protein, or a truncated form of the protein. The membrane scaffold protein is not intended to encompass various functional membrane proteins, including but not limited to ion channels and other transmembrane receptors, porins, certain cell adhesion molecules, and electron transfer proteins such as NADH dehydrogenase and ATP synthase.

[0024] As used herein, the term "nanopore protein" refers to polypeptide subunits and polymers of the subunits that, when the appropriate higher-order structure forms, can produce an aperture through a membrane. A nanopore protein can refer to a single polypeptide subunit of a polymeric nanopore protein or different oligomeric forms of a single polypeptide subunit. A "mixture of nanopore proteins" refers to a solution that can contain a heterogeneous combination of single and / or oligomeric forms of a nanopore protein. A "native nanopore protein" refers to the native, higher-order state of subunit oligomerization that can form a functional nanopore in a membrane. Exemplary nanopore proteins (i.e., biological nanopores) include α-hemolysin, Mycobacterium smegmatis porin A (MspA), aerolysin, phi29, gramicidin A, maltoporin, OmpG, OmpF, OmpC, Vibrio cholerae cytolysin, PhoE, Tsx, and F-pili.

[0025] A preferred nanopore protein is α-hemolysin (α-HL). α-HL is the major cytotoxic agent released by Staphylococcus aureus and is the first identified member of the pore-forming β-barrel toxin family. This toxin consists mainly of β-sheets (68%) and only about 10% α-helices. The hla gene on the Staphylococcus aureus chromosome encodes a 293-residue protein monomer that forms a heptameric oligomer in the cell membrane to form a complete β-barrel pore. Thus, the native α-HL nanopore protein is an assembly of seven α-HL protein monomers, i.e., an oligomer.

[0026] Conventional biological mutagenesis can be used to optimize any protein component of the nanopore-nanodisc complex for use in the compositions or methods described herein. In some embodiments, the process of isolating the nanodisc-nanopore complex can benefit from a polyhistidine affinity tag (i.e., "His-tag") linked to the MSP or nanopore protein, which is used to purify the complex on an immobilized metal affinity column (e.g., on a nickel affinity column). α-HL or MSP with a terminal 6x His tag can be expressed, recombinantly produced, and purified as shown by SDS-PAGE gels. The biological function of the purified His-tagged protein is expected to be similar to that of the untagged protein. Other mutations can also be introduced for purification purposes. For example, cysteine moieties can be introduced into the protein sequence by mutagenesis and used for chemical conjugation to the thiol-reactive moiety of an affinity tag (e.g., maleimide or iodoacetamide). Exemplary affinity tags include biotin (which can mediate purification via solid-phase streptavidin), DNA and RNA (which can mediate purification via solid-phase nucleic acids with complementary sequences), epitopes (which can mediate purification via solid-phase antibodies or antibody fragments), or other ligands (which can mediate purification via the solid-phase receptors for those ligands).

[0027] Protein engineering and mutagenesis techniques can be used to alter the structure of biological pores and customize their properties for specific applications. In certain embodiments, α-hemolysin can, for example, be mutated inside the pore to produce variants with improved stability and / or with altered surface charge to optimize the detection of target analytes. Suitable α-HL variants include those disclosed in the published PCT applications WO2016069806, WO2018002125, and WO2019166458, as well as U.S. Patent Nos. 15,274,770 and 10,351,908, which are incorporated herein by reference. In certain embodiments, suitable α-HL variants can include one or more of the following mutations: A1K / R, D2N, S3K, D4K / N, K8R, T12K / R, N17K / R, D24A, V26D, H35D / E / G / L, K37S, N47K, E70K, S99K, Y101D, S106K, T109K, E111N / S, M113A / S, D127G, D128G / K, T129G, T131G, L135I, T145S, K147N / S, V149K, P151K, T233R, E287R, and M298A.

[0028] As used herein, a "membrane" is a component of a sensor or detection system or device, and not a component of a nanopore-nanodisc complex. The membrane is a thin film that separates two compartments or reservoirs (e.g., a cis compartment and a trans compartment) and prevents the free diffusion of ions and other molecules between the two compartments or reservoirs. Suitable membranes are amphiphilic layers formed from amphiphilic molecules, i.e., molecules that have both hydrophilic and lipophilic properties. Such amphiphilic molecules can be naturally occurring (e.g., phospholipids) or synthetic. Exemplary amphiphilic materials include various phospholipids such as 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), palmitoyl-oleoyl-phosphatidyl-choline (POPC), dioleoyl-phosphatidyl-methylester (DOPME), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin. Exemplary synthetic amphiphilic molecules include, for example, poly(n-butyl methacrylate-phosphocholine), poly(amide ester)-phosphocholine, poly(lactide)-phosphocholine, polyethylene glycol-poly(ε-caprolactone) di- or triblock, polyethylene glycol-poly(lactide) di- or triblock, and polyethylene glycol-poly(lactide-co-glycolide) di- or triblock.

[0029] Preferably, the membrane is a lipid bilayer. The lipid bilayer is a model of the cell membrane and has been widely used for various experimental purposes. The membrane can also be a solid-state membrane, i.e., a layer prepared from a solid material in which one or more orifices are formed. The membrane can be a single layer (such as a coating or thin film on a support substrate), or it can be a freestanding element. Examples of materials for thin film solid-state membranes include silicon nitride, aluminum oxide, titanium oxide, and silicon oxide.

[0030] Figure 1Summarizes three basic steps of an exemplary method for forming a nanopore sensor assembly for a detection device according to the present invention; details of each step are further discussed herein. In step 1, an aqueous mixture of a nanopore protein, a suitable lipid, and a suitable membrane scaffold protein is formed to provide a sample of nanopore-nanodisc complexes. In one embodiment, the nanopore is α-HL, the suitable lipid is DPhPC, and the membrane scaffold protein is MSPD1. The sample includes a population of nanopore-nanodisc complexes, each nanopore-nanodisc complex containing a native nanopore protein; however, not every complex in the sample necessarily includes a correctly assembled native nanopore protein, and thus, in some embodiments, the sample can be described as a "heterogeneous sample", and it may be advantageous to perform one or more purification steps to provide a sample enriched in the target nanopore-nanodisc complexes. In steps 2A and 2B, nanopore-nanodisc complexes having appropriate physical properties can optionally be separated or purified from the aqueous mixture. In this embodiment, two consecutive purification steps are performed: size exclusion chromatography (SEC, step 2A) and immobilized metal affinity chromatography (IMAC, step 2B). The purification steps enrich the population of nanopore-nanodisc complexes that include the native nanopore protein. It should be understood that any suitable purification protocol known in the art can be applied according to the methods described herein. In step 3, the purified nanopore-nanodisc complexes are applied to a lipid bilayer assembly (i.e., membrane) of a detection cell to assimilate the native nanopore protein into the membrane, thereby forming a functional nanopore sensor. Advantageously, according to this method, a sample enriched in native, oligomeric proteins is applied to the membrane, thereby increasing the efficiency of forming a functional sensor. In contrast, prior art methods require correct in-membrane self-assembly of protein subunits to form native higher-order structures, which is a less efficient and potentially error-prone process that can compromise the function of the detection system.

[0031] In some embodiments, the nanopore-nanodisc complex can be, for example, a lipid bilayer disc with a diameter of 7 to 16 nm stabilized by a membrane scaffold protein (MSP). In some embodiments, the MSP is a suitable derivative of apoA-I, such as the commercially available MSP1D1 protein. The present invention contemplates other types of amphiphilic nanodisc "bands", such as amphiphilic peptides. It should be understood that the diameter of the nanopore-nanodisc complex can be less than 7 nm (e.g., less than 6 nm, 5 nm, 4 nm, 2 nm or smaller) or greater than 16 nm (e.g., greater than 18 nm, 20 nm or 25 nm or larger). Generally, the lipid disc area for the methods or compositions described herein is not greater than about 50,000 nm 2 , or in some cases not greater than about 10,000 nm 2 or sometimes not greater than about 1,000 nm 2or even at other times not greater than about 500 nm 2 。The nanopore - nanodisc complex may, but does not necessarily need to, occupy a circular area. Under certain conditions, the nanopore - nanodisc complex can be distinguished from vesicles or liposomes because nanodiscs do not have an aqueous cavity, and can be distinguished from micelles because nanodiscs have a bilayer. It should be understood that the nanopore - nanodisc complex can also be made of other materials. For example, nanodiscs can be formed from non - lipid membranes. Those skilled in the art will understand that the optimal physical properties of nanodiscs will be determined by a specific target application, such as the physical properties of the target protein and other components of the system into which the target protein is incorporated. For example, when applying a nanopore - nanodisc assembly to a lipid bilayer, the nanodisc incorporating the nanopore protein should have dimensions suitable for maintaining the membrane solubility and transmembrane pore structure of the native protein. In certain embodiments, where the nanodisc is assembled with DPhPC, MSP1D1, and α - HL, the diameter of the complex is expected to be about 9.7 nm and the thickness is about 4.6 to 5.6 nm.

[0032] As described herein, lipid nanodiscs can consist of a lipid bilayer molecule surrounded by two parallel belt - shaped MSPs, where the amphiphilic helix of MSP stabilizes the hydrophobic fatty acids at the edge of the lipid disc. Particularly useful lipid nanodiscs and compositions and methods for their manufacture are set forth in, for example, U.S. Patent Nos. 7,083,958 and 7,662,410, which are incorporated herein by reference. In certain embodiments of the present invention, useful lipids for forming nanodiscs include 1,2 - diphytanoyl - sn - glycero - 3 - phosphocholine (DPhPC) and 1,2 - dimyristoyl - sn - glycero - 3 - phosphocholine (DMPC).

[0033] In certain embodiments, lipid nanodiscs can be prepared by mixing MSP with detergent-solubilized phospholipids. As described herein, self-assembly of the nanodiscs occurs during removal of the detergent from the mixture. It has been demonstrated that the presence of MSP limits the shape and size of the lipid nanodiscs and provides a narrow size distribution (+ / -3%) in detergent-free aqueous solution, excellent reproducibility, and extraordinary stability. The ratio of MSP to detergent can be selected to achieve the desired size and properties of the nanodiscs. For example, the number of structural units of MSP can be varied to adjust the nanodisc diameter from 9.8 nm to 12.9 nm, as described in Denisov et al., J. Am. Chem. Soc. 126, 3477-3487 (2004), which is incorporated herein by reference. Exemplary methods for incorporating membrane proteins into nanodiscs are described in Raschle et al., J. Am. Chem. Soc. 131, 17777-17779 (2009). Similar methods can be used to insert protein nanopores (such as α-HL, MspA, aerolysin, etc.) into lipid nanodiscs. In some embodiments, the nanopore-nanodisc complex is formed in an aqueous buffer consisting of 20 mM Tris, pH 7.4, 0.5 M EDTA, 100 mM NaCl, and 14 mM to 40 mM cholate. In certain embodiments, the buffer contains 19 mM cholate. In some embodiments, DPhPC lipid is added to an aqueous mixture in a solution containing 50 mM DPhPC, 20 mM Tris, pH 7.4, and 100 mM sodium cholate. In some embodiments, the final concentration of cholate in the nanodisc assembly reaction is greater than about 14 mM; in one specific embodiment, the final concentration of cholate is about 19 mM.

[0034] In specific embodiments, the nanopore-nanodisc complex is formed in a mixture of membrane scaffold protein (MSP), detergent-solubilized phospholipids (such as DPhPC), and nanopore protein. In this mixture, MSP self-assembles with the detergent-solubilized phospholipids to form nanodiscs that embed the α-HL nanopore protein. Self-assembly occurs when the detergent is removed from the mixture, for example, using Bio- (Bio-Rad, Hercules Calif.). In some embodiments, the molar ratio of nanopore protein to MSP protein to lipid will be from (about 0.5 to about 5) to (about 1 to about 15) to (about 50 to about 200). The optimal ratio can be determined empirically and will depend on the specific protein and lipid components of the target complex, as well as the particular method for forming the nanodisc complex. In one exemplary embodiment, the molar ratio of α-HL protein to MSP1D1 protein to DPhPC lipid is about 1 to 6 to 120 (i.e., 1:6:120). In another embodiment, the molar ratio of α-HL protein to MSP1D1 protein to DPhPC lipid is about 1:6:101,

[0035] A population of nanopore-nanodisc complexes containing a native nanopore protein can be purified from the mixture by conventional size-exclusion chromatography (SEC), which is well known in the art. The size of the target nanopore-nanodisc complex will determine the characteristics and details of the chromatography column and the chromatography protocol. In one embodiment, a chromatography column designed to purify complexes having an M r of about 10,000 - 600,000 is used to collect the target nanopore-nanodisc complex. Methods known in the art (e.g., gel electrophoresis and Western blotting) can be used to confirm that the appropriate fractions are retained from the SEC column eluate. In certain embodiments, additional purification steps are employed to further enrich the nanopore-nanodisc complexes containing a native nanopore protein. For example, immobilized metal affinity chromatography (e.g., nickel-based affinity matrix) can be used to specifically retain complexes in which the MSP or the nanopore protein has been engineered to express a polyhistidine affinity tag. Such methods have been described in detail in the art.

[0036] The nanopore-nanodisc complexes described herein exhibit improved stability in aqueous buffer and can also be lyophilized (e.g., for storage and transportation) and reconstituted as needed (e.g., for forming nanopore sensors or detection systems). As used herein, the term "buffer" refers to an aqueous solution capable of maintaining the pH of the solution at a nearly constant value. Buffers achieve this by including a weak acid and its conjugate base, such that the pH does not change substantially upon addition of a small amount of acid or base. Representative buffers include citric acid, acetic acid, dipotassium phosphate (K 2 HPO 4 ), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), and borate. Commonly used buffers include, but are not limited to, TAPS, bicine, tris, tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, SSC, MES, and succinic acid. In some embodiments, the nanopore-nanodisc complexes can be stored in an aqueous buffer at 4°C.

[0037] The nanopore-nanodisc complexes described herein can be components of a composition. For example, these components can be dried (e.g., as a powder) or in a stable buffer (e.g., chemically stable, thermally stable). For example, the dried components can be prepared by lyophilization, vacuum and centrifugation-assisted drying, and / or ambient drying. In various embodiments, the composition containing the nanopore-nanodisc complex is in lyophilized form in a single container. In other embodiments, the composition is an aqueous solution comprising the nanopore-nanodisc complex, which is stable when stored at 4 °C.

[0038] As used herein, the term "lyophilization" in connection with a formulation according to the present invention refers to the process of stabilizing a composition by freeze-drying methods known in the art. The solvent (e.g., water) is removed by sublimation after freezing under vacuum, and residual water is desorbed at elevated temperature. In the pharmaceutical field, lyophilized compositions typically have a residual moisture of about 0.1% (w / w) to 5% (w / w) and exist as a powder or a physically stable cake. Lyophilized products are characterized by rapid dissolution upon addition of a reconstitution medium.

[0039] As used herein, the term "reconstitution formulation" refers to a formulation that has been lyophilized and redissolved by the addition of a diluent. The diluent can contain, but is not limited to, water, sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20 or polysorbate 80), pH-buffered solution (e.g., phosphate-buffered solution), and combinations thereof.

[0040] The present disclosure provides the use of the nanopore-nanodisc complexes described herein in the manufacture of a system for data acquisition (e.g., a sensor or a detection device). In an exemplary system, a lipid bilayer membrane is formed across an orifice in a solid support cell such as PTFE. The lipid bilayer membrane can be formed according to the following steps: i) coating the support cell with a thin film of lipid (e.g., 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, "DPhPE") dissolved in hexane, ii) air-drying the coated cell to remove hexane, iii) coating the support cell with lipid by dissolving PE in 1-hexadecene and depositing the solution on the coated support cell with a pipette, and iv) moving a bubble over the orifice in the support cell to form a lipid bilayer membrane over the orifice. To insert a nanopore into the membrane, the nanopore-nanodisc complex is applied to the lipid bilayer membrane, and then the nanopore protein is assimilated (i.e., inserted) into the membrane. In certain embodiments, a native nanopore is inserted into the membrane by mechanical force, such as by electroporation or by using a bubble.

[0041] The detection system includes a membrane that separates a cis chamber and a trans chamber. Standard electrodes (e.g., Ag / AgCl) on the cis and trans sides of the nanopore provide a current source. The ionic current between two ion-sensitive electrodes is measured using a current-sensing circuit, and this ionic current passes through the nanopore in a solution containing a suitable electrolyte (e.g., >1M KCl). The electrodes complete the circuit through a transimpedance amplifier, and the voltage output provided by this transimpedance amplifier is proportional to the ionic current within a frequency range. Data from the nanopore can be acquired using an Axopatch 200B amplifier. This type of system is consistent with conventional systems used in the field of nanopores to evaluate analytical capabilities. Incorporating a native nanopore protein into the membrane produces a functional sensor that enables current to flow through the membrane. In this way, by monitoring the ionic current in the system, it can be detected that the native nanopore protein has been correctly incorporated into the membrane. For example, when the native nanopore is correctly incorporated, the expected current at -100 mV may be approximately 200 pA.

[0042] In some embodiments, the detection system can include a nanopore array having any suitable number of nanopores. In some cases, the array includes about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, about 10,000, about 15,000, about 20,000, about 40,000, about 60,000, about 80,000, about 100,000, about 200,000, about 400,000, about 600,000, about 800,000, about 1,000,000, etc. nanopores. In some cases, the array includes at least 200, at least 400, at least 600, at least 800, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 10,000, at least 15,000, at least 20,000, at least 40,000, at least 60,000, at least 80,000, at least 100,000, at least 200,000, at least 400,000, at least 600,000, at least 800,000, or at least 1,000,000 nanopores, near the sensor circuit or sensing electrode. One or more nanopores can be associated with a separate electrode and a sensing integrated circuit or multiple electrodes and a sensing integrated circuit. In some embodiments, a transimpedance amplifier array implemented in CMOS is arranged to measure an array of independent sensor currents in parallel. An example of such an amplifier array has been disclosed by Kim et al. (e.g., see Kim, B.N., Herbst, A.D., Kim, S.J., Minch, B.A., & Lindau, M. 2013. Parallel Recording of Neurotransmitters Release from Chromaffin Cells using a 10x10 CMOS IC Potentiostat Array with On-Chip Working Electrodes. Biosensors and Bioelectronics, 41, 736-744). The nanopore device can include a plurality of individually addressable sensing electrodes. Each sensing electrode can include a membrane adjacent to the electrode, and one or more nanopores in the membrane.

[0043] In certain embodiments, each lipid nanodisc in the lipid nanodiscs applied to the membranes of the arrays described herein will have no more than one protein nanopore assimilated therein. Alternatively, a separate nanodisc can include more than one protein nanopore.

[0044] The detection device of the present disclosure can be used to detect any one of a variety of analytes, including but not limited to ions, nucleic acids, nucleotides, polypeptides, bioactive small molecules, lipids, sugars, etc. Thus, one or more of these analytes can be present in or pass through the orifice of the protein nanopore in the device described herein.

[0045] In a preferred embodiment, the present disclosure further provides a system and method for nucleic acid sequencing based on "expansion sequencing". The "expansion sequencing" (SBX) protocol developed by Stratos Genomics (see, e.g., Kokoris et al., U.S. Pat. No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion") is based on the polymerization of unnatural monomeric substrates, called "XNTPs". Generally, SBX uses this biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an "Xpandomer". The transcribed sequence is encoded in a high signal-to-noise reporter along the Xpandomer backbone, with the reporters spaced approximately 10 nm apart and designed for high signal-to-noise, well-differentiated reactions. These differences provide a significant performance enhancement in terms of the sequence read efficiency and accuracy of the Xpandomer relative to native DNA. An overview of the SBX process is described in Figure 2A , Figure 2B , Figure 2C and Figure 2D .

[0046] XNTPs are expandable, 5'-triphosphate-modified unnatural substrates that are compatible with template-dependent enzymatic polymerization. A highly simplified XNTP is shown in Figure 2A , which highlights the unique features of these unnatural substrates: XNTP 200 has two distinct functional regions; namely: a selectively cleavable phosphoramidate bond 210 that links the 5'-α-phosphate 215 to the nucleobase 205; and a tether 220 attached at certain positions within the nucleoside triphosphate amide, which allows for control of the expansion through cleavage of the phosphoramidate bond. The tether of the XNTP contains linker arm portions 225A and 225B separated by a selectively cleavable phosphoramidate bond. Each linker is attached to one end of a reporter construct 230 through a linking group (LG), as disclosed in U.S. Patent No. 8,324,360 to Kokoris et al., which is incorporated herein by reference in its entirety. XNTP 200 is illustrated in a "constrained configuration", which is characteristic of the XNTP substrate and the daughter strand after polymerization. The constrained configuration of the polymerized XNTP is a precursor to the expanded configuration, as seen in the Xpandomer product. The transition from the constrained configuration to the expanded configuration occurs upon cleavage of the P-N bond of the phosphoramidate within the main backbone of the daughter strand.

[0047] The synthesis of Xpandomer polymers is summarized in Figure 2B and Figure 2C . During assembly, monomer XNTP substrates 245 (XATP, XCTP, XGTP, and XTTP) are polymerized at the extendable end of the nascent daughter strand 250 through a template-directed polymerization process using the single-stranded template 240 as a guide. Typically, this process starts from a primer and proceeds in the 5′ to 3′ direction. Typically, a DNA polymerase or other polymerase is used to form the daughter strand, and conditions are selected to obtain a complementary copy of the template strand. After synthesizing the daughter strand, the coupled tether contains a constrained Xpandomer that further contains the daughter strand. The tether in the daughter strand has a "constrained configuration" of the XNTP substrate. The constrained configuration of the tether is a precursor to the extended configuration, as seen in the Xpandomer product.

[0048] As Figure 2C shown, the transition from the constrained configuration 260 to the unfolded configuration 265 is caused by the cleavage of an aminophosphonate bond (represented by an unshaded ellipse for simplicity) that is selectively cleavable within the major backbone of the daughter strand. In this example, the tether contains one or more reporters or reporter constructs 230A, 230C, 230G, or 230T that are specific to the nucleobases to which they are attached, thereby encoding the sequence information of the template. In this way, the tether provides a means to extend the length of the Xpandomer and reduce the linear density of the parental strand sequence information.

[0049] Figure 2D Illustrate that Xpandomer 265 translocates from the cis reservoir 275 to the trans reservoir 285 through the nanopore 280. As Figure 1 shown, the α-HL nanopore-nanodisc assembly is incorporated into a lipid bilayer that separates and electrically isolates two electrolyte reservoirs. A typical electrolyte has 1 molar KCl buffered to a pH of 7.0. The α-HL nanopore is oriented to capture the Xpandomer first from the stem side. This orientation is advantageous when using translocation control methods because it reduces the blocking artifacts that occur when first entering the vestibule. When a small voltage (usually 100 mV) is applied across the bilayer, the nanopore restricts the flow of ionic current and is the main resistance in the circuit. After passing through the nanopore, each reporter construct in the reporter constructs of the linearized Xpandomer (labeled "G", "C", and "T" in this illustration) generates a unique and reproducible electrical signal (represented by the superimposed trace 290) that is specific to the nucleobase to which it is attached.

[0050] Example

[0051] Example 1

[0052] Assembly and Purification of α-Hemolysin Nanopores in Nanodisc Carriers

[0053] Formation of nanodiscs.

[0054] This example describes the reconstitution of the native α-hemolysin nanopore protein in lipid nanodiscs and the purification of the nanopore-nanodisc complex to assimilate the native nanopore protein in a lipid membrane.

[0055] The nanopore-nanodisc complex was formed by incubating α-hemolysin protein, MSP protein, and DPhPC lipid together at a molar ratio of 1:6:101. The reaction buffer consisted of 20 mM Tris, pH 7.4, 0.5 mM EDTA, 100 mM NaCl, and 30 mM cholate. The wild-type α-hemolysin protein was obtained from Sigma and a stock solution of 20 μM (calculated for the heptameric form) was prepared in 50% glycerol / 50% water. A 50 mM stock solution of DPhPC (obtainable from Avanti Polar Lipids) was prepared in 20 mM pH 7.4 Tris solution supplemented with 100 mM sodium cholate. The MSP1D1 protein (with an N-terminal his-tag) was obtained from Sigma and a 202 μM stock solution was prepared according to the manufacturer's instructions. The 134 μL nanodisc assembly mixture included 0.675 mM DPhPC, 6.67 μM α-HL, and 40 μM MSP. The final concentration of cholate was determined to be >14 mM, which the inventors found to be preferable for the assembly of the α-HL / DPhPC / MSP nanodisc complex. The assembly mixture was incubated at room temperature for 60 minutes. To remove the detergent, 78.8 mg of biobeads SM-2 (obtainable from BioRad) was added and the mixture was shaken at 1200 rpm at room temperature for 2.5 hours. The beads were removed by passing the mixture through a 45 μ filter.

[0056] Purification of nanopore-nanodisc complexes.

[0057] To isolate the nanopore-nanodisc complex containing the native heptameric α-HL protein, size exclusion chromatography (SEC) was first performed using a Superdex 200 Increase column (commercially available from GEH) selected based on the predicted size of the target complex. The column was equilibrated with MSP buffer (20 mM Tris, pH 7.4, 100 mM NaCl, and 0.5 mM EDTA), then 105 μL of the nanodisc assembly mixture was added, and the flow rate was adjusted to 0.5 mL / min at 160 psi. The column trace is shown in Figure 13, and the two fractions collected were designated "1" and "2". The presence of the heptameric α-HL protein in fraction 2 was confirmed by gel electrophoresis.

[0058] Next, perform the Ni-NTA purification step. Prepare a resin column (commercially available from Qiagen) by adding 50 μL of resin to an empty Ni-NTA spin column, and spin the column at 700 x g for two minutes to remove the stock buffer. Then equilibrate the column with 400 μL of EQ buffer containing 20 mM Tris, pH 7.5, and 10 mM imidazole. Remove the EQ buffer by spinning the column at 700 x g for two minutes. Then add the F2 nanodisc sample to the column, and mix the column contents by fixing the column on an end-over-end rotator for 15 minutes. Centrifuge the column at 700 x g for two minutes, and wash three times with 700 μL of wash buffer containing 20 mM Tris, pH 7.5, and 25 mM imidazole. Then elute the His-tagged protein / nanodisc assembly by adding 150 μL of buffer containing 20 mM Tris, pH 7.5, and 250 mM imidazole. Incubate the column for 5 minutes, and then collect the eluate by spinning the column at 700 x g for two minutes.

[0059] The efficacy of the purification step was monitored and evaluated by gel electrophoresis analysis of the following samples: the loaded sample (1 μL of the sample applied to the SEC column); sample 1 (15 μL of fraction 1 collected from the SEC column between 16:30 - 17:55); sample 2 (15 μL of fraction 2 collected from the SEC column between 19:00 - 20:45); the FT sample (15 μL of the column flow-through); samples W1, W2, and W3 (15 μL each of the first, second, and third wash samples from the IMAC); and sample E1 (15 μL of the sample eluted from the IMAC). A representative gel is shown in Figure 3 The arrows indicate the positions of the heptameric α-HL protein and the MSP protein. These results confirm the successful assembly and purification of the α-HL nanopore-nanodisc complex containing the native nanopore protein. The faint bands on the gel represent the monomeric α-HL protein, likely the result of dissociation of the native heptameric oligomer during the run of the protein sample in the gel.

[0060] The invention has been described herein in general and broad terms. Each narrower species and subgeneric group that falls within the general disclosure also forms part of the invention. This includes the general description of the invention, with the proviso or negative limitation of removing any subject matter, whether or not the excised material is specifically recited herein.

[0061] It should also be understood that the singular forms "a", "an", and "the" as used in this application and the appended claims include plural referents unless the context clearly dictates otherwise. The term "X and / or Y" means "X" or "Y" or "X" and "Y", and the letter "s" following a noun denotes both the plural and singular forms of that noun. Further, in instances where the invention is described in terms of Markush groups, it is intended and will be recognized by those skilled in the art that the invention encompasses and is thus also described in terms of any individual member and any subgroup of members of the Markush group, and the applicant reserves the right to amend the application or claims to expressly refer to any individual member or any subgroup of members of the Markush group.

[0062] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should also be understood that unless specifically defined herein, the terminology used herein will be given its conventional meaning as known in the relevant art.

[0063] References throughout this specification to "one embodiment", "an embodiment", and variations thereof mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in different places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0064] As used in this specification and the appended claims, the singular forms "a", "an", "the", and "said" include plural referents, i.e., one or more, unless the context clearly dictates otherwise. For example, the term "sensor" refers to one or more sensors, and the term "detection device comprising a sensor" refers to a detection device including at least one sensor, where the detection device comprising a sensor may have, for example, 1 sensor, 10 sensors, 10 2 sensors, 10 3 sensors, 10 4 sensors, 10 5 sensors, 10 6 sensors, or more than 10 6A plurality of sensors. A plurality of sensors means more than one sensor. It should also be noted that the connecting terms "and" and "or" are generally used in the broadest sense to include "and / or", unless the content and context clearly specify inclusivity or exclusivity in a particular case. Thus, the use of an alternative (e.g., "or") should be understood to mean any one of the alternatives, both, or any combination thereof. Additionally, the combination of "and" and "or" when referred to herein as "and / or" is intended to cover embodiments that include all relevant items or ideas, as well as one or more other alternative embodiments that include less than all relevant items or ideas.

[0065] Unless the context otherwise requires, throughout the specification and the subsequent claims, the word "comprising" and its synonyms and variations, such as "having" and "including", and its variations, such as "containing", are to be interpreted in an open, inclusive sense, e.g., "including but not limited to". The term "consisting essentially of" limits the scope of the claim to the specified materials or steps, or those that do not materially affect the basic and novel features of the claimed invention.

[0066] Any headings used in this document are only for expediting the review by the reader and should not be construed as limiting the invention or the claims in any way. Thus, the headings and abstracts of the present disclosure provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0067] Where numerical ranges are provided herein, it should be understood that each intermediate value between the upper and lower limits of the range (with a base of one-tenth of the lower limit unit, unless the context clearly specifies otherwise) and any other stated or intermediate value within the range are included in the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also covered by the invention, subject to any expressly excluded limitations within the specified range. When the range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the invention.

[0068] For example, unless otherwise stated, any concentration range, percentage range, ratio range, or integer range provided herein should be understood to include any integer values within the range and, where appropriate, fractional values thereof (e.g., one-tenth and one-hundredth of an integer). Additionally, unless otherwise stated, any numerical range cited herein in relation to any physical characteristic (such as polymer subunits, dimensions, or thickness) should be understood to include any integer within the range. As used herein, unless otherwise stated, the term "about" means ±20% of the indicated range, value, or structure.

[0069] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned and / or listed in the application data sheet in this specification, including but not limited to U.S. Provisional Patent Application No. 62 / 928,207 filed on October 30, 2019, are hereby incorporated by reference in their entirety. For the purpose of describing and disclosing materials and methods such as those described in the publications, these documents may be incorporated by reference and may be used in conjunction with the presently described invention. The publications provided above and discussed throughout are for their disclosure prior to the filing date of the present application only. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate any cited publication by virtue of prior invention.

[0070] All patents, publications, scientific articles, websites, and other documents and materials cited or mentioned herein are indicative of the level of skill of those of ordinary skill in the art to which the present invention pertains, and each such cited document and material is hereby incorporated by reference to the extent that it were individually incorporated by reference in its entirety or set forth in its entirety herein. The applicant reserves the right to physically incorporate into the written description of this application any and all materials and information from any such patents, publications, scientific articles, websites, electronically available information, and other cited materials or documents.

[0071] Generally, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments and the full scope of equivalents authorized by these claims. Thus, the claims are not limited by the present disclosure.

[0072] In addition, the written description portion of the patent includes all claims. In addition, all claims, including all original claims and all claims from any and all priority documents, are hereby incorporated by reference in their entirety into the written description portion of the specification, and the applicant reserves the right to physically incorporate into the written description of this application or any other portion any and all such claims. Thus, for example, in no case may the patent be construed as allegedly not providing a written description of the claims because the exact language of the claims is not set forth in these phrases in the written description portion of the patent.

[0073] The claims will be construed in accordance with the law. However, notwithstanding any claim or perception of ease or difficulty in construing any claim or portion thereof, in no case shall any adjustment or modification of any claim or portion thereof during the prosecution of one or more applications leading to this patent be construed as having forfeited any right to any and all equivalents that do not form part of the prior art.

[0074] Other non-limiting embodiments are within the following claims. The patent should not be construed as limited to the specific examples or non-limiting embodiments or methods specifically and / or expressly disclosed herein. In no event shall the patent be construed as being limited by any statement made by any examiner or any other official or employee of the Patent and Trademark Office, unless such statement is expressly adopted by the applicant in a responsive writing without limitation or reservation.

Claims

1. A method for preparing a detection device comprising one or more native nanopore proteins, the method comprising the steps of: (a) forming an aqueous mixture comprising a nanopore protein, a membrane scaffold protein (MSP), and a first lipid to produce a sample of nanodisc-nanopore protein complexes, wherein the population of nanodisc-nanopore protein complexes in the sample each comprises a native nanopore protein; (b) providing a solid support comprising one or more orifices, wherein a membrane is formed over each of the orifices, wherein the membrane comprises a second lipid, and wherein the membrane separates a cis chamber from a trans chamber in the detection device; and (c) contacting one or more membranes with a population of nanopore-nanodisc complexes comprising the native nanopore protein to assimilate the native nanopore protein into each of the membranes, wherein the aqueous mixture further comprises a detergent, wherein the final concentration of the detergent is from 14 mM to 40 mM, wherein the first lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), the MSP is MSP1D1, the nanopore protein is α-hemolysin (α-HL), the detergent is cholate, and the second lipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), and wherein the molar ratio of lipid to MSP to nanopore protein is 101:6:1 or 120:6:

1.

2. The method according to claim 1, further comprising the step of purifying the population of nanopore-nanodisc complexes comprising the native nanopore protein from the aqueous mixture prior to the step of contacting the one or more membranes with the population of nanopore-nanodisc complexes comprising the native nanopore protein.

3. The method according to claim 2, wherein the step of purifying the population of nanopore-nanodisc complexes comprising the native nanopore protein comprises one or both of size exclusion chromatography and affinity chromatography.

4. The method according to claim 1, wherein the solid support comprises a plurality of orifices, wherein a membrane is formed over each of the plurality of orifices, and wherein each of the membranes is contacted with the nanopore-nanodisc complexes comprising the native nanopore protein.

5. A method for sequencing a polymer, the method comprising using a detection device prepared by the method according to any one of claims 1 to 4.

6. The method according to claim 5, wherein the polymer is Xpandomer.

7. A method for forming a native nanopore protein in a membrane, the method comprising the steps of: (a) forming an aqueous mixture comprising a nanopore protein, a membrane scaffold protein (MSP), and a first lipid to produce a sample of nanodisc-nanopore protein complexes, wherein the population of nanodisc-nanopore protein complexes in the sample each comprises a native nanopore protein; (b) providing a membrane comprising a second lipid; and (c) contacting the membrane with a population of nanopore-nanodisc complexes comprising the native nanopore protein to assimilate the native nanopore protein into the membrane, wherein the aqueous mixture further comprises a detergent, and wherein the final concentration of the detergent is greater than 14 mM and equal to or less than 40 mM, wherein the first lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), the MSP is MSP1D1, the nanopore protein is α-hemolysin (α-HL), the detergent is cholate, and the second lipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), and wherein the molar ratio of lipid to MSP to nanopore protein is 101:6:1 or 120:6:

1.

8. The method according to claim 7, further comprising, prior to the step of contacting the membrane with a population of nanopore-nanodisc complexes comprising the native nanopore protein, the step of purifying the population of nanopore-nanodisc complexes comprising the native nanopore protein from the aqueous mixture.

9. The method according to claim 8, wherein the step of purifying the population of nanopore-nanodisc complexes comprises one or both of size exclusion chromatography and immobilized metal affinity chromatography.

10. A composition comprising a nanopore-nanodisc complex in an aqueous buffer, wherein the nanopore-nanodisc complex comprises a native nanopore protein, a membrane scaffold protein (MSP), and a lipid, and wherein the aqueous buffer comprises a detergent, wherein the native nanopore protein is α-hemolysin (α-HL), the MSP is MSP1D1, the lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), and the detergent is cholate, and wherein the molar ratio of lipid to MSP to nanopore protein is 101:6:1 or 120:6:1, and the concentration of cholate is from more than 14 mM to 40 mM.

11. A composition comprising a lyophilized powder comprising a nanopore-nanodisc complex, wherein the nanopore-nanodisc complex comprises a native nanopore protein, a membrane scaffold protein (MSP), and a lipid, wherein the native nanopore protein is α-hemolysin (α-HL), the MSP is MSP1D1, and the lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), and wherein the molar ratio of lipid to MSP to nanopore protein is 101:6:1 or 120:6:1.

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