Methods and compositions for the assembly of biological nanopores - Patents.com

Abstract

Methods and compositions are provided for the fabrication and use of sensing devices based on one or more natural biological nanopores. Uses include, but are not limited to, nucleic acid detection and sequencing. The present invention relates generally to new methods and compositions for making protein-based nanopore sensors, and more specifically to methods for assembling natural nanopore proteins within lipid nanodiscs used as carriers for delivering nanopores to lipid membrane components of sensor systems, and particularly for their use in nanopore-based nucleic acid sequencing methods.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 928,207, filed October 30, 2019, which is hereby incorporated by reference in its entirety for all purposes. [Technical field]

[0002] The present invention relates generally to new methods and compositions for making protein-based nanopore sensors, and more specifically to methods for assembling native nanopore proteins within lipid nanodiscs that are used as carriers for delivering nanopores to lipid membrane components of sensor systems, and in particular for their use in nanopore-based nucleic acid sequencing methods. [Background technology]

[0003] In the past two decades, nanopore sensors have emerged as a powerful tool and have had a profound impact on science and biotechnology. Nanopore technology is generally divided into biological nanopores and solid-state nanopores according to their materials. Solid-state nanopores are traditionally fabricated by drilling nanoscopic pores using semiconductor or microfluidic techniques such as ion or electron beam sculpting in silicon or graphene-based membranes such as Si, SiN, or SiO2. However, most nanopore applications, such as DNA sequencing, small molecule sensing, drug screening, molecular sieving, and biomolecular analysis, require high-precision geometry, sensitivity, and reproducibility that cannot be achieved with solid-state pores.

[0004] The most important and most highly desirable application of nanopores is DNA sequencing. However, a major problem associated with the extremely fast translocation speed of DNA through the nanopore (a few nucleotides pass through the nanopore in a few microseconds) results in few data points for each base, preventing 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 the sequence of DNA onto measurable polymers called "Xpandomers" (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 along the Xpandomer backbone in high signal-to-noise reporters about 10 nm apart, designed for high signal-to-noise, highly differentiated response. These differences provide a significant performance improvement in sequence read efficiency and accuracy of Xpandomers compared to natural DNA. Xpandomer can enable several next-generation DNA sequencing detection technologies and is well suited for nanopore sequencing.

[0005] Alpha-hemolysin (α-HL) is the most widely used biological nanopore for single molecule analysis, mainly due to its small internal diameter and structural reproducibility. Alpha-HL is a monomeric polypeptide that self-assembles in lipid bilayer membranes to form a transmembrane heptameric pore with a vestibule of 2.6 nm diameter and a limiting aperture (narrowest point of the pore) of 1.5 nm diameter. The limiting aperture of the Alpha-HL nanopore allows linear molecules with dimensions comparable to single-stranded DNA to pass through, or "translocate," while molecules with diameters larger than about 2.0 nm, e.g., double-stranded DNA, are excluded from translocation. Despite its advantages in DNA sequencing, Alpha-HL (and other oligomeric transmembrane protein nanopores) still have inherent structural limitations, e.g., due to the reduced stability of native oligomers in aqueous solutions and the incomplete assembly of native proteins in lipid membranes, creating a need for improved methods and compositions for making biological nanopore sensors.

[0006] The present invention fulfills these needs and, as described below, provides the advantages of further related improvements.

[0007] Not all of the subject matter described in the Background section is necessarily prior art, and it should not be assumed that it is prior art merely as a result of its description in the Background section. Along these lines, an awareness of a prior art problem described in the Background section or related to such subject matter should not be treated as prior art unless it is expressly stated to be prior art. Instead, the discussion of any subject matter in the Background section should be treated as part of the inventor's approach to a particular problem that may also be inventive in itself. Summary of the Invention

[0008] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims.

[0009] Briefly, the present disclosure provides methods and compositions for improved fabrication of nanopore-based sensors. In certain embodiments, the methods and compositions enable fabrication of biological nanopore-based sensors with improved assimilation of native nanopore structures.

[0010] In one aspect, the invention provides a method of making a detection device comprising one or more native nanopore proteins, the method comprising: (a) forming an aqueous mixture comprising the nanopore protein, a membrane scaffolding protein (MSP), and a first lipid to produce a sample of Nanodisc-nanopore protein complexes, wherein a population of Nanodisc-nanopore protein complexes in the sample each comprises the native nanopore protein, (b) providing a solid support comprising one or more apertures, wherein a membrane is formed over each of the apertures, the membrane comprising a second lipid, the membrane separating a cis chamber from a trans chamber in the detection device, and (c) contacting the one or more membranes with the population of nanopore-Nanodisc complexes comprising the native nanopore protein to assimilate the native nanopore protein into each of the membranes. In one embodiment, the method further comprises purifying the population of nanopore-Nanodisc complexes comprising the native nanopore protein from the aqueous mixture prior to contacting the one or more membranes with the population of nanopore-Nanodisc complexes comprising the native nanopore protein. In a further embodiment, purifying the population of nanopore-Nanodisc complexes comprising the native nanopore protein comprises one or both of size-exclusion and affinity chromatography. In another embodiment, the aqueous mixture further comprises a detergent, and the final concentration of the detergent is 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 openings, a membrane is formed over each of the plurality of openings, and each of the membranes is contacted with a nanopore-Nanodisc complex that comprises a native nanopore protein.

[0011] In another aspect, the invention provides a method of sequencing a polymer comprising the use of any of the detection devices described above. In certain embodiments, the polymer is an Xpandomer.

[0012] In another aspect, the invention provides a method of forming a native nanopore protein in a membrane, the method comprising: (a) forming an aqueous mixture comprising a nanopore protein, a membrane scaffolding protein (MSP), and a first lipid to generate a sample of Nanodisc-nanopore protein complexes, each of the population of Nanodisc-nanopore protein complexes comprising the native nanopore protein; (b) providing a membrane comprising a second lipid; and (c) contacting the membrane with the population of nanopore-Nanodisc complexes comprising the native nanopore protein to assimilate the native nanopore protein of the membrane. In one embodiment, the method further comprises purifying the population of nanopore-Nanodisc complexes comprising the native nanopore protein from the aqueous mixture prior to contacting the membrane with the population of nanopore-Nanodisc complexes comprising the native nanopore protein. In certain embodiments, purifying the population of nanopore-Nanodisc complexes comprises one or both of size exclusion chromatography and immobilized metal affinity chromatography. In another embodiment, the aqueous mixture further comprises a detergent, and the final detergent concentration is 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.

[0013] In another aspect, the invention provides a composition comprising a nanopore-Nanodisc complex in an aqueous buffer, the nanopore-Nanodisc complex comprising a native nanopore protein, a membrane scaffolding protein (MSP), and a lipid, and 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 greater than 14 mM to 40 mM.

[0014] In another aspect, the invention provides a composition comprising a lyophilized powder comprising a nanopore-Nanodisc complex, the nanopore-Nanodisc complex comprising a native nanopore protein, a membrane scaffolding 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]

[0015] [Figure 1] 1 is a flow chart illustrating one embodiment of a method for making a biological nanopore-based detection system.

[0016] [Figure 2A] 2A, 2B, 2C, and 2D are summarized schematic diagrams showing the key features of generalized XNTP and their use in sequencing by extension (SBX). [Figure 2B] 2A, 2B, 2C, and 2D are summarized schematic diagrams showing the key features of generalized XNTP and their use in sequencing by extension (SBX). [Figure 2C] 2A, 2B, 2C, and 2D are summarized schematic diagrams showing the key features of generalized XNTP and their use in sequencing by extension (SBX). [Figure 2D] 2A, 2B, 2C, and 2D are summarized schematic diagrams showing the key features of generalized XNTP and their use in sequencing by extension (SBX).

[0017] [Diagram 3] 1 is a SEC trace showing the A280 of eluted samples over time.

[0018] [Figure 4] 1 is a gel showing protein samples taken from various stages of the nanopore purification process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention may be more readily understood by reference to the following detailed description of preferred embodiments of the invention and examples contained herein. Unless otherwise explained, 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.

[0020] Biological nanopore proteins that have found use in nucleic acid sequencing techniques include those based on natural transmembrane proteins in which individual polypeptide subunits self-assemble in the membrane and form a pore when oligomerized into their native conformation. Conventional biological nanopore sensors, such as αHL nanopores, are typically assembled by applying an aqueous solution of solubilized protein to the micron-sized membrane components 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 conformation. Reconstitution of native membrane proteins in lipid bilayers presents several technical challenges due, for example, to the low solubility and stability of proteins in aqueous solutions and the difficulty of efficiently and consistently assembling the appropriate native conformation in lipid substrates. The present disclosure addresses these challenges by providing methods and compositions for fabricating nanopore sensors, where native oligomeric nanopore structures are assembled in lipid nanodiscs prior to assimilation of the nanopore in the membrane. Nanodiscs incorporating native nanopore proteins (e.g., appropriately sized complexes and / or incorporation of heterologous detection "tags") can be optionally purified from the mixture to obtain a more homogenous sample of native nanopores. The purified nanopore-Nanodisc complexes can then be applied to lipid bilayer membranes to assimilate the native protein structure into the membrane to form a functional nanopore sensor or detector. A further advantage provided by the present invention is that the structure of the native nanopore protein is greatly stabilized when formed within the Nanodisc, thus providing improved compositions for, for example, storage and transport of native nanopore proteins.

[0021] Biomarkers are well known in the art. In some embodiments, nanodiscs are nanoscale disk-shaped phospholipid bilayers stabilized by two surrounding amphipathic helical protein "belts" called membrane scaffolding proteins (MSPs) and soluble in aqueous solution. Nanodiscs can be used as a vehicle to incorporate membrane proteins (MPs) of interest into the bilayer to preserve the structure and activity of the MPs, and have traditionally been used for biophysical, enzymatic or structural investigations of MPs (for a review, see, e.g., Bayburt and Sligar, FEBS Lett.; 584(9):172-1727 (2010)). In this approach, membrane protein targets and / or phospholipids are transiently solubilized with detergent in the presence of the surrounding amphipathic helical MSPs. Once the detergent is removed by dialysis or adsorption to hydrophobic beads, the target MPs simultaneously assemble with phospholipids into a disk-shaped bilayer with a size controlled by the length of the MSPs. Thus, the resulting Nanodiscs provide a native-like phospholipid bilayer environment that keeps membrane proteins in solution, provides stability and functional requirements of the incorporated targets, and also allows control of the oligomeric state of the target membrane proteins. Nanodiscs are known to be robust and can be frozen or lyophilized along with the incorporated MPs. The inventors have found that Nanodiscs offer several advantages as nanopore delivery and storage vehicles, as discussed further herein.

[0022] 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, membrane scaffold proteins have a hydrophobic face that can associate with the non-polar interior of the phospholipid bilayer and a hydrophilic face that favorably interacts with polar solvents such as aqueous buffers. Membrane scaffold protein sequences can be naturally occurring, engineered using recombinant technology, or constructed de novo. Naturally occurring membrane scaffold proteins include apolipoproteins, which are components of lipoproteins. Known classes of apolipoproteins include A (including, for example, apo AI and apo A-II), B, C, D, E, and H. Non-naturally occurring membrane scaffold proteins include MSP1 and MSP2, which are described in U.S. Pat. No. 7,691,414, which is incorporated herein by reference in its entirety. An exemplary commercially available non-naturally occurring MSP is MSP1D1, available, for example, from Sigma. Membrane scaffold proteins can be full-length proteins or truncated forms of the proteins. Membrane scaffold proteins are not intended to encompass the various functional membrane proteins, including, but not limited to, ion channels and other transmembrane receptors, porins, certain cell adhesion molecules, and electron transport proteins such as NADH dehydrogenase and ATP synthase.

[0023] As used herein, the term "nanopore protein" refers to polypeptide subunits and multimers of subunits that can create an opening through a membrane when the appropriate conformation is formed. Nanopore protein may refer to a single polypeptide subunit of a multimeric nanopore protein or different oligomeric forms of a single polypeptide subunit. A "mixture of nanopore proteins" refers to a solution that may contain a heterogeneous combination of single and / or oligomeric forms of nanopore proteins. A "native nanopore protein" refers to a native conformation 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-pilus.

[0024] A preferred nanopore protein is α-hemolysin (α-HL). α-HL is the major cytotoxic substance released by the bacterium Staphylococcus aureus and is the first identified member of the pore forming beta-barrel toxin family. The toxin is composed mostly of beta-sheets (68%) with only about 10% α-helices. The hla gene on the S. aureus chromosome encodes a 293-residue protein monomer that forms a heptameric oligomer in the cell membrane to form a complete beta-barrel pore. Thus, the native α-HL nanopore protein is an assembly of seven α-HL protein monomers, i.e., an oligomer.

[0025] 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 either the MSP or the nanopore protein, which is used for purification of the complex on an immobilized metal affinity column (e.g., on a nickel affinity column). As demonstrated by SDS-PAGE gels, α-HL or MSP with a terminal 6×His tag can be expressed, reconstituted, and purified. The biological functionality of the purified His-tagged protein is expected to be similar to that of the non-tagged protein. Other mutations can also be introduced for purification purposes. For example, a cysteine ​​moiety can be introduced into the protein sequence by mutagenesis and used for chemical conjugation to a thiol-reactive moiety of the 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 having complementary sequences), epitopes (which can mediate purification via solid-phase antibodies or antibody fragments), or other ligands (which can mediate purification via solid-phase receptors for those ligands).

[0026] Protein engineering and mutagenesis techniques can be used to modify the structure of biological pores and tailor their properties for specific applications. In certain embodiments, α-hemolysin can be mutated to generate variants with improved stability and / or altered surface charge, for example, within the pore, to optimize detection of the analyte of interest. Suitable α-HL variants include those disclosed in published PCT applications WO2016069806, WO2018002125 and WO2019166458, as well as U.S. Pat. Nos. 15,274,770 and 10,351,908, which are incorporated herein by reference. In certain embodiments, suitable α-HL mutants may 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.

[0027] As used herein, a "membrane" is a component of a sensor or detection system or device, and not a component of the nanopore-nanodisc complex. A membrane is a thin film that separates two compartments or reservoirs (e.g., the cis and trans chambers) and prevents the free diffusion of ions and other molecules between them. A suitable membrane is an amphiphilic layer formed of amphipathic molecules, i.e., molecules that have both hydrophilic and lipophilic properties. Such amphipathic molecules can be either naturally occurring, such as phospholipids, or synthetic. Exemplary amphiphiles include various phospholipids, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidylmethylester (DOPME), 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin. Exemplary synthetic amphiphilic molecules include molecules such as poly(n-butyl methacrylate-phosphorylcholine), poly(ester amide)-phosphorylcholine, polylactide-phosphorylcholine, polyethylene glycol-poly(caprolactone)-di- or tri-block, polyethylene glycol-polylactide-di- or tri-block, and polyethylene glycol-poly(lactide-glycolide) di- or tri-block.

[0028] Preferably, the membrane is a lipid bilayer. Lipid bilayers are models of cell membranes and are widely used for experimental purposes. The membrane can also be a solid membrane, i.e. a layer prepared from a solid material with one or more openings formed therein. The membrane can be a layer, such as a coating or film on a support substrate, or can be a free-standing element. Materials used for thin solid membranes include silicon nitride, aluminum oxide, titanium oxide, silicon oxide, etc.

[0029] FIG. 1 summarizes three basic steps of an exemplary method for forming a nanopore sensor component of a detection device according to the present invention, the details of each step being further described 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 contains a collection of nanopore-Nanodisc complexes each containing a native nanopore protein, but because not all complexes in the sample necessarily contain the properly assembled native nanopore protein, in certain embodiments the sample may be described as a "heterogeneous sample" and it may be advantageous to carry out one or more purification steps to provide a sample enriched for the nanopore-Nanodisc complexes of interest. In steps 2A and 2B, nanopore-Nanodisc complexes having suitable physical properties may optionally be isolated or purified from the aqueous mixture. In this embodiment, two sequential 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 containing the native nanopore protein. It is understood that any suitable purification protocol known in the art can be used according to the methods described herein. In step 3, the purified nanopore-nanodisc complexes are applied to the lipid bilayer component, i.e., membrane, of the detection cell to allow assimilation of the native nanopore protein into the membrane to form a functional nanopore sensor. Advantageously, this methodology increases the efficiency of forming a functional sensor since a sample enriched in native oligomeric proteins is applied to the membrane. In contrast, prior art methodologies require correct intramembrane self-assembly of protein subunits to form native conformations, a process that is less efficient and potentially prone to errors that can compromise the functionality of the detection system.

[0030] In certain embodiments, the nanopore-nanodisc complex can be, for example, a lipid bilayer disc of 7-16 nm diameter stabilized by a membrane scaffolding protein (MSP). In some embodiments, the MSP is a suitable derivative of apoA-I, such as the commercially available MSP1D1 protein. Other types of amphiphilic nanodisc "belts," such as amphipathic peptides, are contemplated by the present invention. It will be understood that the nanopore-nanodisc complex can have a diameter of less than 7 nm (e.g., less than 6 nm, 5 nm, 4 nm, 2 nm or less in diameter) or greater than 16 nm (e.g., greater than 18 nm, 20 nm, or 25 nm or more in diameter). Typically, the area of ​​the lipid discs used in the methods or compositions described herein is about 50,000 nm 2 Less than or equal to 10,000 nm, or in some cases about 10,000 nm 2 Less than or sometimes about 1,000 nm 2 or less, or at still other times, about 500 nm 2 The nanopore-nanodisc complex can, but need not, occupy a circular region. In certain conditions, the nanopore-nanodisc complex can be distinguished from a vesicle or liposome due to the absence of an aqueous lumen in the nanodisc, and from a micelle due to the presence of a bilayer within the nanodisc. It will be appreciated that the nanopore-nanodisc complex can also be made from other materials. For example, nanodiscs can be formed from non-lipid membranes. One of skill in the art will appreciate that the optimal physical properties of the nanodisc, such as the physical properties of the target protein and other components of the system in which the target protein is incorporated, will be determined by the particular application(s) of interest. For example, nanodiscs incorporating nanopore proteins must have a size suitable to preserve the membrane solubility and transmembrane pore structure of the native protein when the nanopore-nanodisc assembly is applied to lipid bilayers. In certain embodiments where the nanodiscs are assembled with DPhPC, MSP1D1, and α-HL, the complex is expected to be about 9.7 nm in diameter and about 4.6-5.6 nm thick.

[0031] As described herein, lipid nanodiscs can be composed of a bilayer of lipid molecules surrounded by two parallel belt-like MSPs, the amphipathic helices of which stabilize the hydrophobic fatty acids at the edge of the lipid disc. Particularly useful lipid nanodiscs and compositions and methods for their manufacture are described, for example, in U.S. Patent Nos. 7,083,958 and 7,662,410, which are incorporated herein by reference. In certain embodiments of the present invention, lipids useful for forming nanodiscs include 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

[0032] In certain embodiments, lipid nanodiscs may be prepared by mixing MSPs with detergent-stabilized phospholipids. Self-assembly of the nanodiscs occurs during removal of the detergent from the mixture, as described herein. The presence of MSPs has been demonstrated to restrict the shape and size of lipid nanodiscs, providing a narrow size distribution (+ / - 3%), excellent reproducibility and exceptional stability in detergent-free aqueous solutions. The ratio of MSPs to detergents can be selected to achieve the desired size and properties of the nanodiscs. For example, the number of structural units of MSPs can be varied to allow tuning of 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, and aerolysin, 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 a particular embodiment, the buffer contains 19 mM cholate. In some embodiments, DPhPC lipid is added to the 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, and in one particular embodiment, the final concentration of cholate is about 19 mM.

[0033] In certain embodiments, the nanopore-nanodisc complex is formed with a mixture of membrane scaffolding protein (MSP), detergent-solubilized phospholipid (e.g., DPhPC), and nanopore protein. In the mixture, the MSP self-assembles with the detergent-solubilized phospholipid 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-Beads® (Bio-Rad, Hercules, Calif.). In some embodiments, the molar ratio of nanopore protein to MSP protein to lipid is 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 depends on the particular protein and lipid components of the complex of interest, as well as the particular method of forming the Nanodisc complex. In an 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.

[0034] A population of nanopore-Nanodisc complexes containing native nanopore proteins can be purified from the mixture by conventional size-exclusion chromatography (SEC), as is well known in the art. The size of the nanopore-Nanodisc complexes of interest will determine the characteristics and details of the column and chromatography protocol. In one embodiment, the size of the nanopore-Nanodisc complexes is between about 10,000 and 600,000 M. rThe nanopore-Nanodisc complexes of interest are collected using a column designed to purify complexes having the MSP-NANODISC complex. Methods known in the art (e.g., gel electrophoresis and Western blotting) can be used to confirm that appropriate fractions are retained from the SEC column eluate. In certain embodiments, additional purification steps are used to further enrich for nanopore-Nanodisc complexes containing native nanopore proteins. For example, immobilized metal affinity chromatography (e.g., nickel-based affinity matrices) can be used to specifically retain complexes in which either the MSP or the nanopore protein has been engineered to express a polyhistidine affinity tag. Such methodologies are well described in the art.

[0035] The nanopore-nanodisc complexes described herein exhibit improved stability in aqueous buffers, and can be lyophilized, for example, for storage and transport, and reconstituted as needed, for use in forming, for example, a nanopore sensor or detection system. As used herein, the term "buffer" refers to an aqueous solution capable of maintaining the pH of the solution at a nearly constant value. The buffer achieves this by including a weak acid and its conjugate base, such that the pH does not change substantially after the addition of small amounts of acid or base. Representative buffers include citric acid, acetic acid, dipotassium phosphate (K2HPO4), 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 aqueous buffers at 4°C.

[0036] The nanopore-Nanodisc complexes described herein can be components of a composition. The components can be, for example, dried (e.g., powder) or dried in a stable buffer (e.g., chemically stabilized, thermally stabilized). Dry components can be prepared, for example, by lyophilization, vacuum and centrifuge assisted drying, and / or ambient drying. In various embodiments, the composition comprising the nanopore-Nanodisc complexes is in lyophilized form in a single container. In other embodiments, the composition is an aqueous solution comprising the nanopore-Nanodisc complexes that is stable when stored at 4°C.

[0037] The term "lyophilized" as used herein in connection with the formulations according to the invention refers to a method in which a composition is stabilized by freeze-drying methods known in the art. The solvent (e.g. water) is removed by sublimation under vacuum and freezing after the departure of the residual water at high temperature. In the pharmaceutical field, lyophilized compositions usually have a residual moisture content of about 0.1-5% (w / w) and exist as a powder or a physically stable cake. Lyophilized products are characterized by their rapid dissolution after the addition of a reconstitution medium.

[0038] As used herein, the term "reconstituted formulation" refers to a formulation that is lyophilized and reconstituted by the addition of a diluent. The diluent can include, but is not limited to, water, a sodium chloride solution (e.g., 0.9% (w / v) NaCl), a glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., 0.01% polysorbate 20 or polysorbate 80), a pH buffer solution (e.g., phosphate buffer solution), and combinations thereof.

[0039] The present disclosure provides for the use of the nanopore-nanodisc complexes described herein in the manufacture of systems (e.g., sensors or detection devices) for data collection. In an exemplary system, a lipid bilayer membrane is formed across the opening of, for example, a PTFE solid support cell. The lipid bilayer membrane can be formed according to the following steps: i) priming the support cell with a thin coat of lipid (e.g., 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, "DPhPE") dissolved in hexane, ii) air-drying the coated cells to remove the hexane, iii) coating the lipid on the support cell by dissolving PE in 1-hexadecene and depositing the solution with a pipette on the primed support cell, and iv) moving an air bubble over the opening of the support cell to form a lipid bilayer membrane over the opening. To insert the nanopore into the membrane, the nanopore-nanodisc complex is applied to the lipid bilayer membrane, and the nanopore protein assimilates (i.e., inserts) into the membrane. In certain embodiments, the native nanopore is inserted into the membrane by mechanical forces, for example, by electroporation, or by using air bubbles.

[0040] The detection system includes a membrane that separates the cis and trans chambers. Standard electrodes, e.g., Ag / AgCl, on the cis and trans sides of the nanopore provide a current source. A current sensing circuit is used to measure the ionic current passing through the nanopore in a solution containing an appropriate electrolyte, e.g., KCl >1M, between two ion-sensitive electrodes. The electrodes complete the circuit through a transimpedance amplifier that provides a voltage output proportional to the ionic current over 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 to evaluate analytical capabilities in nanopore technology. Assimilation of a native nanopore protein into the membrane creates a functional sensor that allows current to flow across the membrane. In this way, proper assimilation of the native nanopore protein into the membrane can be detected by monitoring the ionic current in the system, e.g., the expected current at -100 mV upon correct assimilation of the native nanopore can be approximately 200 pA.

[0041] In some embodiments, the detection system may include an array of nanopores 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 10000, about 15000, about 20000, about 40000, about 60000, about 80000, about 100000, about 200000, about 400000, about 600000, about 800000, about 1000000, etc. In some cases, the array comprises 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 in proximity to a sensor circuit or sensing electrode. One or more nanopores may be associated with an individual electrode and sensing integrated circuit, or with multiple electrodes and sensing integrated circuits. In some embodiments, an array of transimpedance amplifiers implemented in CMOS is configured to measure an array of independent sensor currents in parallel. An example of such an amplifier array is disclosed by Kim et al. (see, e.g., Kim, BN, Herbst, AD, Kim, SJ, Minch, BA, & 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 may include multiple individually addressable sensing electrodes. Each sensing electrode may include a membrane adjacent to the electrode and one or more nanopores in the membrane.

[0042] In certain embodiments, each of the lipid nanodiscs applied to the membranes of the arrays described herein has no more than one protein nanopore incorporated therein. Alternatively, an individual nanodisc can contain two or more protein nanopores.

[0043] The detection devices of the present disclosure can be used to detect any of a variety of analytes, including, but not limited to, ions, nucleic acids, nucleotides, polypeptides, biologically active small molecules, lipids, sugars, etc. Thus, one or more of these analytes can be present within or pass through the opening of a protein nanopore in the devices described herein.

[0044] In a preferred embodiment, the present disclosure further provides a system and method for sequencing nucleic acids based on "sequencing by expansion". The sequencing by expansion (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 known as "XNTPs". In general, 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 along the Xpandomer backbone in high signal-to-noise reporters about 10 nm apart, designed for high signal-to-noise, highly differentiated response. These differences provide a significant performance improvement in sequence read efficiency and accuracy of Xpandomer compared to natural DNA. A generalized overview of the SBX process is shown in Figures 2A, 2B, 2C, and 2D.

[0045] XNTPs are extendable 5' triphosphate modified non-natural substrates compatible with template-dependent enzymatic polymerization. A highly simplified version of XNTPs is shown in FIG. 2A, which highlights the unique features of these non-natural substrates. XNTPs 200 have two distinct functional regions: a selectively cleavable phosphoramidate bond 210 that links the 5'α-phosphate 215 to the nucleobase 205, and a tether 220 that is attached within the nucleoside triphosphoramidate at a position that allows controlled extension by intranucleotidic cleavage of the phosphoramidate bond. The XNTP tether is composed of linker arm moieties 225A and 225B separated by a selectively cleavable phosphoramidate bond. Each linker is attached to one end of a reporter 230 via a linking group (LG) as disclosed in U.S. Pat. No. 8,324,360 to Kokoris et al., which is incorporated herein by reference in its entirety. XNTP200 is shown in a "constrained configuration" characteristic of XNTP substrates and daughter strands after polymerization. The constrained configuration of polymerized XNTPs is the precursor to the extended configuration, as seen in the Xpandomer product. The transition from the constrained to the extended configuration occurs upon cleavage of the phosphoramidate P--N bond in the primary backbone of the daughter strand.

[0046] The synthesis of the Xpandomer polymer is summarized in Figures 2B and 2C. During assembly, monomeric XNTP substrates 245 (XATP, XCTP, XGTP, and XTTP) are polymerized on the extendable end of the nascent daughter strand 250 by a process of template-directed polymerization using a single-stranded template 240 as a guide. Generally, the process starts from a primer and proceeds in the 5' to 3' direction. Generally, a DNA polymerase or other polymerase is used to form the daughter strand, and conditions are selected to yield a complementary copy of the template strand. After the daughter strand is synthesized, the coupled tether contains a constrained Xpandomer that further comprises 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.

[0047] As shown in Figure 2C, the transition from the constrained configuration 260 to the extended configuration 265 results from cleavage of a selectively cleavable phosphoramidate bond (shown by an unshaded oval for simplicity) in the primary backbone of the daughter strand. In this embodiment, the tethers contain one or more reporter constructs or reporter constructs 230A, 230C, 230G, or 230T specific to the nucleobases to which they are linked, thereby encoding the sequence information of the template. In this way, the tethers provide a means to extend the length of the Xpandomer and reduce the linear density of the sequence information of the parent strand.

[0048] Figure 2D shows the Xpandomer 265 translocating through the nanopore 280 from the cis reservoir 275 to the trans reservoir 285. As shown in Figure 1, the α-HL nanopore-nanodisk assembly is assimilated into a lipid bilayer membrane that separates and electrically isolates the two reservoirs of electrolyte. A typical electrolyte is 1 molar KCl buffered to a pH of 7.0. The α-HL nanopore is oriented to capture the Xpandomer from the stem side first. This orientation is advantageous to use the translocation control method since it results in fewer blockage artifacts when first entering the antrum. When a small voltage, typically 100 mV, is applied across the bilayer, the nanopore constrains the flow of ionic current and is the primary resistance in the circuit. Upon passing through the nanopore, each of the linearized Xpandomer reporter constructs (labeled in this figure as "G," "C," and "T") generates a distinct and reproducible electronic signal (shown by the overlaid trace 290) specific to the nucleobase to which it is linked. EXAMPLES

[0049] Example 1 Assembly and purification of α-hemolysin nanopores on nanodisc supports Nanodisc formation

[0050] This example describes the reconstitution of native α-hemolysin nanopore protein in lipid nanodiscs and purification of the nanopore-nanodisc complex for assimilation of the native nanopore protein in lipid membranes.

[0051] Nanopore-Nanodisc complexes were formed by incubating α-hemolysin protein, MSP protein, and DPhPC lipid together in 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. Wild-type α-hemolysin protein was obtained from Sigma, and a 20 μM stock solution (calculated for the heptameric form) was prepared in 50% glycerol / 50% water. A 50 mM stock solution of DPhPC (available from Avanti Polar Lipids) was prepared in 20 mM Tris, pH 7.4, supplemented with 100 mM sodium cholate. 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 contained 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 we found to favor the assembly of α-HL / DPhPC / MSP Nanodisc complexes. The assembly mixture was incubated at room temperature for 60 minutes. To remove the detergent, 78.8 mg Biobeads SM-2 (available from BioRad) was added and the mixture was shaken at 1200 rpm for 2.5 hours at room temperature. The beads were removed by passing the mixture through a 45 μ filter. Nanopore-nanodisc complex

[0052] To isolate nanopore-Nanodisc complexes containing native heptameric α-HL protein, size-exclusion chromatography (SEC) was first performed using a Superdex 200 Increase column (commercially available from GEH), which was selected based on the predicted size of the complex of interest. The column was equilibrated with MSP buffer (20 mM Tris, pH 7.4, 100 mM NaCl, and 0.5 mM EDTA), and 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 FIG. 13, with the two fractions collected designated as "1" and "2". The presence of heptameric α-HL protein in fraction 2 was confirmed by gel electrophoresis.

[0053] Next, a Ni-NTA purification step was performed. Column resin (commercially available from Qiagen) was prepared by adding 50 μL of resin to an empty Ni-NTA spin column, and the column was spun at 700×g for 2 min to remove the storage buffer. The column was then equilibrated with 400 μL of EQ buffer containing 20 mM Tris, pH 7.5 and 10 mM imidazole. The EQ buffer was removed by spinning the column at 700×g for 2 min. The F2 Nanodisc sample was then added to the column, and the column contents were mixed by fixing the column on an end-over-end rotator for 15 min. The column was centrifuged at 700×g for 2 min and washed three times with 700 μL of wash buffer containing 20 mM Tris, pH 7.5 and 25 mM imidazole. The His-tagged protein / Nanodisc assemblies were then eluted by adding 150 μL of buffer containing 20 mM Tris, pH 7.5 and 250 mM imidazole. The column was incubated for 5 minutes and then the eluate was collected by centrifuging the column at 700 xg for 2 minutes.

[0054] The effectiveness of the purification process was monitored and assessed by analysis of the following samples by gel electrophoresis: load sample (1 μL of sample applied to the SEC column); sample 1 (15 μL of fraction 1 collected from the SEC column from 16:30 to 17:55); sample 2 (15 μL of fraction 2 collected from the SEC column from 19:00 to 20:45); FT sample (15 μL of column flow-through); samples W1, W2 and W3 (15 μL each of the first, second and third wash samples from IMAC); and sample E1 (15 μL of sample eluted from IMAC). A representative gel is shown in Figure 3. Arrows indicate the position of the heptameric α-HL protein and the MSP protein. These results confirm the successful assembly and purification of α-HL nanopore-nanodisc complexes containing native nanopore proteins. The faint band on the gel representing monomeric α-HL protein may be the result of dissociation of native heptameric oligomers when the protein samples are run in the gel.

[0055] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation that removes any subject matter from the genus, regardless of whether the excised material is specifically described herein.

[0056] It is also to be understood that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise, and the term "X and / or Y" means "X" or "Y," or both "X" and "Y," and the letter "s" following a noun indicates both the plural and the singular form of the noun. Furthermore, when features or aspects of the invention are described in terms of a Markush group, the invention is intended to encompass and be described in terms of any individual members of the Markush group and any subgroups of members, as will be recognized by those of skill in the art, and applicants reserve the right to amend this application or claims to specifically refer to any individual member or any subgroup of members of the Markush group.

[0057] 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 be further understood that, unless specifically defined herein, terms used herein are to be given their conventional meaning as known in the relevant art.

[0058] References throughout this specification to "one embodiment" or "an embodiment" and variations thereof mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0059] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents, i.e., one or more, unless the content and context clearly dictate 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 that includes at least one sensor, and a detection device comprising a sensor may, for example, include one sensor, ten sensors, ten 2 Sensors, 10 3 Sensors, 10 4 Sensors, 10 5 Sensors, 10 6 10 sensors or 6 A sensor may have more than one sensor. A plurality of sensors refers to more than one sensor. It should also be noted that the conjunctive terms "and" and "or" are generally used in the broadest sense to include "and / or," unless the content and context clearly dictate inclusiveness or exclusiveness, as the case may be. Thus, the use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. Furthermore, when described herein as "and / or," the "and" and "or" configurations are intended to encompass embodiments that include all associated items or ideas, as well as one or more other alternative embodiments that include fewer than all associated items or ideas.

[0060] Unless the context requires otherwise, throughout the specification and claims which follow, the word "comprise" and its synonyms and variations, such as "have" and "include," and variations thereof, such as "comprises" and "comprising," are to be construed in an open and inclusive sense, e.g., "including, but not limited to." The term "consisting essentially of" limits the claim to certain materials or steps or those that do not materially affect the basic and novel characteristics of the claimed invention.

[0061] Any headings used within this document are merely utilized to facilitate the reader's review thereof and should not be construed as limiting the scope of the invention or the claims in any way. Thus, the headings and abstracts of the disclosure provided herein are merely for convenience and do not interpret the scope or meaning of the embodiments.

[0062] Where a range of values ​​is provided herein, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in the stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the invention.

[0063] For example, any concentration range, percentage range, ratio range, or integer range provided herein should be understood to include any integer within the stated range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers), unless specifically stated otherwise. Also, any numerical range described herein for any physical characteristic, such as polymer subunits, size, or thickness, should be understood to include any integer within the stated range, unless specifically stated otherwise. As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless specifically stated otherwise.

[0064] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet, including but not limited to U.S. Provisional Patent Application No. 62 / 928,207, filed October 30, 2019, are incorporated herein by reference in their entirety. Such documents may be incorporated by reference, for example, for the purpose of describing and disclosing the materials and methodologies described in the publications that may be used in connection with the inventions described herein. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publications by prior invention.

[0065] All patents, publications, scientific articles, websites, and other documents and materials referenced or mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is incorporated by reference herein to the same extent as if it were individually incorporated by reference in its entirety or in its entirety as if set forth herein. Applicant reserves the right to physically incorporate into this specification any and all materials and information from such patents, publications, scientific articles, websites, electronically available information, and other referenced materials or documents.

[0066] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but rather to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

[0067] Moreover, the description portion of this patent includes all claims. Moreover, all claims, including all original claims and all claims from any and all priority documents, are incorporated by reference in their entirety into the specification portion of this specification, and the applicant reserves the right to physically incorporate any and all such claims into the specification or other portions of this application. Thus, for example, a patent may not be construed under any circumstances as not providing a specification of a claim for an allegation that the exact wording of the claim is not set forth haec verba in the specification portion of the patent.

[0068] The claims are to be interpreted according to law. However, regardless of whether a claim or a portion thereof is asserted or recognized to be easy or difficult to interpret, under no circumstances should any adjustment or modification of a claim or a portion thereof during the prosecution of an application or application leading to a patent be construed as a waiver of any and all equivalents thereof that do not form part of the prior art.

[0069] Other non-limiting embodiments are within the scope of the following claims. The patent should not be construed as limited to the particular examples or non-limiting embodiments or methods specifically and / or expressly disclosed herein. Under no circumstances should this patent be construed as limited by any statements made by any examiner or other officer or employee of the Patent and Trademark Office, unless such statements are specifically and expressly qualified or reserved in the applicant's reply brief.

Claims

1. 1. A method of making a sensing device comprising one or more native nanopore proteins, comprising: (a) forming an aqueous mixture comprising a nanopore protein, a membrane scaffolding protein (MSP), and a first lipid to produce a sample of Nanodisc-nanopore protein complexes, wherein each population of Nanodisc-nanopore protein complexes in the sample comprises a native nanopore protein; (b) providing a solid support comprising one or more openings, a membrane formed over each of said openings, said membrane comprising a second lipid, said membrane separating a cis chamber from a trans chamber in said detection device; and (c) contacting the one or more membranes with the population of nanopore-Nanodisc complexes comprising the native nanopore protein to assimilate the native nanopore protein into each of the membranes; Including, the aqueous mixture further comprises a detergent, the detergent having a final concentration of 14 mM±20% to 40 mM±20%; the first lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), the MSP is MSP1D1 or a mutant of MSP1D1, the nanopore protein is α-hemolysin (α-HL) or a mutant of α-HL, the detergent is cholate, and the second lipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE); the lipid to MSP to nanopore protein molar ratio is 101±20%:6±20%:1±20% or 120±20%:6±20%:1±20%; The method.

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

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

4. 2. The method of claim 1, wherein the solid support comprises a plurality of openings, a membrane is formed over each of the plurality of openings, and each of the membranes is in contact with the nanopore-Nanodisc complex that comprises the native nanopore protein.

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

6. The method of claim 5 , wherein the polymer is an Xpandomer.

7. 1. A method for forming a native nanopore protein in a membrane, comprising: (a) forming an aqueous mixture comprising a nanopore protein, a membrane scaffolding protein (MSP), and a first lipid to produce a sample of Nanodisc-nanopore protein complexes, wherein each of the population of Nanodisc-nanopore protein complexes comprises a native nanopore protein; (b) providing a membrane comprising a second lipid; and (c) contacting the membrane with the population of nanopore-Nanodisc complexes comprising the native nanopore protein to assimilate the native nanopore protein into the membrane; Including, further comprising purifying said population of nanopore-Nanodisc complexes comprising said native nanopore protein from said aqueous mixture prior to contacting said membrane with said population of nanopore-Nanodisc complexes comprising said native nanopore protein; the aqueous mixture further comprises a detergent, the detergent having a final concentration of greater than 14 mM to 40 mM; the first lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), the MSP is MSP1D1 or a mutant of MSP1D1, the nanopore protein is α-hemolysin (α-HL) or a mutant of α-HL, the detergent is cholate, and the second lipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE); the lipid to MSP to nanopore protein molar ratio is 101±20%:6±20%:1±20% or 120±20%:6±20%:1±20%; The method.

8. 8. The method of claim 7, wherein purifying the population of nanopore-nanodisc complexes comprises one or both of size exclusion chromatography and immobilized metal affinity chromatography.

9. 1. A composition comprising a nanopore-Nanodisc complex in an aqueous buffer, the nanopore-Nanodisc complex comprising a native nanopore protein, a membrane scaffolding protein (MSP), and a lipid, the aqueous buffer comprising a detergent; the native nanopore protein is α-hemolysin (α-HL) or a mutant of α-HL, the MSP is MSP1D1 or a mutant of MSP1D1, the lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), and the detergent is cholate; the lipid to MSP to nanopore protein molar ratio is 101±20%:6±20%:1±20% or 120±20%:6±20%:1±20% and the cholate concentration is greater than 14 mM to 40 mM; The composition.

10. 1. A composition comprising a lyophilized powder comprising a nanopore-Nanodisc complex, the nanopore-Nanodisc complex comprising a native nanopore protein, a membrane scaffolding protein (MSP), and a lipid; the native nanopore protein is α-hemolysin (α-HL) or a mutant of α-HL, the MSP is MSP1D1 or a mutant of MSP1D1, and the lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); the lipid to MSP to nanopore protein molar ratio is 101±20%:6±20%:1±20% or 120±20%:6±20%:1±20%; The composition.

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