WO2018213372A1 - Nucleic acid-lined nanodiscs - Google Patents

Abstract

Provided herein, in some embodiments, are nucleic acid-lined nanodiscs and methods and kits for producing the nucleic acid-lined nanodiscs.

Description

NUCLEIC ACID-LINED NANODISCS

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 62/506,796, filed May 16, 2017, which is incorporated by reference herein in its entirety.

FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under Contract No. W91 INF- 12-1- 0420 awarded by the Army Research Office, Contract No. 1317694 awarded by National Science Foundation, and Contract Nos. F32GM113406 and AI037581 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

A nanodisc is a synthetic model membrane system that may be used in studies of membrane proteins. Typically, a nanodisc is composed of a lipid bilayer of phospholipids with a hydrophobic edge wrapped by two amphipathic/amphiphilic proteins, referred to as membrane scaffolding proteins (MSPs).

SUMMARY

Provided herein, in some aspects, are compositions, methods and kits for producing large nanodiscs (e.g., having a diameter of 20-100 nm) lined by nucleic acid nano structures. Smaller, conventional nanodiscs, each of which is composed of a lipid bilayer wrapped by at least two copies of a MSP ("MSP-lined nanodisc"), typically are used in vitro to study membrane proteins. MSP-lined nanodiscs, however, are limited by their size (e.g., -15 nm diameter), which is limited by the length of the MSPs. The larger, more stable nanodiscs of the present disclosure are useful for studying larger membrane proteins (e.g., complexes), lipid rafts, and virus entry, for example, which are difficult to study using conventional MSP-lined nanodisc. Thus, present disclosure provides, in some embodiments, compositions, kits, and methods for producing large (e.g., greater than 20 nm) nanodiscs, the circumference of which are determined by the circumference of a nucleic acid nanostructure lining.

In some embodiments, a method of the present disclosure includes docking smaller MSP- lined nanodiscs within the cavity of a nucleic acid nanostructure. The MSPs of the smaller nanodiscs are, for example, functionalized at introduced amino acids (e.g., 2, 3, 4, 5, 6 or more cysteines or other amino acids) with nucleic acid "anti-handle" strands, which are capable of hybridizing to complementary nucleic acid "handle" strands lining the interior surface/cavity of the nucleic acid nanostructure. These smaller MSP-lined nanodiscs can be fused to form larger nanodiscs, for example, by reacting the smaller MSP-lined nanodiscs with lipid molecules and detergent, followed by reconstitution into the larger nucleic acid-lined nanodiscs. The detergent is then removed via dialysis, for example.

In other embodiments, a method of the present disclosure includes docking amphiphilic peptides, which can fold into smaller nanodiscs, within the cavity of a nucleic acid nanostructure. These peptides can be assembled inside the nucleic acid nanostructure through hybridization between nucleic acid anti-handles conjugated onto a peptide terminal domain and nucleic acid handles lining the interior surface/cavity of the nucleic acid nanostructure.

Experimental results provide herein shows that these larger nucleic acid-lined nanodiscs may be used (e.g., in combination with cryo-electron microscopy (EM)) to study viral entry (e.g., poliovirus entry) processes, which may be useful, for example, for therapeutic drug design to treat virus infection. These nanodiscs are also useful for studying the structure and/or function of membrane proteins, such as membrane protein receptors.

Thus, some aspects of the present disclosure provide a nucleic acid-lined nanodisc comprising: a lipid bilayer; and a cylindrical nucleic acid nanostructure, wherein the lipid bilayer is surrounded by the cylindrical nucleic acid nanostructure (see, e.g., FIG. 1A).

In some embodiments, wherein the lipid bilayer is attached to the cylindrical nucleic acid nanostructure, and optionally wherein the lipid bilayer is non-covalently attached to the cylindrical nucleic acid nanostructure. For example, the lipid bilayer may be attached to the cylindrical nucleic acid nanostructure through hybridization of lipid bilayer-linked nucleic acid handle strands bounds to nanostructure-linked nucleic acid anti-handle strands (See, e.g., FIG. 1A).

In some embodiments, the nucleic acid-lined nanodisc has an outer diameter of at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 150 nm, or at least 200 nm. For example, the nucleic acid-lined nanodisc may have an outer diameter of 50 nm to 250 nm. In some embodiments, the nucleic acid-lined nanodisc has an outer diameter of 200 to 1000 nm.

In some embodiments, the nucleic acid-lined nanodisc comprises a protein inserted in the lipid bilayer. The protein may be, for example, a membrane protein, such as a transmembrane protein. In some embodiments, the nucleic acid-lined nanodisc comprises a heterogeneous population of proteins (e.g., two or more different types of proteins) inserted in the lipid bilayer (e.g., traverses the lipid bilayer).

In some embodiments, a virus (e.g., a poliovirus) is bound to the nucleic acid lined nanodisc. In some embodiments, the nanodisc further comprises a pore.

In some embodiments, the cylindrical nucleic acid nanostructure is assembled from

DNA.

Also provided herein is a method of producing a nucleic acid-lined nanodisc, comprising (a) attaching membrane scaffold protein (MSP)-lined nanodiscs to a cylindrical nucleic acid nanostructure, wherein each MSP-lined nanodisc comprises a lipid bilayer and MSPs; (b) producing a nucleic acid nanostructure lined with the MSP-lined nanodiscs; (c) combining in a solution lipid molecules and detergent with the nucleic acid nanostructure lined with the MSP- lined nanodiscs; and (d) producing a nucleic acid-lined nanodisc.

In some embodiments, step (d) comprises removing the detergent from the solution. For example, the detergent may be removed by dialysis.

In some embodiments, the MSP-lined nanodiscs have an outer diameter of 5 nm to 15 nm. For example, the MSP-lined nanodiscs may have an outer diameter of 10 to 12 nm. In some embodiments, the MSP-lined nanodiscs have an outer diameter of 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, or 15 nm.

In some embodiments, nucleic acid handle strands are attached to an interior surface of the cylindrical nucleic acid nanostructure, nucleic acid anti-handle strands are attached to the MSP-linked nanodiscs, and the nucleic acid handle strands bind to the nucleic acid anti-handle strands. A pair of handle and anti-handle strands are any two single- stranded nucleic acids that bind to each other to bring two entities close to each other (e.g., a handle strand is linked to one entity while the anti-handle strand is linked to another entity).

In some embodiments, 5-100 nucleic acid handle strands are attached to the interior surface of the nucleic acid nanostructure. In some embodiments, at least three nucleic acid anti- handle strands are attached to each of the MSP-lined nanodiscs.

In some embodiments, the lipid molecules comprise at least one of l-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and cholesterol. In some embodiments, the lipid molecules comprise a mixture of POPC, POPG, and cholesterol (e.g., 5% to 15%, such as 10% cholesterol).

In some embodiments, the detergent comprises octyl glucoside. Other detergents may be used. Also provided herein is a nucleic acid-lined nanodisc produced by any one of the methods of the present disclosure. In some embodiments, the nucleic acid-lined nanodisc has an outer diameter of at least 50 nm, at least 100 nm, or at least 200 nm.

Further provided herein is an array comprising a plurality of the nucleic acid-lined nanodiscs of the present disclosure bound to each other, wherein the lipid bilayer can rotate freely (e.g., 360°) within the cylindrical nucleic acid nanostructure (see, e.g., FIG. 10, right image). In some embodiments, the lipid bilayer is attached to the cylindrical nucleic acid nanostructure through hybridization of two opposing lipid bilayer-linked nucleic acid handle strands bounds to two opposing nano structure-linked nucleic acid anti-handle strands.

In some embodiments, the nucleic acid-lined nanodiscs are bound to each other through molecular interactions between molecules bound to an exterior surface of the cylindrical nucleic acid nanostructures. The molecules may be handle and anti-handle strands (e.g., strand pairs) or proteins that bind to each other, such as a ligand-ligand or ligand-receptor binding pair (e.g., streptavidin and biotin).

Also provided herein is a two-dimensional array comprising a plurality of membrane scaffold protein (MSP)-lined nanodiscs, wherein nucleic acid strands are linked to an exterior surface of the MSP nanodiscs, and wherein the MSP nanodiscs are linked to each other though binding of the nucleic acids strands to each other (see, e.g., FIG. 10, left image).

Further provided herein is a composition comprising two nucleic acid-lined nanodiscs of the present disclosure. In some embodiments, one of the nucleic acid-lined nanodiscs is stacked (e.g., vertically) on top of the other of the nucleic acid-lined nanodiscs (see, e.g., FIG. 11).

In some embodiments, the nucleic acid-lined nanodisc further comprises a plurality of V- shaped heterodimers, wherein each of the V-shaped heterodimers comprises two monomers linked to each other (see, e.g., FIG. 12). In some embodiments, the two monomers are linked to each other by a nucleic acid bridge strand (e.g., a single-stranded nucleic acid).

In some embodiments, the lipid bilayer further comprises one or more flippase(s). For example, the one or more flippase(s) may be selected from ABCA1, ABCA4, and ABCA7.

In some embodiments, the cylindrical nucleic acid nanostructure further comprises one or more one or more translocase(s). For example, the one or more translocase(s) may be selected from ABC translocases.

In some embodiments, a styrene maleic acid (SMA) co-polymer is lined to an interior surface of the cylindrical nucleic acid nanostructure (see, e.g., FIG. 15). In some embodiments, the SMA co-polymer is terminally modified with a cyano group and is coupled to a nucleic acid strand that is modified with an azido group. BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1B. DNA-corralled nanodisc reconstitution. (FIG. 1A) Addition of detergent and free lipid molecules (POPC/POPG plus 10% cholesterol) to small nanodisc-decorated barrels (90 nm outer diameter) followed by dialysis results in the reconstitution of DNA-corralled nanodisc (DCND). Each DNA barrel is decorated with ssDNA overhang as handles to hybridize with ssDNA chemically conjugated small nanodisc. (FIG. IB) Negative- stain TEM images of DNA-origami barrel, with 70 nm inner diameter, before (top left) and after (top right) assembly with small nanodiscs. TEM images after dialysis to remove detergent, forming integrated large sized nanodiscs (bottom right) and after ultracentrifugation (UC) to remove free lipid vesicles (bottom left). Scale bar, 100 nm

FIG. 2. A proposed model for the assembly of the large bilayer within DNA-origami barrels (90 nm outer diameter). Bringing the 11-nm nanodiscs close to each other to within several nanometers is a critical first step. Adding excess lipids solubilized in detergent destabilizes and induces fusion of the neighboring nanodiscs. Next, the intermediate-sized nanodiscs fuse, perhaps aided by transient deformation of the barrel into an ellipsoid contour, to yield a single large bilayer nanodisc. Scale bar, 50 nm.

FIGs. 3A-3C. Reconstruction of hVDAC-1 and RC clusters within DCND. (FIG. 3A) Addition of lipids solubilized in detergent and hVDAC- 1 to small nanodisc-decorated DNA- origami barrels (90 nm outer diameter), followed by dialysis, leads to reconstitution of hVDACl clusters within DCND. Multimeric assemblies of voltage-dependent anion channel (VDAC) can be formed within the nanodiscs (right). (FIG. 3B) Typical DCND containing multimeric assemblies of VDAC. (FIG. 3C) Comparison of empty, RC and VDAC containing DCND. Scale bar, 50 nm.

FIG. 4. Poliovirus interactions with DCND containing CD 155 receptor. (Left) Negative- stain TEM images of DCND (60 nm outer diameter) containing CD 155 ectodomain interacting with poliovirus. Scale bar, 100 nm. (Right) TEM images showing individual viral particles tethered to DCND. Some images show the bending of the bilayer and the creation of a pore in nanodisc by the poliovirus (top right). Scale bar, 50 nm.

FIGs. 5A-5B. Coupling of DNA oligos to nanodisc. (FIG. 5A) SDS-PAGE of SMCC and SPDP coupling. SMCC coupling resulted in better yield. (FIG. 5B) Size exclusion chromatography was performed to purify oligo-nanodiscs from free oligos and aggregates.

FIG. 6. TEM characterization of DCND reconstituted inside 90-nm barrel. Negative- stain images show the formation of integrated large sized nanodiscs inside the DNA barrel. The image also shows the formation of free lipid vesicles outside the DNA barrels, which can be removed after ultracentrifugation. The POPC/POPG lipid mixture was used in the reconstitution of DCND.

FIGs. 7A-7C. TEM characterization of 60-nm DNA-origami-barrel without a bilayer. (FIG. 7A) Negative stain EM for the 60 nm. (FIG. 7B) Cryo-EM of empty 60 nm DCND particles (lacking membranes). The image shows the side and top views side by side. (FIG. 7C) The dimensions of DNA origami barrel.

FIG. 8 (top) shows a negative- stain image by TEM analysis for the 60 nm DCND (outer diameter). FIG. 8 (bottom) shows the dimensions of the 60 nm DCND.

FIGs. 9A-9D. Cryo-EM analysis of the 60 nm DCND with and without poliovirus. (FIG. 9A) Membrane-free DNA barrel. (FIG. 9B) DCND particles. The yellow arrows point to the lipid bilayer boundaries. (FIG. 9C) Poliovirus plus DCND. The bilayer is partially separated from the DNA. (FIG. 9D) The bilayer is tilted within the DNA barrel.

FIG. 10. Formation of array of MSP nanodiscs. Left, oligonucleotide-functionalities on 10 nm MSP nanodiscs enable self-assembly into 2D arrays. Middle left, negative-stain TEM image of an array. Right, 87-nm outer diameter DNA-origami barrels programmed with exterior functionalities that enable hexagonal-lattice formation, and modified interior functionalities that allow detachment of all but two opposed handle- anti-handle connections to the enclosed 60 nm MSP nanodisc. This enables free rotation of the nanodisc within the barrel.

FIG. 11. Left, schematic of DNA-barrel-scaffolded double-decker MSP nanodisc. Right, cryoEM images of single-layer DNA-origami barrel component.

FIG. 12. Proposed DNA-arena-scaffolded lipid-nanodisc reconstitution. Left, schematic of DNA-origami arena formed as a self-limiting ring from V-blocks each assembled as a heterodimer of DNA-origami blocks (blue and orange). The opening angle of the rigid V-block determines the size the assembled arena. The interior of the arena is decorated with ssDNA handles for capture of the 10-nm-diameter MSP nanodiscs as in FIG. 10. Right, negative- stain TEM images of ~200-nm inner-diameter arenas. Current efforts are underway to reconstitute large lipid nanodiscs within the interior of these DNA-origami arenas.

FIG. 13 Procedures for preparing nanodiscs containing parallel oriented membrane proteins.

FIG. 14. Controlled reconstitution of ABC transporters into DNA-corralled nanodiscs. The formation of ABC complex with defined stoichiometry and orientation by base-pairing of designated, complementary oligonucleotides attached to each ABC flippase through UV cleavable linker. The DNA-templated complex assembly will be carried out in detergent. After inserting into nanodisc, the DNA oligo attached to ABC flippases will be cleaved if desired. FIG. 15. DNA-origami barrel decorated with SMA.

FIG. 16 is a schematic depicting some of the advantages associated with nucleic acid- lined nanodiscs, including their ability to release or crosslink the lipid bilayer and their increased stability. Note that the nucleic acid (e.g., DNA) scaffold prevents the bilayers from coalescence and improves water solubility.

FIG. 17 (top) shows a method of making nucleic acid-lined nanodiscs using oligo peptides. FIG. 17 (bottom) also shows a schematic depicting the stoichiometric control of membrane protein reconstitution of the nucleic acid-lined nanodiscs, both in terms of type and number/copies of proteins.

FIG. 18 shows negative staining TEM image of 30-nm, 60-nm, and 90-nm DNA barrel origami scaffolded nanodisc reconstitution using short peptides.

DETAILED DESCRIPTION

Provided herein, in some aspects, are large (e.g., having a diameter of greater than 20 nm) nanodiscs, the diameter of which is determined by a nucleic acid nanostructure scaffold. Also provided herein are modular methods for manufacturing these large-sized nanodiscs using DNA-origami barrels as scaffolding corrals. Large-sized nanodiscs can be produced, in some embodiments, by first decorating the inside of DNA barrels with small lipid-bilayer nanodiscs, which open up when adding extra lipid to form large nanodiscs of diameters, for example, of -45 or -70 nm as prescribed by the enclosing barrel dimension. Densely packed membrane protein arrays can then be reconstituted within these large nanodiscs for potential structure

determination. The large nanodiscs of the present disclosure are assembled within a (e.g., cylindrical/barrel-like) nucleic acid nanostructure through a nucleation process that uses smaller MSP-lined nanodisc "seeds," additional supporting lipids and detergent. Once reconstituted and dialyzed (or otherwise purified), these larger nucleic acid-lined nanodiscs may be used to study, for example, large membrane protein complexes, lipid rafts, and the processes by which virus, e.g., polioviruses, enter cells.

Smaller, MSP-lined nanodiscs that are often used to study membrane protein, address many of the challenges associated with membrane protein solubilization and liposomal preparations. With nanodiscs, generally, the target membrane protein is transiently solubilized with a detergent in the presence of phospholipids and an encircling amphipathic helical protein belt, referred to as a membrane scaffold protein (MSP). When the detergent is removed, by dialysis or adsorption to hydrophobic beads, for example, the target membrane protein simultaneously assembles with phospholipids into a discoidal bilayer with the size controlled by the length of the MSP. The resultant nanodiscs thus keep target membrane proteins in solution, provide a native-like phospholipid bilayer environment that provides stability and functional requirements of the incorporated target membrane protein, and permit control of the oligomeric state of the target membrane protein. Unlike these smaller, MSP-lined nanodiscs, the larger nucleic acid-lined nanodiscs provided herein are not limited by the length of the MSP, but rather are controlled by the circumference and/or shape of the nucleic acid nanostructure within which the smaller functionalized MSP-lined nanodiscs assemble. A "nucleic acid-lined nanodisc," as used herein, refers to a lipid bilayer surrounded by a nucleic acid nanostructure (e.g., FIG. 1A, far right schematic). A "lipid bilayer" is a polar membrane made of two layers of lipid molecules, the structure of which is well known.

Nucleic Acid Nanostructures

MSP-lined nanodisc nucleation, or amphiphilic peptide nucleation, in some

embodiments, may be localized within a nucleic acid nanostructure. A "nucleic acid

nanostructure," as used herein, is an engineered nanostructure (e.g., having a size of less than 1 μιη) assembled from nucleic acids comprising nucleotide domains (regions) that hybridize to each other. Typically, nucleic acid nanostructures are also rationally-designed and artificial (e.g., non-naturally occurring). Nucleic acid nanostructures can self-assemble as a result of sequence complementarity encoded in nucleic acid strands that form that nanostructure. By pairing up complementary segments (through nucleotide base pairing), the nucleic acid strands self-organize under suitable conditions into a predefined nanostructure. Nucleic acid

nanostructures may be formed from a plurality (at least two) of nucleic acid strands encoded to hybridize to each other (see, e.g., N.C. Seeman, Nature 421, 427 (2003); International

Publication No. WO2013/022694; and International Publication No. WO2014/018675, each of which is incorporated herein by reference), or a nucleic acid nanostructure may be formed from a single strand of nucleic acid (see, e.g., International Application No. PCT/US2016/20893, incorporated herein by reference). Nucleic acid nanostructures typically have dimension (e.g., are two-dimensional or three-dimensional).

In some embodiments, a nucleic acid nanostructure has a length in each spatial dimension, and is rationally designed to self-assemble (is programmed) into a pre-determined, defined shape (e.g., a cylinder/barrel) that would not otherwise assemble in nature. The use of nucleic acids to build nanostructures is enabled by strict nucleotide base pairing rules (e.g., A binds to T, G binds to C, A does not bind to G or C, T does not bind to G or C), which result in portions of strands with complementary base sequences binding together to form strong, rigid structures. This allows for the rational design of nucleotide base sequences that will selectively assemble (self-assemble) to form nanostructures. A cylindrical nucleic acid nanostructure (having a barrel-like structure), as depicted in Fig. 1A, is an example of a 3D nucleic acid nanostructure.

Nucleic acid nanostructures are typically nanometer-scale structures (e.g., having a length scale of 1 to 1000 nanometers (nm)), although the term "nanostructure" may also encompass micrometer- scale structures (e.g.,1 to 10 micrometers (μιη)). In some embodiments, a micrometer- scale structure is assembled from more than one nanometer- scale or micrometer- scale structure. In some embodiments, a nucleic acid nanostructure has a length scale of 1 to 1000 nm, 1 to 900 nm, 1 to 800 nm, 1 to 700 nm, 1 to 600 nm, 1 to 500 nm, 1 to 400 nm, 1 to 300 nm, 1 to 200 nm, 1 to 100 nm or 1 to 50 nm. In some embodiments, a nucleic acid nanostructure has a length scale of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μιη. In some embodiments, a nucleic acid nanostructure has a length scale of greater than 1000 nm. In some embodiments, a nucleic acid nanostructure has a length scale of 1 μιη to 2 μιη. In some embodiments, a nucleic acid nanostructure has a length scale of 200 nm to 2 μιη, or more.

In some embodiments, a nucleic acid nanostructure assembles from a plurality of different nucleic acids (e.g., single-stranded nucleic acids, also referred to as single-stranded tiles, or SSTs (see, e.g., Wei, B. et al. Nature, 485, 623-626, 2012, incorporated herein by reference). For example, a nucleic acid nanostructure may assemble from at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 nucleic acids. In some embodiments, a nucleic acid nanostructure assembles from at least 100, at least 200, at least 300, at least 400, at least 500, or more, nucleic acids. The term "nucleic acid" encompasses "oligonucleotides," which are short, single-stranded nucleic acids (e.g., DNA) having a length of 10 nucleotides to 200 nucleotides (also referred to in the art as "staple strands"). In some embodiments, an oligonucleotide has a length of 10 to 20 nucleotides, 10 to 30 nucleotides, 10 to 40 nucleotides, 10 to 50 nucleotides, 10 to 60 nucleotides, 10 to 70 nucleotides, 10 to 80 nucleotides, 10 to 90 nucleotides, 10 to 100 nucleotides, 10 to 150 nucleotides, or 10 to 200 nucleotides. In some embodiments, an oligonucleotide has a length of 20 to 50, 20 to 75 or 20 to 100 nucleotides. In some embodiments, an oligonucleotide has a length of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nucleotides.

In some embodiments, a nucleic acid nanostructure is assembled from single-stranded nucleic acids, double-stranded nucleic acids, or a combination of single-stranded and double- stranded nucleic acids (e.g., includes an end terminal single-stranded overhang). Nucleic acid nanostructures may assemble, in some embodiments, from a plurality of heterogeneous nucleic acids (e.g., oligonucleotides). "Heterogeneous" nucleic acids may differ from each other with respect to nucleotide sequence. For example, in a heterogeneous plurality that includes nucleic acids A, B, and C, the nucleotide sequence of nucleic acid A differs from the nucleotide sequence of nucleic acid B, which differs from the nucleotide sequence of nucleic acid C. Heterogeneous nucleic acids may also differ with respect to length and chemical compositions (e.g., isolated v. synthetic).

The fundamental principle for designing self-assembled nucleic acid structures (e.g., nucleic acid nanostructures) is that sequence complementarity in nucleic acid strands is encoded such that, by pairing up complementary segments, the nucleic acid strands self-organize into a predefined nanostructure under appropriate physical conditions. From this basic principle (see, e.g., Seeman N.C. J. Theor. Biol. 99: 237, 1982, incorporated by reference herein), researchers have created diverse synthetic nucleic acid structures (e.g., nucleic acid nanostructures) (see, e.g., Seeman N.C. Nature 421: 427, 2003; Shih W.M. et al. Curr. Opin. Struct. Biol. 20: 276, 2010, each of which is incorporated by reference herein). Examples of nucleic acid (e.g., DNA) nanostructures, and methods of producing such structures, that may be used in accordance with the present disclosure are known and include, without limitation, lattices (see, e.g., Winfree E. et al. Nature 394: 539, 1998; Yan H. et al. Science 301: 1882, 2003; Yan H. et al. Proc. Natl. Acad, of Sci. USA 100; 8103, 2003; Liu D. et al. J. Am. Chem. Soc. 126: 2324, 2004; Rothemund P.W.K. et al. PLoS Biology 2: 2041, 2004, each of which is incorporated by reference herein), ribbons (see, e.g., Park S.H. et al. Nano Lett. 5: 729, 2005; Yin P. et al. Science 321: 824, 2008, each of which is incorporated by reference herein), tubes (see, e.g., Yan H. Science, 2003; P. Yin, 2008, each of which is incorporated by reference herein), finite two-dimensional and three dimensional objects with defined shapes (see, e.g., Chen J. et al. Nature 350: 631, 1991;

Rothemund P. W. K., Nature, 2006; He Y. et al. Nature 452: 198, 2008; Ke Y. et al. Nano. Lett. 9: 2445, 2009; Douglas S. M. et al. Nature 459: 414, 2009; Dietz H. et al. Science 325: 725, 2009; Andersen E. S. et al. Nature 459: 73, 2009; Liedl T. et al. Nature Nanotech. 5: 520, 2010; Han D. et al. Science 332: 342, 2011, each of which is incorporated by reference herein), and macroscopic crystals (see, e.g., Meng J. P. et al. Nature 461: 74, 2009, incorporated by reference herein).

Examples of nucleic acid (e.g., DNA) nanostructures include, but are not limited to, DNA origami structures, in which a long scaffold strand (e.g., at least 500 nucleotides in length) is folded by hundreds (e.g., 100, 200, 200, 400, 500 or more) of short (e.g., less than 200, less than 100 nucleotides in length) auxiliary staple strands into a complex shape (Rothemund, P. W. K. Nature 440, 297-302 (2006); Douglas, S. M. et al. Nature 459, 414-418 (2009); Andersen, E. S. et al. Nature 459, 73-76 (2009); Dietz, H. et al. Science 325, 725-730 (2009); Han, D. et al. Science 332, 342-346 (2011); Liu, W. et al. Angew. Chem. Int. Ed. 50, 264-267 (2011); Zhao, Z. et al. Nano Lett. 11, 2997-3002 (2011); Woo, S. & Rothemund, P. Nat. Chem. 3, 620-627 (2011); T0rring, T. et al. Chem. Soc. Rev. 40, 5636-5646 (2011), incorporated by reference herein). Staple strands are complementary to and bind to two or more noncontiguous regions of a scaffold strand.

In some embodiments, a scaffold strand is 100-10000 nucleotides in length. In some embodiments, a scaffold strand is at least 100, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, or at least 10000 nucleotides in length. The scaffold strand may be naturally occurring or non- naturally occurring. In some embodiments, a single- stranded nucleic acid for assembly of a nucleic acid nanostructure has a length of 500 base pairs to 10 kilobases, or more. In some embodiments, a single- stranded nucleic acid for assembly of a nucleic acid nanostructure has a length of 4 to 5 kilobases, 5 to 6 kilobases, 6 to 7 kilobases, 7 to 8 kilobases, 8 to 9 kilobases, or 9 to 10 kilobases. Staple strands are typically shorter than 200 nucleotides in length; however, they may be longer or shorter depending on the application and depending upon the length of the scaffold strand (a staple strand is typically shorter than the scaffold strand). In some

embodiments, a staple strand may be 15 to 100 nucleotides, or 15 to 200 nucleotides, in length. In some embodiments, a staple strand is 25 to 50 nucleotides in length.

In some embodiments, a nucleic acid nanostructure may be assembled in the absence of a scaffold strand (e.g., a scaffold-free structure). For example, a number of oligonucleotides (e.g., less than 200 nucleotides or less than 100 nucleotides in length) may be assembled to form a nucleic acid nanostructure.

In some embodiments, a nucleic acid nanostructure may be assembled to form a self- limiting ring structure referred to herein as a 'DNA-arena-scaffolded lipid nanodisc' . These assembled structures involve the linking of multiple copies of DNA origami heterodimers into a pre-determined, defined shape (e.g., a cylinder/barrel). Each DNA origami heterodimer includes two distinct DNA origami units linked by DNA hybridization at low magnesium concentration to form a V-shaped heterodimer. In some embodiments, the diameter of this assembled

nanostructure is 200-1000 nm. In some embodiments, a cylindrical nucleic acid nanostructure has a diameter of 200-300 nm, 200-400 nm, 200-500 nm, 200-600 nm, 200-700 nm, 200-800 nm, 200-900 nm, or 200-1000 nm. In some embodiments, the diameter of this assembled nanostructure is greater than 1000 nm. some embodiments, the diameter of this assembled nanostructure is less than 200 nm.

In some embodiments, a nucleic acid nanostructure may be asymmetric with respect to lipid distribution. The incorporation of protein scramblases and flippases such as ABCA1, ABCA4 and ABCA7 into the lipid bilayer of the nanostructure allows for maintenance of the asymmetry.

In some embodiments, a nucleic acid nanostructure is assembled from single-stranded tiles (SSTs) (see, e.g., Wei B. et al. Nature 485: 626, 2012, incorporated by reference herein) or nucleic acid "bricks" (see, e.g., Ke Y. et al. Science 388: 1177, 2012; International Publication Number WO 2014/018675 Al, published January 30, 2014, each of which is incorporated by reference herein). For example, single- stranded 2- or 4-domain oligonucleotides self-assemble, through sequence- specific annealing, into two- and/or three-dimensional nanostructures in a predetermined (e.g., predicted) manner. As a result, the position of each oligonucleotide in the nanostructure is known. In this way, a nucleic acid nanostructure may be modified, for example, by adding, removing or replacing oligonucleotides at particular positions. The nanostructure may also be modified, for example, by attachment of moieties, at particular positions. This may be accomplished by using a modified oligonucleotide as a starting material or by modifying a particular oligonucleotide after the nanostructure is formed. Therefore, knowing the position of each of the starting oligonucleotides in the resultant nanostructure provides addressability to the nanostructure.

Other methods for assembling nucleic acid nanostructures are known in the art, any one of which may be used herein. Such methods are described by, for example, Bellot G. et al, Nature Methods, 8: 192-194 (2011); Liedl T. et al, Nature Nanotechnology, 5: 520-524 (2010); Shih W.M. et al, Curr. Opin. Struct. Biol., 20: 276-282 (2010); Ke Y. et al, J. Am. Chem. Soc, 131: 15903-08 (2009); Dietz H. et al, Science, 325: 725-30 (2009); Hogberg B. et al, J. Am. Chem. Soc, 131: 9154-55 (2009); Douglas S.M. et al, Nature, 459: 414-418 (2009); Jungmann R. et al, J. Am. Chem. Soc, 130: 10062-63 (2008); Shih W.M., Nature Materials, 7: 98-100 (2008); and Shih W.M., Nature, 427: 618-21 (2004), each of which is incorporated herein by reference in its entirety.

While cylindrical nucleic acid nanostructures are described throughout the present disclosure, it should be understood that a nucleic acid nanostructure may be assembled into one of many defined and predetermined shapes, including without limitation a cylinder (barrel), a capsule, a hemi-sphere, a cube, a cuboidal, a tetrahedron, a cylinder, a cone, an octahedron, a prism, a sphere, a pyramid, a dodecahedron, and a tube. A nucleic acid nanostructure may be a geometric shape (easily recognizable, e.g., circle, triangle, rectangle, etc.) or may be an abstract shape (e.g., free-form, non-geometric curves, random angles, and/or irregular lines). It should be understood that a nucleic acid nanostructure is distinct from condensed nucleic acid (e.g., DNA having a solid or dense core) and may have a void volume (e.g., it may be partially or wholly hollow). In some embodiments, the void volume may be at least 10%, at least 15%, at least 20%, 25 %, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the volume of the nanostructure. The "void volume" of a nucleic acid nanostructure is the cumulative empty space (space not occupied by nucleic acid) within a nucleic acid nanostructure.

In some embodiments, nucleic acid nanostructures are rationally designed. A nucleic acid nanostructure is "rationally designed" if the nucleic acids that form the nanostructure are selected based on pre-determined, predictable nucleotide base pairing interactions that direct nucleic acid hybridization (for a review of rational design of DNA nanostructures, see, e.g., Feldkamp U., et al. Angew Chem Int Ed Engl. 2006 Mar 13;45(12): 1856-76, incorporated herein by reference). For example, nucleic acid nanostructures may be designed prior to their synthesis, and their size, shape, complexity and modification may be prescribed and controlled using certain select nucleotides {e.g., oligonucleotides) in the synthesis process. The location of each nucleic acid in the structure may be known and provided for before synthesizing a nanostructure of a particular shape. A cylindrical nucleic acid nanostructure, rationally designed to resemble the shape of a cylinder (e.g., a barrel shape- see, e.g., Fig. 1 A), is one example of a particular nucleic acid nanostructure.

A nucleic acid nanostructure, in some embodiments, may be assembled from more than one two-dimensional or three-dimensional nucleic acid nanostructure or more than one three- dimensional nucleic acid nanostructure (e.g., more than one "pre-assembled" nucleic acid nanostructure that is linked to one or more other "pre-assembled" nucleic acid nanostructure).

"Self-assembly" with respect to nucleic acids refers to the ability of nucleic acids (and, in some instances, pre-formed nucleic acid structures (e.g., nucleic acid nanostructures)) to anneal to each other, in a sequence- specific manner, in a predicted manner and without external control. In some embodiments, nucleic acid nanostructure self-assembly methods include combining nucleic acids (e.g., single-stranded nucleic acids, or oligonucleotides) in a single vessel and allowing the nucleic acids to anneal to each other, based on sequence complementarity. In some embodiments, this annealing process involves placing the nucleic acids at an elevated temperature and then reducing the temperature gradually in order to favor sequence- specific binding. Various nucleic acid structures (e.g., nucleic acid nanostructures) or self-assembly methods are known and described herein.

Nucleic acid nanostructures, in some embodiments, do not include coding nucleic acid. That is, in some embodiments, nucleic acid nanostructures are "non-coding" nucleic acid nanostructures (the structures are not formed from nucleic acids that encode other molecules). In some embodiments, less than 50% of the nucleic acid sequence in a nucleic acid nanostructure include coding nucleic acid. For example, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% of a nucleic acid nanostructure may include coding nucleic acid sequence.

Nucleic acid nanostructures may comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA), modified DNA, modified RNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), or any combination thereof. In some embodiments, a nucleic acid nanostructure is a DNA nanostructure. In some embodiments, a DNA nanostructure consists of DNA.

A cylindrical nucleic acid nanostructure is a nucleic acid nanostructure that forms an exterior surface and an interior compartment (having an interior surface). A cylindrical nucleic acid (e.g., DNA) nanostructure may be comprised, for example, of one or more smaller stacked cylindrical nanostructured (e.g., each with two open ends). In some embodiments, a cylindrical nucleic acid nanostructure comprises at least two or at least three smaller (e.g., shorter) cylindrical nanostructures linked together to form one large cylinder. An entire cylindrical nucleic acid nanostructure, in some embodiments, may be made using a single (or two or three) long scaffold strand and shorter staple strands (e.g., using the DNA origami method). Other nucleic acid nanostructure assembly methods may be used and are described elsewhere herein.

Generally, cylindrical nanostructures have an internal void volume that can be calculated by the formula: V = r a, where r is the radius of the cylinder and a is the height of the cylindrical part. The surface area formula is SA = 2 ra + 2 r .

The diameter (e.g., inner diameter) of a cylindrical nucleic acid nanostructure may be, for example, 10-200 nm. In some embodiments, a cylindrical nucleic acid nanostructure has a diameter of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, or 200 nm. In some embodiments, a cylindrical nucleic acid nanostructure has a diameter of 20-30 nm, 20-40 nm, 20-50 nm, 20-60 nm, 20-70 nm, 20-80 nm, 20-90 nm, 20-100 nm, 20-150 nm, 20-200 nm, 30-40 nm, 30-50 nm, 30-60 nm, 30-70 nm, 30-80 nm, 30-90 nm, 30-100 nm, 30-150 nm, or 30-200 nm. In some embodiments, a cylindrical nucleic acid nanostructure has a diameter of greater than 200 nm or less than 10 nm.

MSP-Lined Nanodiscs and Amphiphilic Peptides

The nucleic acid-lined nanodiscs of the present disclosure are produced, for example, by assembling multiple smaller MSP-lined nanodiscs within the interior (void/space) of a nucleic acid nanostructure (e.g., a cylindrical nanostructure). A "MSP-lined nanodisc," as used herein, refers to a discoidal, nanoscale phospholipid bilayer, stabilized by at least one membrane scaffold protein (MSP). For example, conventional MSP-lined nanodisc nanodiscs are composed of a nanometer- sized phospholipid bilayer encircled by two copies of a helical, amphipathic membrane scaffold proteins (MSPs) (Densiov et al. J Am Chem Soc. 2004;

126:3477-87 and Bayburt et al, Nano Lett. 2002;2:853-6). MSP-lined nanodiscs typically have a diameter (e.g., outer diameter) of 5-15 nm, although in some instances, the diameter of a MSP- lined nanodisc may be larger. The larger nucleic acid-line nanodiscs of the present disclosure are produced using multiple (e.g., at least two) smaller (e.g., 5 nm-15 nm) MSP-lined nanodiscs, in some embodiments. In other embodiments, the nucleic acid-line nanodiscs of the present disclosure are produced using multiple amphiphilic peptides instead of MSP-lined nanodiscs.

The nucleic acid-lined nanodiscs of the present disclosure may also be produced by assembling multiple amphiphilic peptides within the interior of a nucleic acid nanostructure. "Amphiphilic peptide," as used herein, refers to a protein having at least one hydrophobic and at least one hydrophilic region. Examples of amphiphilic peptides include, but are not limited to, hydrocarbon-based surfactants (e.g., sodium dodecyl sulfate, benzalkonium chloride,

cocamidopropyl betaine, and 1-octanol), phospholipids, cholesterol, glycolipids, fatty acids, bile acids, and saponins.

As indicated above, MSP-lined nanodiscs are composed of lipid molecules and membrane scaffold proteins (MSPs).

Lipid molecules. A variety of lipid molecules may be used to form MSP-lined nanodiscs and/or larger nucleic acid-lined nanodiscs, as discussed below. In some embodiments, lipid molecules include phospholipids. "Phospholipids" include phosphatidic acids,

phosphoglycerides, and phosphosphingolipids. Phosphatidic acids include a phosphate group coupled to a glycerol group, which may be monoacylated or diacylated. Phosphoglycerides (or glycerophospholipids) include a phosphate group intermediate an organic group (e.g., choline, ethanolamine, serine, inositol) and a glycerol group, which may be monoacylated or diacylated. Phosphosphingolipids (or sphingomyelins) include a phosphate group intermediate an organic group (e.g., choline, ethanolamine) and a sphingosine (non-acylated) or ceramide (acylated) group. The term "phospholipid" also includes salts (e.g., sodium, ammonium) of phospholipids. For phospholipids that include carbon-carbon double bonds, individual geometrical isomers (cis, trans) and mixtures of isomers are included.

Non-limiting examples of phospholipids include phosphatidylcholines,

phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, and phosphatidic acids, and their lysophosphatidyl (e.g., lysophosphatidylcholines and lysophosphatidylethanolamine) and diacyl phospholipid (e.g., diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, diacylphosphatidylserines, diacylphosphatidylinositols, and diacylphosphatidic acids) counterparts.

In some embodiments, the acyl groups of the phospholipids are the same. In other embodiments, the acyl groups of the phospholipids are different. In some embodiments, the acyl groups are derived from fatty acids having C 10-C24 carbon chains (e.g., acyl groups such as lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl groups). Representative

diacylphosphatidylcholines (i.e., l,2-diacyl-sn-glycero-3-phosphocholines) include

distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC),

dipalmitoylphosphatidylcholine (DPPC), dilinoleoylphosphatidylcholine DLPC),

palmitoyloleoylphosphatidylcholine (POPC), palmitoyllinoleoylphosphatidylcholine, stearoyllinoleoylphosphatidylcholine stearoyloleoylphosphatidylcholine,

stearoylarachidoylphosphatidylcholine, didecanoylphosphatidylcholine (DDPC),

dierucoylphosphatidylcholine (DEPC), dilinoleoylphosphatidylcholine (DLOPC),

dimyristoylphosphatidylcholine (DMPC), myristoylpalmitoylphosphatidylcholine (MPPC), myristoylstearoylphosphatidylcholine (MSPC), stearoylmyristoylphosphatidylcholine (SMPC), palmitoylmyristoylphosphatidylcholine (PMPC), palmitoylstearoylphosphatidylcholine (PSPC), stearoylpalmitoylphosphatidylcholine (SPPC), and stearoyloleoylphosphatidylcholine (SOPC).

Examples of diacylphosphatidylethanolamines (i.e., l,2-diacyl-sn-glycero-3- phosphoethanolamines) include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dilauroylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylethanolamine (DMPE), dierucoylphosphatidylethanolamine (DEPE), and

palmitoyloleoylphosphatidylethanolamine (POPE) .

Examples of diacylphosphatidylglycerols (i.e., l,2-diacyl-sn-glycero-3- phosphoglycerols) include, but are not limited to, dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dierucoylphosphatidylglycerol (DEPG), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), and palmitoyloleoylphosphatidylglycerol (POPG).

Examples of diacylphosphatidylserines (i.e., l,2-diacyl-sn-glycero-3- phosphoserines) include, but are not limited to, dilauroylphosphatidylserine (DLPS), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylserine (DPPS), and distearoylphosphatidylserine (DSPS).

Examples of diacylphosphatidic acids (i.e., l,2-diacyl-sn-glycero-3-phosphates) include, but are not limited to, dierucoylphosphatidic acid (DEPA), dilauroylphosphatidic acid (DLPA), dimyristoyiphosphatidic acid (DMPA), dioleoylphosphatidic acid (DOPA),

dipalmitoylphosphatidic acid (DPPA), and distearoylphosphatidic acid (DSPA).

Examples of phospholipids include, but are not limited to, phosphosphingolipids such as ceramide phosphoryllipid, ceramide phosphorylcholine, and ceramide phosphorylethanolamine.

MSP-lined nanodiscs may comprise one or more types of phospholipids. Thus, in some embodiments, a MSP-lined nanodisc may comprise two, three, four, or more, different types of phospholipids.

In some embodiments, a phospholipid is a native lipid extracts. In some embodiments, a phospholipid is a headgroup-modified lipid, e.g., e.g., alkyl phosphates (e.g. 16:0 Monomethyl PE; 18: 1 Monomethyl PE; 16:0 Dimethyl PE; 18: 1 Dimethyl PE; 16:0 Phosphatidylmethanol; 18: 1 Phosphatidylmethanol; 16:0 Phosphatidylethanol; 18: 1 Phosphatidylethanol; 16:0-18: 1 Phosphatidylethanol; 16:0 Phosphatidylpropanol; 18: 1 Phosphatidylpropanol; 16:0

Phosphatidylbutanol; and 18: 1 Phosphatidylbutanol); MRI imaging reagents (e.g.14:0 PE-DTPA (Gd); DTPA-BSA (Gd); 16:0 PE-DTPA (Gd); 18:0 PE-DTPA (Gd); bis(14:0 PE)-DTPA (Gd); bis(16:0 PE)-DTPA (Gd); and bis(18:0 PE)-DTPA (Gd)); chelators (e.g., 14:0 PE-DTPA (Gd); DTPA-BSA (Gd); 16:0 PEDTPA (Gd); 18:0 PE-DTPA (Gd); bis(14:0 PE)-DTPA (Gd); bis(16:0 PE)-DTPA (Gd); and bis(18:0 PE)-DTPA (Gd)); glycosylated lipids (e.g., 18: 1 Lactosyl PE); pH sensitive lipids (e.g., N-palmitoyl homocysteine; 18: 1 DGS; 16:0 DGS; and DOBAQ); antigenic lipids (e.g., DNP PE and DNP Cap PE); adhesive lipids (e.g., 16:0 DG Galloyl); and

functionalized lipids (e.g., 16:0 PA-PEG3-mannose; 16:0 Caproylamine PE; 18: 1 Caproylamine PE; 16:0 MPB PE; 18: 1 MPB PE; 16:0 Ptd Ethylene Glycol; 18: 1 Ptd Ethylene Glycol; 16:0 Folate Cap PE; 16:0 Cyanur PE; 16:0 Biotinyl PE; 18: 1 Biotinyl PE; 16:0 Biotinyl Cap PE; 16:0 Cyanur Cap PE; 18: 1 Biotinyl Cap PE; 16:0 Dodecanoyl PE; 18: 1 Dodecanyl PE; 16:0 Glutaryl PE; 18: 1 Glutaryl PE; 16:0 Succinyl PE; 18: 1 Succinyl PE; 16:0 PDP PE; 18: 1 PDP PE; 16:0 Ptd Thioethanol; 18: 1 Dodecanylamine PE; 16:0 Dodecanylamine PE; 16:0 PE MCC; 18: 1 PE MCC; 16:0 hexynoyl PE; and 16:0 azidocaproyl PE). In some embodiments, a phospholipid may further comprise a protein/molecule of interest (e.g., a membrane protein, a receptor, a transmembrane protein or channel, hydrophobic small molecules, hydrophobic drugs, RNA, and/or peptides). Other molecules of interest may be used.

Additional lipids that may be used as provided herein include lipids of the Archaea and other extremophilic microorganisms (see, e.g., de Rosa M. et al. Biosensors & Bioelectronics 9 (1994) 669-675, incorporated herein by reference). Lipids of the liver iron concentration originate from the formation of two or four ether links between two vicinal hydroxyl groups of a glycerol or more complex polyol, and C20, C25, or C40 isoprenoidic alcohols. Non-limiting examples of archaean-type lipids include those with archaeol (diether) and/or caldarchaeol (tetraether) core structures (Kaur G. et al. Drug Deliv 23(7) (2016) 2497-2512, incorporated herein by reference). In some embodiments, the lipids are extracted from the thermophilic archaeobacterium Sulfolobus solfatarius (Cavagnetto F et al. Biochimica et Biophysica Acta, 1106 (1992) 273-281, incorporated herein by reference).

Membrane Scaffold Proteins (MSPs). The diameter of a MSP-lined nanodisc is typically determined by the size of the MSP that wraps around the phospholipid bilayer. MSPs may be used to stabilize a phospholipid bilayer in a lipid nanodisc. Typically, MSPs are amphipathic alpha helical proteins ("belts") that bind the phospholipid bilayer periphery, surrounding the bilayer. MSPs generally have hydrophobic faces that associate with the nonpolar interior of the phospholipid bilayer as well as hydrophilic faces, which interact with the aqueous exterior environment. In some embodiments, the MSPs do not completely encircle the MSP-lined nanodisc. In other embodiments, the MSPs do completely encircle the MSP-lined nanodisc. In some embodiments, a MSP-lined nanodisc is associated with 1, 2, 3, 4, 5, 6, 7, or more MSPs. MSPs may be naturally occurring (for example, apolipoproteins A, (A-I and A-II), B, C, D, E, and H), or engineered (for example, using recombinant technologies). Examples of MSP constructs include, but are not limited to MSP1, MSP1TEV, MSP1D1, MSP1D1-D73C, MSPIDI(-), MSP1E1, MSP1E1D1, MSP1E2, MSP1E2D1, MSP1E3, MSP1E3D1, MSP1E3D1- D73C, MSP1D1-22, MSP1D1-33, MSP1D1-44, MSP2, MSP2N2, MSP2N3, MSP1FC,

MSP1FN. Other examples of MSPs are known and commercially available (see, e.g., sigmaaldrich.com/life- science/biochemicals/biochemical-products .html?TablePage= 107362161 ) .

MSP-lined nanodiscs, in some instances, may be composed of MSP variants. In some embodiments, the MSP variants comprise introduced unnatural amino acids, for example, cysteine derivatives. Other examples of unnatural amino acids include, but are not limited to, alanine derivatives, alicyclic amino acids, arginine derivatives, aromatic amino acids, asparagine derivatives, aspartic acid derivatives, beta-amino acids, 2,4-diaminobutyric acid (DAB), 2,3- diaminopropionic acid, glutamic acid derivatives, glutamine derivatives, glycine derivatives, homo-amino acids, isoleucine derivatives, leucine derivatives, linear core amino acids, lysine derivatives, methionine derivatives, N-methyl amino acids, norleucine derivatives, norvaline derivatives, ornithine derivatives, penicillamine derivatives, phenylalanine derivatives, phenylglycine derivatives, proline derivatives, pyroglytamine derivatives, serine derivatives, threonine derivatives, tryptophan derivatives, tyrosine derivatives, and valine derivatives. Other examples of unnatural amino acids are known and commercially available (see, e.g.,

sigmaaldrich.com/chemistry/chemistry-products.html?TablePage=16274965).

For example, NanodiscWidth 11 nm (NW11) constructs may be used to engineer covalently circularized MSP-lined nanodiscs (see, e.g., Nasr, M. L. et al. Nature Methods, 14(1): 49-54, 2017, incorporated herein by reference in its entirety). The N and C termini of NW11 variants are covalently linked to each other to form a stable barrier. These MSP constructs contain the consensus sequence recognized by sortase A (LPGTG; SEQ ID NO: 2) near the C terminus and a single glycine residue at the N terminus (after TEV cleavage). The presence of these two sites ensures covalent linkage between the N and C termini of a protein while still conserving the function to form lipid nanodiscs.

NW11 Sequence:

MGSSHHHHHHENLYFQGSTFSKLREQLGPVTQEFWCNLEKETEGLRQEMSKDLEEVKA KVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGECMRDRA R AH VD ALRTHLAP YS DELRQRLA ARLE ALKENGG ARLAE YH AKATEHLS TLS EKAKP A LCDLRQGLLPVLESFKVSFLSALEEYTKKLNTQLPGTGAAALEHHHHHH (SEQ ID NO: 1).

Attaching MSP-lined Nanodiscs or Amphiphilic Peptides to Nucleic Acid Nanostructures

MSP-lined nanodiscs, or amphiphilic peptides, of the present disclosure may be attached, in a prescribed manner, to a nucleic acid nanostructure (e.g., the interior of a cylindrical nucleic acid nanostructure. In some embodiments, MSP-lined nanodiscs or amphiphilic peptides are coupled to a nucleic acid nanostructure via nucleic acid hybridization. For example, MSP-lined nanodiscs or amphiphilic peptides and the interior surface of a nucleic acid nanostructure may be "functionalized" with single- stranded (partially or wholly single-stranded) nucleic acids. These nucleic acid strands are referred to as "handle strands" and complementary "anti-handle strands." Thus, in some embodiments, a MSP-lined nanodisc comprises (e.g., is attached to) nucleic acid anti-handle strands that are complementary to nucleic acid handle strands attached to the interior surface of a nucleic acid nano structure. Through nucleotide base pairing the handle strands bind to the anti-handle strands, thereby linking the MSP-lined nanodiscs or amphiphilic peptides to the interior surface of the nucleic acid nanostructures. The terms "handle strand" and "anti- handle strand" are used to connote complementarity between two single-stranded nucleic acids. Thus, a nucleic acid strand located on a MSP-lined nanodisc or an amphiphilic peptide may be referred to as an anti-handle strand, which is complementary to and binds to a handle strand located on the interior surface of a nucleic acid nanostructure. Likewise, a nucleic acid strand located on a MSP-lined nanodisc or an amphiphilic peptide may be referred to as a handle strand, which is complementary to and binds to an anti-handle strand located on the interior surface of a nucleic acid nanostructure.

A handle strands (or anti-handle strands) may be attached to a nucleic acid nanostructure or a MSP-lined nanodisc or an amphiphilic peptide in a covalent or non-covalent manner. In some embodiments, a handle strand (or anti-handle strand) is attached to a nucleic acid nanostructure through hybridization to nucleotides within the nanostructure, while in other embodiments, a handle strand (or anti-handle strand) is attached to a nucleic acid nanostructure through interacting binding partner molecules (e.g., ligand-receptor binding molecules). In some instances, the binding partner molecules are biotin and streptavidin. Likewise, in some embodiments, a handle strand (or an anti-handle strand) is attached to a MSP-lined nanodisc through interacting binding partner molecules. Other binding partner molecules are apparent to those of ordinary skill in the art and may be used herein, including high affinity protein/protein binding pairs such as antibody/antigen and ligand/receptor binding pairs, hydrophobic interactions, π-π stacking or electrostatic interactions. In some embodiments, MSP-lined nanodiscs or amphiphilic peptides are located with a nucleic acid nanostructure through spatial confinement.

A MSP-lined nanodisc or amphiphilic peptide may be attached to more than one handle strand (or anti-handle strand). In some embodiments, a MSP-lined nanodisc or amphiphilic peptide is linked to 2, 3, 4, 5, or more, handle strands (or anti-handle strands).

The interior surface of a nucleic acid nanostructure may be attached to more than one handle strand (or anti-handle stand), depending in part on the diameter of the nanostructure. Thus, a nucleic acid-lined nanodisc having a diameter (e.g., outer diameter) of 20-200 nm may be composed of a nucleic acid nanostructure comprising 5-100 handle strands (or anti-handle strands). In some embodiments, a nucleic acid nanostructure has 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 handle strands (or anti-handle strands). In some embodiments, a nucleic acid nanostructure has 10-20, 10-30, 10-40 or 10-50 handle strands (or anti-handle strands).

In some embodiments, nucleic acid nanostructures and/or MSP-lined nanodiscs and/or amphiphilic peptides are coupled to handle strands (or anti-handle strands) through -SH groups, click chemistry, -N¾ groups, -COOH groups, π-π stacking, coordinating interaction

(Ni²⁺/polyhistidine), or through electrostatic interactions.

The length of a handle strand (or anti-handle strand) may vary. In some embodiments, a handle strand or anti-handle strand (or at least the single- stranded region of the handle/anti- handle strand) may have a length of 15 to 50 nucleotides. In some embodiments, a handle strand (or anti-handle strand) may have a length of 15, 20, 25, 30, 35, 40, or 50 nucleotides. Depending on the application, a handle strand (or anti-handle strand) may be have a length that is greater than 50 nucleotides.

MSP-lined nanodiscs or amphiphilic peptides may be arranged on or within a nucleic acid nanostructure to form a particular configuration or shape. In some embodiments, MSP- lined nanodiscs or amphiphilic peptides may be arranged on or within a cylindrical nucleic acid nanostructure to line the interior surface of the cylindrical nucleic acid nanostructure, as shown in Figs. 1A and 5. Particular configurations may be prescribed, for example, by positioning handles or other binding partner molecules at prescribed positions on or in the nucleic acid nanostructure.

In some embodiments, the handle strands are positioned so that they are equidistant from one another along the interior surface of the nucleic acid nanostructure. In other embodiments, the handle strands are positioned at different distances from one another along the interior surface of the nucleic acid nanostructure. A nucleic acid nanostructure may comprise, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more nucleic acid handle strands. In some embodiments, a nucleic acid nanostructure may have 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20 nucleic acid handle strands.

In some embodiments, the handle strands may be able to be disengaged from the anti- handle strand via strand displacement. In some embodiments, this capability will enable that only two handle strands positioned 180° apart will be attached to an anti-handle strand. In some embodiments, the nanodisc will be capable of free rotation within the nucleic acid nanostructure. In some embodiments, MSP-lined nanodiscs or amphiphilic peptides are attached (e.g., each attached) to 2, 3, 4, 5 or more anti-handle strands. The diameter (e.g., outer diameter) of a nucleic acid-lined nanodisc (e.g., a cylindrical nucleic acid-lined nanodisc) end product may be, for example, 10-200 nm. In some

embodiments, a nucleic acid-lined nanodisc has a diameter of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, or 200 nm. In some embodiments, a nucleic acid-lined nanodisc has a diameter of 20-30 nm, 20-40 nm, 20-50 nm, 20-60 nm, 20-70 nm, 20-80 nm, 20-90 nm, 20-100 nm, 20-150 nm, 20-200 nm, 30-40 nm, 30-50 nm, 30-60 nm, 30-70 nm, 30-80 nm, 30-90 nm, 30-100 nm, 30-150 nm, or 30-200 nm. In some embodiments, a nucleic acid-lined nanodisc has a diameter of greater than 200 nm or less than 10 nm.

In some embodiments, MSP-lined nanodiscs are assembled into arrayed sheets by linking individual MSP-lined nanodiscs together through complementary nucleic acid handle and anti- handle strands.

Methods of Producing Nucleic Acid-lined Nanodiscs

The present disclosure, in some aspects, provides methods of producing nucleic acid- lined lipid nanodiscs. The following description is an example of a method of producing nucleic acid-lined lipid nanodiscs and is not intended to be limiting.

In some embodiments, methods for producing a nucleic acid-lined lipid nanodisc comprise incubating in a first reaction buffer (i) a nucleic acid nanostructure comprising an interior surface to which nucleic acid handle strands are attached, and (ii) at least two membrane scaffold protein (MSP)-lined nanodiscs, each comprising a lipid bilayer, at least two MSPs, and nucleic acid anti-handle strands that are complementary to the nucleic acid handle strands, to produce a nucleic acid nanostructure lined with MSP-lined nanodiscs.

The first reaction buffer may contain, for example, TE buffer (e.g., IX TE buffer), Tris (e.g., 5-15 mM), EDTA (e.g., 0.5-2 mM), and/or Mg²⁺ (e.g., 5-20 mM).

The first reaction may be incubated over a period of time of 30 min to 4 hours, for example, at temperatures ranging (e.g., gradually decreasing) from 37 °C to 4 °C. In some embodiments, the first reaction is incubated at a temperature of 25°C - 45°C. In some

embodiments, the first reaction is incubated for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.

The concentration of MSP-lined nanodiscs may range from 10-1000 nM. For example, a 10-50, 10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800 or 10-900 nM

concentration of MSP-lined nanodiscs may be used. In some embodiments, the concentration of MSP-lined nanodiscs is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nM. The concentration of nucleic acid nanostructure may range from 1-100 nM. For example, a 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, or 1-90 nM concentration of nucleic acid nanostructure may be used. In some embodiments, the concentration of nucleic acid

nanostructure is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nM.

Methods typically further comprise incubating in a second reaction buffer (i) the nucleic acid nanostructure lined with MSP-lined nanodiscs (e.g., at a concentration of 5-15 nM), (ii) lipid molecules, and (iii) detergent (e.g. 0.5-5%), to produce a nucleic acid-lined nanodisc.

The second reaction buffer may contain, for example, TE buffer (e.g., IX TE buffer), Tris (e.g., 5-15 mM), EDTA (e.g., 0.5-2 mM), and/or Mg²⁺ (e.g., 5-20 mM).

The second reaction may be incubated over a period of time of 30 min to 2 hours, for example, at room temperature (e.g., 25 °C).

Example of lipid molecules are provided elsewhere herein. In some embodiments, the lipid molecules comprise liposomes, e.g., comprising POPC:POPG:cholesterol:DGS-NTA(Ni), e.g., at molar ratios of 51:34: 10:5.

Examples of detergents include, but are not limited to, Decyl β-D-maltopyranoside, Deoxycholic acid, Digitonin, n-Dodecyl β-D-glucopyranoside, n-Dodecyl β-D-maltoside, N- Lauroylsarcosine sodium salt, Sodium cholate, Sodium deoxycholate, Undecyl β-D-maltoside, Triton X-100, CHAPS, 5-Cyclohexylpentyl β-D-maltoside, n-dodecyl phosphatidylcholine, n- octyl^-d-glucoside, and Brij 97.

Dialysis, in some embodiments, may be used to remove detergent from the end product.

Kits

Also provided herein are kits for producing a nucleic acid-lined lipid nanodiscs. The kits may comprise any one or more of the following components: nucleic acid nanostructure, nucleic acid handle strands, membrane scaffold protein (MSP)-lined nanodiscs, nucleic acid anti-handle strands that are complementary to the nucleic acid handle strands, lipid molecules, and detergent.

In some embodiments, the nucleic acid handle strands are attached to the interior surface of the nucleic acid nanostructure, and/or the nucleic acid anti-handle strands are attached to each of the MSP-lined nanodiscs.

Applications

The nucleic acid-lined nanodiscs can be used for a wide variety of applications, including, but not limited to, modeling lipid bilayers and studying different aspects of processes involving membranes. In some embodiments, the nucleic acid-lined nanodiscs may be used for structural and/or functional studies of large membrane proteins (e.g., mammalian respiratory complex) in bilayers, studies of cell-free expression for large membrane proteins and complexes, studies of membrane pores (e.g., proapoptotic proteins, BAX and BAK pores, anthrax pore, among others), studies of the fusion of synaptic vesicle membranes with planar bilayer membranes, studies of lipid rafts, or studies of virus entry into cells and screening of potential inhibitors against virus entry. Nucleic acid-lined nanodiscs may also be used for residual dipolar coupling (RDC) measurements and vaccination (As there are many copies of membrane proteins per disc/avidity). Many other applications of the nucleic acid-lined nanodiscs will be apparent to one of ordinary skill in the art.

Additional Aspects

1. A nucleic acid-lined nanodisc comprising a lipid bilayer having a hydrophobic edge surrounded by a cylindrical nucleic acid nano structure.

2. The nucleic acid-lined nanodisc of paragraph 1, wherein the lipid bilayer is attached to the nanostructure.

3. The nucleic acid-lined nanodisc of paragraph 1 or 2, wherein

(a) the nucleic acid nanostructure comprises an interior surface to which nucleic acid handle strands are attached; and

(b) the lipid bilayer comprises membrane scaffold protein (MSP)-lined nanodiscs, each MSP-lined nanodisc comprising a lipid bilayer and at least two MSPs,

wherein membrane scaffold proteins of (b) comprise amino acids (e.g., cysteines) attached to nucleic acid anti-handle strands that are complementary to and hybridized to the nucleic acid handle strands to form the nucleic acid-lined nanodisc.

4. The nucleic acid-lined nanodisc of paragraph 1 or 2, wherein:

(a) the nucleic acid nanostructure comprises an interior surface to which nucleic acid handle strands are attached; and

(b) amphiphilic peptides linked to nucleic acid anti-handle strands that are complementary to and hybridized to the nucleic acid handle strands to form the nucleic acid-lined nanodisc.

5. The nucleic acid-lined nanodisc of any one of paragraphs 1-4, wherein the nucleic acid nanostructure has a cylindrical shape.

6. The nucleic acid-lined nanodisc of any one of paragraphs 1-5 having a diameter of at least 20 nanometers. 7. The nucleic acid-lined nanodisc of paragraph 6 having a diameter of at least 40 nanometers

8. The nucleic acid-lined nanodisc of paragraph 7 having a diameter of at least 60 nanometers

9. The nucleic acid-lined nanodisc of paragraph 8 having a diameter of at least 80 nanometers.

10. The nucleic acid-lined nanodisc of paragraph 9 having a diameter of 20-200 nanometers.

11. The nucleic acid-lined nanodisc of any one of paragraphs 1-10, wherein the nucleic acid nanostructure comprises deoxyribonucleic acid (DNA).

12. The nucleic acid-lined nanodisc of paragraph 11, wherein the nucleic acid nanostructure is synthesized using a DNA origami method.

13. The nucleic acid-lined nanodisc of paragraph 11, wherein the nucleic acid nanostructure is synthesized using a single- stranded tiling method.

14. The nucleic acid-lined nanodisc of any one of paragraphs 1-13, wherein 5-100 nucleic acid handle strands are attached to the interior surface of the nucleic acid nanostructure.

15. The nucleic acid-lined nanodisc of any one of paragraphs 1-3 and 5-14, wherein each MSP-lined nanodisc comprises at least three nucleic acid handles.

16. A method for producing a nucleic acid-lined lipid nanodisc, the method comprising

(a) incubating in a first reaction buffer

a nucleic acid nanostructure comprising an interior surface to which nucleic acid handle strands are attached, and

(ii) at least two membrane scaffold protein (MSP)-lined nanodiscs, each comprising a lipid bilayer, at least two MSPs, and nucleic acid anti-handle strands that are complementary to the nucleic acid handle strands, to produce a nucleic acid nanostructure lined with MSP-lined nanodiscs; and

(b) incubating in a second reaction buffer

(iii) the nucleic acid nanostructure lined with MSP-lined nanodiscs of (a),

(iv) lipid molecules, and

(v) detergent, to produce a nucleic acid-lined nanodisc.

17. A method for producing a nucleic acid-lined lipid nanodisc, the method comprising

(a) incubating in a first reaction buffer (i) a nucleic acid nanostructure comprising an interior surface to which nucleic acid handle strands are attached, and

(ii) at least two lipid amphiphilic peptides attached to nucleic acid anti-handle strands that are complementary to the nucleic acid handle strands, to produce a nucleic acid nanostructure lined with amphiphilic peptides; and

(b) incubating in a second reaction buffer

(iii) the nucleic acid nanostructure lined with amphiphilic peptides of (a),

(iv) lipid molecules, and

(v) detergent, to produce a nucleic acid-lined nanodisc.

18. The method of paragraph 16 or 17, wherein the nucleic acid nanostructure has a cylindrical shape.

19. The method of any one of paragraphs 16-18 further comprising dialyzing the second reaction buffer containing the nucleic acid-lined nanodisc to remove the detergent.

20. The method of any one of paragraphs 16-19 further comprising synthesizing the nucleic acid nanostructure.

21. The method of any one of paragraphs 16-20, wherein the first reaction buffer and/or reaction buffer further comprises a buffer and/or salt.

22. The method of any one of paragraphs 16-21, wherein the nucleic acid

nanostructure is present in the first reaction buffer at a concentration of 1-15 nM.

23. The method of any one of paragraphs 16-22, wherein MSP-lined nanodiscs or the amphiphilic proteins are present in the first reaction buffer at a concentration of 20-750 nM.

24. The method of any one of paragraphs 16-23, wherein the lipid molecules comprise at least one of l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and cholesterol.

25. The method of any one of paragraphs 16-24, wherein the detergent comprises octyl glucoside.

26. A kit for producing a nucleic acid-lined lipid nanodisc, comprising

a nucleic acid nanostructure;

nucleic acid handle strands;

membrane scaffold protein (MSP)-lined nanodiscs; and

nucleic acid anti-handle strands that are complementary to the nucleic acid handle strands.

27. The kit of paragraph 26 further comprising lipid molecules and/or detergent. 28. The kit of paragraph 26 or 27, wherein the nucleic acid handle strands are attached to the interior surface of the nucleic acid nanostructure, and/or wherein the nucleic acid anti-handle strands are attached to each of the MSP-lined nanodiscs.

29. A kit for producing a nucleic acid-lined lipid nanodisc, comprising

a nucleic acid nanostructure;

nucleic acid handle strands;

amphiphilic proteins; and

nucleic acid anti-handle strands that are complementary to the nucleic acid handle strands.

30. The kit of paragraph 29 further comprising lipid molecules and/or detergent.

31. The kit of paragraph 29 or 30, wherein the nucleic acid handle strands are attached to the interior surface of the nucleic acid nanostructure, and/or wherein the nucleic acid anti-handle strands are attached to each of the amphiphilic peptides.

EXAMPLES

Example 1

Early attempts to make stable circularized nanodiscs (cNDs) larger than 50 nm in diameter (e.g., to study the early steps of cell entry for large viruses) turned out to be difficult, as these large nanodiscs are susceptible to aggregation. Moreover, it is challenging to express and purify the large scaffold proteins required to make these nanodiscs using E. coli expression systems. To overcome these issues, we applied external DNA origami barrels as scaffolding corrals. Scaffolded DNA origami employs folding of a long scaffold strand with hundreds short staple strands to achieve self-assembly of custom shapes. Using DNA origami, each component strand can be uniquely addressed, providing a molecular billboard to arrange biomolecules with prescribed compositions and stoichiometries for biofunctional study. The mechanical stiffness of self-assembled DNA nanostructures also can confine the precise morphology and dimensions of nanomaterials, including hard inorganic and soft biomaterials, through casting growth. Each DNA barrel we used in this study recruits a number of ~11-nm diameter nanodiscs that are circumscribed by a pair of non-circularized, oligonucleotide-functionalized scaffold proteins (3C-NW11), and directs their reconstitution into a single large nanodisc with a diameter prescribed by the dimension of the enclosing barrel (FIG. 1). We employed two different sized barrels: 90 and 60 outer diameter to reconstitute -70 or -45 nm nanodiscs, respectively (FIGs. IB, FIGs. 6-8).

Our DNA-origami barrels were designed using the software caDNAnol9 and folded in a one-pot reaction using a 20-hour thermal annealing program. Correctly folded structures were isolated by glycerol-gradient centrifugation and verified by negative-stain transmission electron microscopy (TEM) (FIG. IB). The 11 nm nanodiscs were first covalently coupled to

oligonucleotides through Sulfo-SMCC crosslinkers (FIG. 5), and then assembled onto the DNA- origami barrels through hybridization to the single- stranded DNA handles (36, 24 handles for 90-nm, 60-nm barrel respectively) preimmobilized onto the nanostructure (FIG. 1A). The successful hybridization with 11-nm sized nanodiscs was confirmed by negative stain-EM. As shown in FIG. IB, the DNA-origami barrel inner face is lined with small white disk- shaped structures along the interior face of the barrels. Most of the internalized nanodiscs have sizes around 11-nm in diameter, while some nanodiscs have size bigger than 11-nm, which can result from the heterogeneity of uncircularized nanodiscs. In order to direct the reconstitution of the 11- nm nanodiscs into one single large nanodisc, we added excess lipids solubilized in detergent followed by dialysis. After dialysis, we performed isopycnic ultracentrifugation through a sucrose gradient to separate the DNA-barrel-scaffolded lipid nanodiscs from free vesicles (FIG. IB).

In FIG. 2, without being bound by theory, we propose a model to explain the formation of the large bilayer. We hypothesize that bringing the 11-nm nanodiscs to within several nanometers, achieved here by DNA-origami templating, is a first step for the assembly of the larger bilayer. Secondly, a destabilization is developed at some point between the neighboring 11-nm nanodiscs to induce a highly localized rearrangement/fusion of the adjacent bilayers, resulting in one interconnected structure. This destabilization step is facilitated by the detergent that is used to solubilize the lipid mixture. The detergent destabilizes the interaction of MSP with its enclosed lipids thus enabling the merge of neighboring nanodiscs. Thirdly, fusion of smaller nanodiscs should initially create ellipsoid larger nanodiscs, until additional lipids can be recruited. Therefore, flexibility in the DNA-origami barrel (i.e. ability to distort into an ellipsoid as in FIG. 2, third panels) may help facilitate later fusion events. Lastly, additional lipids are used to inflate ellipsoid larger nanodiscs into circular larger nanodiscs.

We were able to assemble DCNDs using different lipids including the neutral lipid DOPC and the negatively charged lipid mixture POPC/POPG with or without cholesterol (FIG. IB, FIG. 6). We obtained the best yield when we used POPC/POPG with 10% cholesterol, as the cholesterol molecule is believed to increase membrane rigidity. Interestingly, the yield did not improve after adding more cholesterol molecules. With the barrel acting as a bumper case to prevent aggregation, the enclosed nanodiscs were stable for at least two months when stored at 4 °C, representing a great improvement in large nanodisc stability. Having established a modular method to reconstitute large stable nanodiscs using DNA barrel, we proceeded to examine whether this nanodisc platform could be used for membrane- protein incorporation for further structural and/or functional studies. Here we chose two model proteins that are known to establish functional assemblies with or without other proteins and co- factors: human Voltage-Dependent Anion Channel 1 (hVDAC-1) and the Rhodobacter sphaeroides photosynthetic reaction center protein (RC). hVDAC-1, a beta barrel protein, is known to oligomerize on the surface of the outer mitochondrial membrane in apoptosis induction. Early EM and AFM studies of mitochondria from Neurospora grassa or potato tubers showed dense packing of VDAC pores or even hexagonal spindle-like arrangements. It is therefore hypothesized that oligomer interactions of VDAC may play a role for its function. On the other hand, RC is a mostly alpha-helical protein that plays a key role in the photochemical conversion of light into chemical energy.

Purified hVDACl and RC in detergent were added to the 11-nm nanodisc-decorated DNA origami along with lipids before the dialysis step (FIG. 3A). After dialysis and purification of hVDACl- and RC-loaded DCND by density gradients, we performed EM analysis to confirm the incorporation. As shown in FIGs. 3B and 3C, EM images verify the incorporation of both proteins into nanodiscs. We also showed that multimeric assemblies are formed (FIG. 3A) within many of the nanodiscs when we added excess amount of either protein before the dialysis step. Presenting membrane proteins at high density, both in freely diffusing as well as crystalline contexts, may prove useful to investigate both structural and functional rearrangements directly from the very same membranes. Often proteins are found functional in 2D crystals allowing a number of studies to collect electron diffraction data and yielding high resolution structures. Our method could be further developed to a general platform for membrane -protein structure determination by subatomic averaging using cryo-electron tomography, electron diffraction or by X-ray free electron laser (XFEL).

We next explored whether our nanodiscs could be used as a model membrane system to study early steps in viral entry. Poliovirus (~30-nm diameter) is the prototype member of the enterovirus genus of the picornavirus family, which are positive-sense, single- stranded RNA viruses with ~7,500-base genomes enclosed by an icosahedral capsid, missing an envelope.31 CD 155 (also known as the poliovirus receptor, PVR) is the receptor to induce poliovirus infection, which catalyzes a conformational rearrangement and expansion of the virus particle32, eventually leading to RNA release across the membrane.

We prepared the 45-nm DCNDs (hosted by 60 nm outer-diameter DNA-origami barrels) containing lipids derivatized with a nitrilotriacetate (NT A) nickel-chelating head group. These DCND were functionalized with the His-tagged CD 155 ectodomain. Next, the receptor- decorated nanodiscs were incubated with poliovirus for 5 minutes at 4 °C. The complex was then heated to 37°C for 15 minutes to initiate receptor-mediated viral uncoating. Negative-stain EM confirmed the binding of the virus to DCND (FIG. 4); in some cases, we observed apparent pore formation in the nanodiscs after the virus initial binding. We also observed by negative-stain and cryo-EM that many of the nanodiscs were partially released from their DNA scaffolds after binding the virus (FIG. 4 and FIG. 9). One potential explanation is the creation of extra membrane tension after receptor binding, which causes dsDNA dissociation between 3CNW11 and DNA barrel.

In conclusion, we have developed a modular method to produce stable nanodiscs with sizes up to -70 nm diameter using DNA-origami barrel as scaffold. We believe our system can potentially create much larger nanodiscs, with more complicated geometry. Additionally, site- specific functionalization of DNA-origami provides a tool to precisely control membrane protein insertion, potentially with precisely controlled stoichiometry and transmembrane orientation. Therefore, our platform could provide an excellent tool for producing homogenous membrane protein complexes in native-like environment with designed composition, stoichiometry, and orientation. We have demonstrated the utility of this model system to reconstitute membrane protein 2D clusters for potential structure determination. We used two membrane proteins, hVDAC-1 and RC, which are known to form functional oligomeric arrays in native membranes. Furthermore, we have established this system to probe an outstanding virology problem of viral entry through host membranes.

Materials and Methods

Expression and Purification of triple cysteine NW11 (3C-NW11)

3C-NW11 construct in pET-28a containing a tobacco etch virus (TEV) protease- cleavable N-terminal His6 tag and a C-terminal sortase-cleavable His6 tag was transformed into BL21-Gold (DE3) competent Escherichia coli cells (Agilent). 3L cell cultures were grown at 37 °C with agitation at 200 r.p.m. in Luria broth (LB) medium supplemented with 50 g/ml kanamycin. Expression was induced at an OD600 of 0.6 with 1 mM IPTG, and cells were grown for another 3h at 37°C. Cells were harvested by centrifugation (7,000 x g, 15 min, 4 °C), and cell pellets were stored at -80 °C. 3C NW11 was purified as follows; Pellets of cells were resuspended in Buffer A (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 8 mM BME) plus 1% triton X100 and lysed by sonication on ice. Lysate was centrifuged (35,000 x g, 50 min, 4 °C), and the supernatant was loaded onto a ΝΪ2+-ΝΤΑ column. Resin was washed with 10 CV of the following buffers: buffer A + 1% Triton X-100, buffer A + 50 mM sodium cholate, buffer A, and buffer A + 30 mM imidazole. Proteins were eluted with buffer A + 500 mM imidazole.

Reconstitution of 3C-NW11 nanodiscs

We used ratio of 1:75 3C-NWl l:lipid to assemble nanodiscs. Lipids (POPC:POPG, 3:2; solubilized in sodium cholate) and 3C-NW11 were incubated on ice for 1 h. After incubation, sodium cholate was removed by incubation with Bio-beads SM-2 (Bio-Rad) for 1 h on ice followed by incubation overnight at 4 °C. The nanodisc preparations were filtered through 0.22 m nitrocellulose-filter tubes to remove the Bio-beads. The nanodisc preparations were further purified by size-exclusion chromatography while monitoring the absorbance at 280 nm on a Superdex 200 10 x 300 column equilibrated with 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 8 mM BME, 0.5 mM EDTA. Fractions corresponding to the size of each nanodisc were collected and concentrated. The purity of nanodisc preparations was assessed using SDS-PAGE.

Nanodisc-DNA conjugation and purification

The bifunctional cross linker Sulfo-SMCC (Thermo Scientific) was dissolved in anhydrous dimethylsulfoxide (DMSO) to give a final concentration of 100 mM. 10 nmoles of DNA oligo (with primary amine modification, /5AmMC6/TAGATGGAGTGTGGTGTGAAG) was incubated with a 100 times molar excess of the crosslinker in buffer B (100 mM NaPi, pH 8.0, 150 mM NaCl and 7.5% DMSO) for 1 h at 23°C. The reaction mix was applied to Amicon filter (Millipore, 3kD) and centrifuged at 7000 rpm for 50 min (repeat 3 times), and then went through a disposable Bio-rad P-6 spin column to remove excessive cross linker. Next, 50 uL of 5 uM nanodisc was incubated with purified DNA oligo-SMCC from the first step at 23 °C in buffer C (containing 100 mM NaPi, pH 7.4, 150 mM NaCl) for 2 h (DNAmanodisc ratio 12: 1). We removed the BME from the nanodisc sample right before the incubation with DNA oligo-SMCC by applying it to Bio-rad P-6 spin column. The oligo-conjugated nanodisc was then purified by size exclusion chromatography (preferred, FIG. 5) or by using Centricon concentrators (30 kDa MW cutoff, Millipore) and centrifuging at 4000 g for 10 min (repeat 5 times).

Design and assembly of DNA origami structures

The DNA origami/crystal nanostructures were designed using the software caDNAno.l DNA Origami was folded by mixing p7308 scaffold at 10 nM with 10-fold excess of staples in folding buffer (containing 5 mM Tris-HCl, 1 mM EDTA, 12 mM MgC12, pH 8) and subjected to a thermal annealing ramp (from 65 °C to 25 °C over 20 h). Well-folded DNA origami was purified by a rate-zonal centrifugation procedure using a 15-45% (v/v) glycerol gradient. Assembly of oligo-conjugated nanodisc with DNA Origami

Excess of oligonucleotide-conjugated nanodisc was incubated with the DNA corrals containing handle strands (5'-CTTCACACCACACTCCATCTA-3'). Nanodisc assembly was performed in buffer containing 5mM Tris-HCl, ImM EDTA, 10 mM MgC12, using an annealing protocol, in which the temperature was gradually decreased from 37 °C to 4 °C over 2 h.

Large lipid nanodisc reconstitution

DNA corrals containing small nanodiscs were mixed with 9X amount liposomes

(POPC:POPG:cholesterol:DGS-NTA(Ni) ratio of 51:34: 10:5, for the poliovirus experiment) then diluted with tris buffer containing octyl glucoside (5 mM Tris, 1 mM EDTA, 12 mM MgC12, pH 8, 0.7% OG). Next, this solution was incubated on a Thermomixer at 300 rpm at room

temperature for 1 h. The entire solution was then transferred into a 7K MWCO Slide-a-Lyzer dialysis cassette (Thermo Scientific). The cassette was dialyzed against 2 L of tris buffer for 48 h. After dialysis, the sample was recovered from the dialysis cassette and concentrated using an Amicon column. Reconstituted nanostructures were separated from excess lipids by equilibrium centrifugation using sucrose gradient (30, 25, 20, 15, 10 from bottom to top). The gradient solutions were layered into ultracentrifuge tubes and centrifuged at 48,000 rpm for 5 h at 4°C. The gradient was fractionated, and aliquot of each fraction was checked for the presence of assembled DCND.

VDAC-1 production

Human VDAC1 was expressed, purified and refolded as detailed previously.2- 3 Briefly, the plasmid containing pET21d:hVDACl (VDACl(l-283)-Leu-Glu-His6) construct was transformed to BL21 (DE3) competent cells. Expression of hVDACl was carried out in LB medium and induced by ImM IPTG at 37 °C for 3-5 hours. Cells were lysed and the inclusion bodies containing hVDACl were collected and solubilized in denaturing buffer (8 M urea, 50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 20 mM imidazole). hVDACl was subsequently purified with Ni-NTA resin and precipitated through dialysis against dialysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 5 mM DTT). The precipitate was collected and dissolved in 6M guanidine hydrochloride buffer. Refolding of hVDACl was carried out at 4 °C by very slow, dropwise dilution into lOx volume of refolding buffer (50 mL; 50 mM NaPi, pH 6.8, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, 1% (43 mM) lauryldimethylamine oxide (LDAO)). The refolded hVDAC 1 was further purified through cation exchange chromatography, from which the fractions containing properly folded hVDACl were pooled and concentrated for nanodisc reconstitution. Transmission electron microscopy

For imaging, particles were adsorbed onto glow discharged carbon-coated TEM grids (Ted Pella) and then stained using a 0.7% (for the poliovirus samples) or 2% aqueous uranyl formate solution. The samples were visualized with a JEOL JEM- 1400 TEM, operated at 80 kV in the bright-field mode.

Cryo electron microscopy

Gatan CP3 system was used to plunge-freeze a glow-discharged Quantifoil grids (EMS, Hatfield, PA) after the application of 3 μΐ of the poliovirus-DCND solution (blot times set to 3, 4 or 5s). Grids were transferred into an FEI F20 electron microscope operating at an acceleration voltage of 200 kV. Micrographs were acquired on a K2 Summit camera (Gatan, Pleasanton, California) in super-resolution mode.

Example 2

CryoEM data collection is more rapid when a larger density of particles can be imaged per micrograph frame. In order to increase the density of guest nanodiscs per frame, we implement lateral clustering of DNA-origami barrels, each hosting a 60 nm MSP nanodisc. We programmed oligonucleotide-functionalized, uncyclized 10-nm-diameter MSP nanodiscs to assemble into arrayed sheets (FIG. 10, left). We extend the concept by displaying six vertical stripes of four ssDNA handles on the outside circumference of our 87-nm outer diameter DNA- origami barrels (FIG. 10, right). Addition of splint strands will drive formation of a hexagonal lattice of these barrels; we program degeneracy such that one of three different azimuthal orientations will be randomly adopted at each position. For sixteen of the eighteen interior handles that engage MSP anti-handles, we design them to disengage on command via strand displacement. The two remaining handle- antihandle interactions are programmed to be 180 degrees apart. This enables free rotation of the enclosed nanodisc. This provides a powerful platform for cryoEM imaging of enclosed bilayer-embedded membrane proteins, as they will be presented at high density and at a variety of orientations without requiring tilting of the stage.

Example 3

In order to assemble double-decker nanodiscs, we program coaxial head-to-head and taiktail stacking interfaces for reversibly inducible dimerization of pairs of DNA-origami barrels, each hosting a guest MSP nanodisc. In the dimeric states, a double-decker arrangement of MSP nanodiscs exists, with spacing prescribed by the design of the DNA-origami components (FIG. 11). In head:head dimers, the "upper" face of each nanodisc is facing outwards, and in taihtail dimers, the "lower" face of each nanodisc is facing outwards. Inducing and reversing

dimerization is driven by addition of ssDNA strands. This enables sequential control over insertion of protein guests into the two faces of each nanodisc bilayer, as one face of each bilayer will be inaccessible in the dimer state. This is similar to the case of insertion of proteins into the outside of liposomes. A difference here is that we are able to split double-decker nanodiscs, and then effectively flip them inside out on command. Furthermore, double-decker bilayers provide a useful tool for the study of phenomena such as bilayer fusion or nuclear-pore formation. Thus, we propose the use of double-decker MSP nanodiscs to study complex formation between outermitochondrial-membrane protein VDACl and inner- mitochondrial membrane protein ANT.

Example 4

Even larger MSP nanodiscs prove useful as they can be used to study entry of

correspondingly larger viruses, and as well have the potential to host larger 2D membrane- protein crystals. For this purpose, we have designed and validated a variant corral architecture referred to as DNA-origami "arenas" (FIG. 12). These are homo-multimeric assemblies based on the self-limiting ring structures recently reported. Here a left monomer and right monomer are folded in separate tubes, and then linked by DNA hybridization at low magnesium

concentration to make a V-shaped heterodimer. The monomers are linked by a hinge and one or two bridges (red); the length of the bridge determines the angle of the V. When the magnesium concentration is increased, then electrostatic repulsion between DNA double helices is reduced, and the yellow plugs can insert into the orange sockets, where the attractive energy is provided by one base pair sticky-end interactions between each helix:helix interface (20 total). The average number of V-heterodimers per ring is determined by the opening angle for the V. The bottom of each V-heterodimer is decorated with seven ssDNA handles that are complementary to the ssDNA handles on the 10 nm MSP nanodiscs from FIG. 4.

Example 5

We engineer asymmetric, DNA-corralled nanodiscs with respect to lipid distribution. We produce and use different subtypes of flippases namely, ABCA1, ABCA4 and ABCA7 to maintain the asymmetry. ABCA1 is known to actively flip phosphatidylcholine,

phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 favorably flips phosphatidylserine. On the other hand, ABCA4 flips phosphatidylethanolamine in the reverse direction. The temporary attachment of designated oligonucleotides to individual membrane proteins allows the formation of stable, detergent- solubilized membrane protein complexes by base-pairing of complementary oligonucleotide sequences, thus facilitating the insertion of the membrane protein complex into nanodisc with defined stoichiometry and composition (FIG. 13). Moreover, this strategy allows the insertion of a membrane protein in parallel or anti-parallel direction with respect to other membrane protein(s) that is part of the complex. Following similar procedures, we co-reconstitute different ABC flippases into large DNA-corralled nanodiscs (FIG. 14). As an alternative, we control the stoichiometry and orientation of ABC translocases in DNA-corralled nanodiscs through tagging each of them with two short oligonucleotide that can be site- specifically hybridized onto the DNA scaffold interior surface.

Full-length ABC flippases are produced using HEK293T cells Expression System. Each subtype of ABC flippase is solubilized in detergents before hybridization with the designated oligo. We use native lipid mixture such as Porcine brain lipid or a mixture of synthetic lipids solubilized in detergent to assemble DNA-corralled nanodiscs. Both native and synthetic lipids are available from Avanti Polar Lipids (Alabaster, AL). After mixing the lipids with DNA barrels decorated with ABC flippases (solubilized in detergent), we gradually remove the detergent by dialysis or by using biobeads. The properly assembled DNA-corralled nanodiscs are further purified by isopycnic ultracentrifugation using a sucrose gradient. ATP is added to the DNA-corralled nanodiscs to initiate the active transport of the different lipids. One could use ATP regeneration system to regenerating ATP if needed; there are several ATP-regeneration systems available and well established.

To confirm the establishment of asymmetry, we supplement the lipid mixture with 0.5- 2% of fluorescently labeled lipids and use fluorescence interference contrast (FLIC) microscopy which is adapted to measure lipid flip-flop across supported membranes. We immobilize DNA- belted nanodiscs on a solid substrate utilizing complementary oligos (one from the DNA scaffold and one from the surface substrate) to allow for the asymmetry analysis by FLIC.

The presence of flippases along the protein of interest in the same nanodisc should not be a problem, as the surface area of the DNA-corralled nanodisc is large and sufficient to incorporate hundreds of copies of a membrane protein. Moreover we can restrict the position of the flippases through attaching them to the sides of the DNA-origami barrel.

We test whether membrane proteins will uni-directionally incorporate into asymmetric nanodiscs or not. We incorporate integral membrane proteins with large ectodomain (such as E.coli ATPase or Ryanodine receptor) so it will be easy to examine the directionality of incorporation by EM. Also, we examine several peripheral membrane proteins to determine whether they end up in a specific orientation (prefer one side of bilayer) in these asymmetric nanodiscs.

Example 6

Styrene maleic acid co-polymers (SMA) have been successfully used to directly extract membrane proteins from native membranes into discoidal SMA lipid particles (SMALPs), without the need of detergents. The structure of SMALPs is stabilized by the intercalation of the hydrophobic styrene groups between the acyl chains of the phospholipids, while the hydrophilic maleic acid groups face the solvent.

An important implication of the preservation of a native environment around an MP in native nanodiscs is the possibility of direct biochemical analysis of native interactions of the protein with surrounding lipids or with other membrane proteins.

One limitation of the SMA extraction method is the SMALPs nanoparticle has a maximal diameter of approximately 15 nm. This means that membrane proteins or complexes that are too large to fit within this limit are very unlikely to be successfully extracted. Another limitation is the SMALPs rather have broad size of distribution. To overcome these issues, we engineer DNA-corralled SMA nanodisc (DCSN). We use SMA co-polymer that is terminally modified with a cyano group and couple it to DNA oligonucleotides that are modified with an azido group. Next we perform click chemistry to form DNA-SMA hybrids. The oligonucleotide then can be site-specifically hybridized onto the DNA origami barrel interior surface as shown in FIG. 15.

First, we test the ability of the SMA decorated DNA-origami barrel to support a bilayer in its cavity. We characterize the DCSN using EM or fluorescent microscopy to confirm the bilayer formation. Once we confirm the bilayer formation, we then test the ability of DNA barrels to extract large complexes from native environments.

Highly pure mitochondria is obtained from HEK293F cells by sequential centrifugation. Next, DCSN is added to pure mitochondria and incubated overnight with slow stirring. The extraction is centrifuged at 100,000 g for 30 min and the supernatant is concentrated by 300 kDa cutoff centrifugal filter (Millipore). Concentrated sample will be loaded and centrifuged on sucrose gradients. Gradients are fractionated and investigated by western blot using primary antibodies that recognizes each component of the complex. The fraction that contains all the components of the complex will be concentrated and the purity is further verified by silver stain- PAGE. Next, the samples are vitrified and subjected to EM analysis. Example 7

This method uses an amphiphilic peptide, which can folded into nanodisc as well. This peptide can be assembled inside DNA barrel through hybridization between ssDNA conjugated onto peptide terminal and ssDNA from DNA barrel. After adding extra lipid and detergent, large sized nanodiscs can be produced through dialysis. 30-nm, 60-nm, and 90-nm barrel templated nanodiscs were produced (see FIGs. 16-18).

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding,"

"composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

What is claimed is: CLAIMS

1. A nucleic acid-lined nanodisc comprising:

a lipid bilayer; and

a cylindrical nucleic acid nanostructure,

wherein the lipid bilayer is surrounded by the cylindrical nucleic acid nanostructure.

2. The nucleic acid-lined nanodisc of claim 1, wherein the lipid bilayer is attached to the cylindrical nucleic acid nanostructure, and optionally wherein the lipid bilayer is non-covalently attached to the cylindrical nucleic acid nanostructure.

3. The nucleic acid-lined nanodisc of claim 2, wherein the lipid bilayer is attached to the cylindrical nucleic acid nanostructure through hybridization of lipid bilayer-linked nucleic acid handle strands bounds to nanostructure-linked nucleic acid anti-handle strands.

4. The nucleic acid-lined nanodisc of any one of claims 1-3, wherein the nucleic acid-lined nanodisc has an outer diameter of at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 150 nm, or at least 200 nm.

5. The nucleic acid-lined nanodisc of claim 4, wherein the nucleic acid-lined nanodisc has an outer diameter of 50 nm to 250 nm.

6. The nucleic acid-lined nanodisc of any one of claims 1-5, wherein the nucleic acid-lined nanodisc comprises a protein inserted in the lipid bilayer, optionally wherein the protein is a membrane protein, and optionally wherein the protein is a transmembrane protein.

7. The nucleic acid-lined nanodisc of claim 6, wherein the nucleic acid-lined nanodisc comprises a heterogeneous population of proteins inserted in the lipid bilayer.

8. The nucleic acid-lined nanodisc of any one of claims 1-5, wherein a virus is bound to the nucleic acid lined nanodisc, and optionally wherein the nanodisc further comprises a pore.

9. The nucleic acid-lined nanodisc of any one of claims 1-5, wherein the cylindrical nucleic acid nanostructure is assembled from DNA.

10. A method of producing a nucleic acid-lined nanodisc, comprising

(a) attaching membrane scaffold protein (MSP)-lined nanodiscs to a cylindrical nucleic acid nanostructure, wherein each MSP-lined nanodisc comprises a lipid bilayer and MSPs;

(b) producing a nucleic acid nanostructure lined with the MSP-lined nanodiscs;

(c) combining in a solution lipid molecules and detergent with the nucleic acid nanostructure lined with the MSP-lined nanodiscs; and

(d) producing a nucleic acid-lined nanodisc.

11. The method of claim 10, wherein step (d) comprises removing the detergent from the solution, and optionally wherein the detergent is removed by dialysis.

12. The method of claim 10 or 11, wherein the MSP-lined nanodiscs have an outer diameter of 5 nm to 15 nm, or wherein the MSP-lined nanodiscs have an outer diameter of 10 to 12 nm.

13. The method of any one of claims 10-12, wherein

nucleic acid handle strands are attached to an interior surface of the cylindrical nucleic acid nanostructure,

nucleic acid anti-handle strands are attached to the MSP-linked nanodiscs, and the nucleic acid handle strands bind to the nucleic acid anti-handle strands.

14. The method of claim 13, wherein 5-100 nucleic acid handle strands are attached to the interior surface of the nucleic acid nanostructure, and/or at least three nucleic acid anti-handle strands are attached to each of the MSP-lined nanodiscs.

15. The method of any one of claims 10-14, wherein the lipid molecules comprise at least one of l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l-palmitoyl-2-oleoyl-sn- glycero-3-phosphoglycerol (POPG), and cholesterol, optionally wherein the lipid molecules comprise a mixture of POPC, POPG, and cholesterol.

16. The method of any one of claims 10-15, wherein the detergent comprises octyl glucoside.

17. A nucleic acid-lined nanodisc produced by the method of any one of claims 10-16, optionally wherein the nucleic acid-lined nanodisc has an outer diameter of at least 50 nm.

18. An array comprising a plurality of the nucleic acid-lined nanodiscs of any one of claims 1-9 bound to each other, wherein the lipid bilayer can rotate freely within the cylindrical nucleic acid nano structure.

19. The array of claim 18, wherein the lipid bilayer is attached to the cylindrical nucleic acid nanostructure through hybridization of two opposing lipid bilayer-linked nucleic acid handle strands bounds to two opposing nano structure-linked nucleic acid anti-handle strands.

20. The array of claim 18 or 19, wherein the nucleic acid-lined nanodiscs are bound to each other through molecular interactions between molecules bound to an exterior surface of the cylindrical nucleic acid nanostructures.

21. A two-dimensional array comprising a plurality of membrane scaffold protein (MSP)- lined nanodiscs, wherein nucleic acid strands are linked to an exterior surface of the MSP nanodiscs, and wherein the MSP nanodiscs are linked to each other though binding of the nucleic acids strands to each other.

22. A composition comprising two nucleic acid-lined nanodiscs of any one of claims 1-9, optionally wherein one of the nucleic acid-lined nanodiscs is stacked on top of the other of the nucleic acid-lined nanodiscs.

23. The nucleic acid-lined nanodisc of any one of claims 1-9 further comprising a plurality of V-shaped heterodimers, wherein each of the V-shaped heterodimers comprises two monomers linked to each other, and optionally wherein the two monomers are linked to each other by a nucleic acid bridge strand.

24. The nucleic acid-lined nanodisc of any one of claims 1-9, wherein the lipid bilayer further comprises one or more flippase(s).

25. The nucleic acid-lined nanodisc of claim 24, wherein the one or more flippase(s) is/are selected from ABCA1, ABCA4, and ABCA7.

26. The nucleic acid-lined nanodisc of any one of claims 1-9, wherein the cylindrical nucleic acid nanostructure further comprises one or more one or more translocase(s).

27. The nucleic acid-lined nanodisc of claim 27, wherein the one or more translocase(s) is/are selected from ABC translocases.

28. The nucleic acid-lined nanodisc of any one of claims 1-9, wherein a styrene maleic acid (SMA) co-polymer is lined to an interior surface of the cylindrical nucleic acid nanostructure.

29. The nucleic acid-lined nanodisc of claim 28, w herein the SMA co-polymer is terminally modified with a cyano group and is coupled to a nucleic acid strand that is modified with an azido group.