DNA (RNA) assembly mediated by charged and charge-switching peptides

Abstract

A positively-charged peptide was able to fold both 2D and 3D DNA origami structures under a physiological environment with high yield. In-situ investigation of the folding dynamics showed that the isothermal folding pathway was catalyzed by the short peptides. By adding phosphate to the arginine groups in the peptide, the positive charges on the peptide could be enzymatically controlled to control the folding of DNA structure pathways. This approach provides a method for creating adaptive DNA nanomachines with dynamic folding pathways under physiological environments.

Description

[0001] DNA (RNA) ASSEMBLY MEDIATED BY CHARGED AND CHARGE-SWITCHING PEPTIDES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 662,334, filed on June 20, 2024, which is incorporated by reference herein in its entirety.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] This application was filed with a Sequence Listing XML in ST.26 XML format in accordance with 37 C.F.R. § 1.831 and PCT Rule 13ter. The Sequence Listing XML file submitted in the USPTO Patent Center, “208192-0020-W001_sequence_listing_xml_16-JUN-2025. xml,” was created on June 16, 2025, contains 384 sequences, has a file size of 340.0 kilobytes (348,160 bytes), and is incorporated by reference in its entirety into the specification.

[0006] BACKGROUND

[0007] Folding DNA origami under physiological environments is highly plausible in biological applications such as delivery and gene therapeutics. Unfortunately, isothermal annealing of DNA origami is limited to specific structures with certain 2D structures. Folding of 3D DNA origami using isothermal methods only presented very low yield. The DNA structures can be easily trapped during the folding process at room temperature, which is referred to as kinetic traps. To overcome these traps, thermal annealing is often required for base pairs to correct the false pairing to reach the lowest energy state.

[0008] What is needed are new methods of forming nucleic acid nanostructures, such as 2D and 3D DNA origami nanostructures.

[0009] SUMMARY

[0010] One embodiment described herein is a method of forming a nucleic acid nanostructure using peptide-assisted assembly, the method comprising: contacting a nucleic acid with one or more charged peptides to form a combination; incubating the combination for a period of time; and forming the nucleic acid nanostructure. In one aspect, the nucleic acid comprises DNA, RNA, or a combination thereof. In another aspect, the nucleic acid comprises a single stranded or a double stranded nucleic acid. In another aspect, the nucleic acid comprises a single-stranded circular DNA scaffold. In another aspect, the nucleic acid comprises an M13mp18 scaffold. In another aspect, the combination further comprises a plurality of nucleic acid staple strands. In another aspect, the combination is incubated under physiological conditions for about 1 hour to about 60 hours. In another aspect, the combination is incubated at about 37 °C to about 40 °C for about 3 hours to about 24 hours. In another aspect, the combination is incubated in a buffer comprising Tris-acetate and NaCI without Mg2+. In another aspect, a concentration of the NaCI in the combination ranges from about 1 mM to about 200 mlVI. In another aspect, the charged peptides comprise positively charged peptides. In another aspect, the charged peptides comprise at least one arginine residue, tyrosine residue, cysteine residue, or combinations thereof. In another aspect, the charged peptides comprise an N-terminal acetyl group. In another aspect, the charged peptides comprise at least one phosphate group. In another aspect, the charged peptides comprise an arginine-to-phosphate ratio of about 0.1 :1 to about 2:1. In another aspect, the charged peptides comprise one or more peptides having the amino acid sequence of any one of SEQ ID NO: 1-7. In another aspect, a concentration of the charged peptides in the combination ranges from about 5 pM to about 75 pM. In another aspect, the combination further comprises tris(2-carboxyethyl)phosphine (TCEP), iodoacetamide (IAA), alkaline phosphatase (ALP), dimethylsulfoxide (DMSO), or combinations thereof. In another aspect, the nucleic acid nanostructure is a 1-dimensional (1 D), 2-dimensional (2D), or 3-dimensional (3D) nanostructure. In another aspect, the nucleic acid nanostructure is a 2D or 3D DNA origami nanostructure.

[0011] Another embodiment described herein is a kit for forming a nucleic acid nanostructure using peptide-assisted assembly, the kit comprising: a nucleic acid scaffold; one or more charged peptides; a buffer comprising Tris-acetate and NaCI; and a plurality of nucleic acid staple strands. Another embodiment described herein are peptide and DNA origami structures disclosed herein.

[0012] Another embodiment described herein are peptide and DNA origami structures disclosed herein.

[0013] Another embodiment described herein is a method for forming DNA-peptide origami structures described herein.

[0014] Another embodiment described herein is a method for isothermal folding of ssRNA nanostructure using positively-charged peptides.

[0015] Another embodiment described herein is a method for programming DNA assembly with charge-switching peptides.

[0016] Another embodiment described herein is a method for using enzymes to program DNA with charge-switching peptides.

[0017] Another embodiment described herein is a method for crystallizing peptides and DNA origami using a hanging drop method. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019] FIG. 1 shows a schematic illustrating the isothermal co-assembly of cationic peptides and 2D DNA origami at 37 °C. The negatively charged phosphate groups on the M13 scaffold and staple strands are neutralized by cationic peptides containing positively charged arginine residues, facilitating the assembly of the DNA origami structures.

[0020] FIG. 2A-C show P3-1 D tile co-assembly. FIG. 2A shows a schematic representation of the 1 D DNA tile assembly based on a four-way junction (4WTJ) design. FIG. 2B shows the chemical structure of peptide AC-RRRY-NH2 (P3) (SEQ ID NO: 1). FIG. 2C shows atomic force microscopy (AFM) image confirming the successful co-assembly of the P3-1 D DNA tile.

[0021] The isothermal co-assembly was carried out by incubating the components with 50 pM Ac-RRRY-NH2peptide in TANa buffer (containing 2 mM NaCI) at 37 °C for 12 hours. The final concentration of each DNA tile strand was 250 nM (4 strands total). AFM characterization was performed on the assembled product without purification.

[0022] FIG. 3A-C show P3-2D DNA origami co-assembly. FIG. 3A shows a schematic illustrating the design of triangular 2D DNA origami created using Cadnano2 software. FIG. 3B shows an agarose gel electrophoresis (AGE) analysis of the cationic peptide-assisted isothermal coassembly of 2D DNA origami in a magnesium-free buffer, performed at varying concentrations of P3. The co-assembly exhibited a narrow range of effective P3 concentrations. High peptide concentrations led to aggregation, while low concentrations resulted in partial DNA origami assembly. FIG. 30 shows atomic force microscopy (AFM) images confirming the successful coassembly of P3 with 2D DNA origami at 50 pM P3. The co-assembly of P3-triangular 2D DNA origami was carried out isothermally by incubating the M13mp18 scaffold strand with a 10-fold excess of staple strands and the peptide Ac-RRRY-NH2(SEQ ID NO: 1). A typical assembly reaction contained 2 nM M13mp18 scaffold, 20 nM of each staple strand (207 in total), and 50 pM Ac-RRRY-NH2. The mixture was incubated at 37 °C for 3 hours in TANa buffer (containing 2 mM NaCI). The resulting origami structures were characterized by agarose gel electrophoresis (AGE) and atomic force microscopy (AFM) without prior purification of the staple strands.

[0023] FIG. 4A-D show P3-3D DNA origami co-assembly. FIG. 4A shows a schematic representation of the 12-helix bundle (12hb) 3D DNA origami design created using Cadnano2 with a square lattice. FIG. 4B shows a coarse-grained oxDNA simulation of the designed 3D DNA origami structure. FIG. 4C shows an agarose gel electrophoresis (AGE) analysis of the isothermal co-assembly of P3-3D DNA origami in magnesium-free buffer, at varying peptide concentrations. The co-assembly exhibited a narrow optimal concentration range for P3: high concentrations induced aggregation, while low concentrations resulted in incomplete assembly. FIG. 4D shows an atomic force microscopy (AFM) images confirmed the successful formation of 3D DNA origami structures at 70 mM NaCI. The isothermal co-assembly of P3-3D DNA origami was performed by incubating the M13mp18 scaffold with a 30-fold excess of staple strands and the cationic peptide AC-RRRY-NH2. A typical reaction mixture contained 4.5 nM M13mp18 scaffold, 135 nM of each staple strand (164 total), and 670 pM Ac-RRRY-NH2(SEQ ID NO: 1). The assembly was carried out at 37 °C for 60 hours in TANa buffer containing 70 mM NaCI. The resulting structures were characterized by agarose gel electrophoresis (AGE) and atomic force microscopy (AFM), without prior purification of the staple strands.

[0024] FIG. 5A-C show P3-1 D RNA tile co-assembly. FIG. 5A shows a schematic illustration of the bKL-8b RNA tile assembly. FIG. 5B-C show atomic force microscopy (AFM) images confirm the successful isothermal co-assembly of the P3-1 D RNA tile facilitated by the cationic peptide AC-RRRY-NH2(SEQ ID NO: 1). Isothermal co-assembly of the P3-1 D RNA tile was conducted by incubating the RNA tile (bKL 8b, single strand, final concentration: 20 ng / pL) with 50 pM Ac- RRRY-NH2in TANa buffer ([NaCI] = 2 mM) at 37 °C for 12 hours. The assembled structures were directly characterized by AFM without purification.

[0025] FIG. 6A-B show P3-2D ssRNA origami co-assembly. FIG. 6A shows a schematic representation of the 2D single-stranded RNA (ssRNA) origami assembly. FIG. 6B shows atomic force microscopy (AFM) image confirming the successful isothermal co-assembly of P3-2D ssRNA origami. Isothermal co-assembly of the P3-2D ssRNA origami was performed by incubating the RNA strand (final concentration: 20 ng / pL) with 50 pM Ac-RRRY-NH2in TANa buffer ([NaCI] = 2 mM) at 37 °C for 12 hours. The assembled structures were characterized by AFM without purification.

[0026] FIG. 7A-C show the influence of peptide charge on 3D DNA origami co-assembly (P3, P4, P5). FIG. 7A shows an agarose gel electrophoresis (AGE) analysis showing the minimum phosphate-to-arginine ([R]:[P]) ratio required for successful co-assembly of P3-3D DNA origami is 1 :1. FIG. 7B shows an AGE analysis of P4- and P5-assisted 3D DNA origami co-assembly reveals that higher peptide charge reduces the required [R]:[P] ratio, with P4 and P5 enabling successful assembly at 0.5:1 and 0.25:1 , respectively. These results demonstrate that peptides with higher net positive charge facilitate more efficient co-assembly of 3D DNA origami, requiring lower peptide concentrations to achieve structural formation. FIG. 7C shows AFM analysis of P4- and P5-assisted 3D DNA origami co-assembly at [R]:[P] ratios of 0.5:1 and 0.25:1. P3-3D DNA Origami Co-assembly. Isothermal co-assembly was performed by incubating the M13mp18 scaffold (4.5 nM), a 30-fold excess of staple strands (135 nM each; 164 total), and 330 pM Ac- RRRY-NH2(P3 peptide) (SEQ ID NO: 1) in TANa buffer containing 0-200 mM NaCI. The mixture was incubated at 37 °C for 60 hours. The assembled structures were characterized by agarose gel electrophoresis (AGE) and atomic force microscopy (AFM), without purification of the staple strands. P4-3D DNA Origami Co-assembly. A similar procedure was used for co-assembly with the higher-charged P4 peptide (Ac-RRRRY-NH2) (SEQ ID NO: 2). The reaction contained 4.5 nM scaffold, 135 nM each staple (164 strands), and 0-188 pM peptide in TANa buffer with 100 mM NaCI. Incubation was carried out at 37 °C for 60 hours, followed by characterization via AGE and AFM without purification. P5-3D DNA Origami Co-assembly. For the most highly charged peptide, P5 (AC-RRRRRY-NH2) (SEQ ID NO: 3), the co-assembly reaction included 4.5 nM scaffold, 135 nM each staple (164 strands), and 0-150 pM peptide. The mixture was incubated at 37 °C for 60 hours in TANa buffer containing 100 mM NaCI. Products were analyzed using AGE and AFM without purification.

[0027] FIG. 8A-B show temperature dependence of P5-3D DNA origami co-assembly. Atomic force microscopy (AFM) images demonstrate that the isothermal co-assembly of P5-3D DNA origami is successful only within a narrow temperature range (37-40 °C). FIG. 8A shows the structure of AC-RRRRRY-NH2 (P5) (SEQ ID NO:3). FIG. 8B shows micrographs of DNA structures assembled at various temperatures.

[0028] Isothermal co-assembly was performed by incubating M13mp18 scaffold (4.5 nM), a 30- fold excess of staple strands (135 nM each; 164 strands), and 50 pM Ac-RRRRRY-NH2(P5 peptide) (SEQ ID NO: 3) in TANa buffer containing 100 mM NaCI. Reactions were incubated at various temperatures (room temperature, 34.5 °C, 37 °C, 39.5 °C, 42 °C, and 44.5 °C) for 60 hours. Assembled structures were analyzed using agarose gel electrophoresis (AGE) and AFM without purification of the staple strands.

[0029] FIG. 9A-B show a phase diagram of charge-unbalanced P3-DNA mixtures. FIG. 9A shows graphs of absorbance versus NaCI concentrations at various ratios. FIG. 9B shows a phase diagram of the ratio versus NaCI concentration. Black dots: aggregates / coacervates (A500 > 0.05); grey dots: A500 < 0.05. Absorbance at 500 nm was used as an indicator of turbidity to determine phase transition points in P3-DNA mixtures. The absorbance at 500 nm was measured as a function of NaCI concentration at various phosphate-to-arginine ([R]:[P]) ratios: 0.25: 1 , 0.5: 1 , 1 :1 , 2:1 , 5:1 , and 10:1. Conditions under which the absorbance dropped below 0.05 were defined as phase transition points, marking the boundary between single-phase and two-phase regions. Data points corresponding to the two-phase region are shown in grey, and those in the single- phase region in blue. A binodal phase boundary was fitted using the Flory-Huggins-Voorn- Overbeek (FH-VO) model to predict phase separation behavior as a function of NaCI concentration.

[0030] To construct the phase diagram, mixtures of P3 peptide (Ac-RRRY-NH2) (SEQ ID NO: 1) and DNA were prepared at [R]:[P] ratios of 0.25:1 , 0.5:1 , 1 :1 , 2:1 , 3:1 , and 5:1. Each mixture was tested at NaCI concentrations of 0, 20, 50, 70, 100, 150, and 200 mM. Turbidity measurements were performed using an automated microplate reader (BioTek). For each condition, 2 pL of sample was loaded onto a TAKE3 microvolume plate, and absorbance at 500 nm was recorded to assess the presence of phase-separated assemblies.

[0031] FIG. 10 shows co-assembly of P3-3D DNA origami near the P3-DNA binodal curve. A coassembly diagram was constructed to evaluate the formation of P3-3D DNA origami as a function of phosphate-to-arginine ([R]:[P]) ratio and NaCI concentration. Successful co-assembly conditions, as determined by agarose gel electrophoresis (AGE), are marked in green, while unsuccessful conditions are marked in grey. Comparison with the P3-DNA phase diagram (FIG. 9) reveals that successful co-assembly occurs predominantly near the binodal phase boundary, indicating a close link between phase behavior and productive DNA origami assembly. For coassembly, the same DNA components and [R]:[P] ratios as those used in the phase diagram study were applied. The phosphate concentration was maintained at 1 mM, with final concentrations of 4.5 nM M13mp18 scaffold and 135 nM of each staple strand (164 total). Reactions were incubated at 37 °C for 60 hours across NaCI concentrations of 0, 20, 50, 70, 100, and 200 mM. The resulting assemblies were analyzed by AGE without purification of staple strands.

[0032] FIG. 11A-C show fast co-assembly of peptide-2D DNA origami via charge-switching peptide P3p responsive to Alkaline phosphatase (ALP). FIG. 11 A shows a schematic illustration showing the enzymatic conversion of Ac-RRRpY-NH2(P3p, +1 charge) (SEQ ID NO: 4) to Ac- RRRY-NH2(P3, +3 charge) (SEQ ID NO: 1) catalyzed by ALP, which triggers the co-assembly of 2D DNA origami. FIG. 11 B shows an agarose gel electrophoresis (AGE) analysis of P3-2D DNA origami co-assembly after 1 hour incubation with increasing ALP concentrations, demonstrating accelerated assembly kinetics. FIG. 11C shows atomic force microscopy (AFM) images of 2D DNA origami following 1 hour incubation at 37 °C without ALP and with 6 U / mLALP, confirming the formation of well-defined origami structures upon enzymatic activation. For rapid coassembly, 2 nM M13mp18 scaffold and a 10-fold excess of staples (207 strands) were mixed with 400 M P3p peptide in TANa buffer (2 mM NaCI). ALP was added at 0, 1 , 2, 4, 6, 8, and 10 U / mL, respectively. Samples were incubated at 37 °C for 1 hour before AGE analysis. Efficient triangular 2D DNA origami assembly was achieved within 1 hour at 37 °C in the presence of 6 U / mLALP.

[0033] FIG. 12A-C show ALP-enabled rapid co-assembly of P3p-3D DNA origami without aggregation. FIG. 12A shows a schematic or representative images showing that ALP enzyme facilitates P3p-3D DNA origami co-assembly at high phosphate-to-arginine ([R]:[P]) ratios without inducing aggregation. Samples were incubated at 37 °C for 12 hours. FIG. 12B shows an agarose gel electrophoresis (AGE) results demonstrating fast co-assembly of P3p-3D DNA origami after 12 hours incubation at 37 “C with ALP. FIG. 12C shows AGE results for P3-3D DNA origami co-assembly after 12 hours incubation at 37 °C under identical [R]:[P] ratios and NaCI concentrations as in (b). The presence of aggregates trapped in the wells indicates unsuccessful assembly due to slower kinetics of P3-3D DNA origami co-assembly. For rapid 3D DNA origami co-assembly, 4.5 nM M13mp18 scaffold and a 30-fold excess of staple strands (164 total) were mixed with varying concentrations of P3p peptide (0.33, 0.66, 1.0, and 1.33 mM) in TANa buffer containing NaCI at 20, 70, 100, and 200 mM. ALP enzyme was added at 4 U / mL. Samples were incubated isothermally at 37 °C for 12 hours and analyzed by AGE. Efficient 3D DNA origami assembly was achieved within 12 hours under these conditions.

[0034] FIG. 13A-E show DMSO-triggered charge switching enables peptide-3D DNA origami coassembly via disulfate (-S-S-). FIG. 13A shows a schematic illustrating the DMSO-induced oxidation of Ac-CRRY-NH2(P2c, +2 charge) (SEQ ID NO: 6) into a disulfide-linked dimer NH2- YRRC-AC-AC-CRRY-NH2(P4C, +4 charge) (SEQ ID NO: 7), resulting in increased charge density through covalent cysteine-cysteine bonds. FIG. 13B shows a schematic showing that Ac-CRRY- NH2protected by TCEP remains reduced and does not undergo oxidation in the absence of 10% DMSO. FIG. 13C shows a schematic depicting the inhibition of disulfide bond formation when AC-CRRY-NH2is pre-treated with iodoacetamide (IAA), which alkylates cysteine residues, preventing oxidation even in the presence of 10% DMSO. FIG. 13D shows an agarose gel electrophoresis (AGE) confirming successful co-assembly of 3D DNA origami triggered by DMSO oxidation of cysteine residues, converting Ac-CRRY-NH2(+2) (SEQ ID NO: 6) to NH2-YRRC-Ac- AC-CRRY-NH2(+4) (SEQ ID NO: 7), at an [R]:[P] ratio of 0.5:1 and NaCI concentration of 70 mM- 100 mM. FIG. 13E shows atomic force microscopy (AFM) images of peptide-3D DNA origami co-assembly without (left) and with (right) 10% DMSO. Scale bar: 1 pm. For DMSO-triggered co-assembly, a charge- switching peptide containing an N-terminal cysteine (Ac-CRRY-NH2) (SEQ ID NO: 7) was designed. Upon oxidation by 10% DMSO, cysteine residues form covalent disulfide bonds (-S-S-), dimerizing the peptide from P2 to P4. Although the total charge remains unchanged, the local charge density doubles from +2 to +4, enhancing electrostatic interactions that promote co-assembly with 3D DNA origami. To prevent premature oxidation, Ac-CRRY-NH2was mixed with tris(2-carboxyethyl)phosphine (TCEP) to maintain cysteine residues in their reduced state. For typical co-assembly, 4.5 nM M13mp18 scaffold and 30-fold excess staples (164 strands) were mixed with 0.25 mM CRRY and 0.25 mM TCEP in 1 * TA buffer containing varying NaCI concentrations. Co-assembly was triggered by adding 10% DMSO and incubating at 37 °C for 60 hours. To confirm that co-assembly was dependent on disulfide bond formation, iodoacetamide (IAA) was used to block cysteine residues. IAA (10 mM) was incubated with 1 mM AC-CRRY-NH2and 1 mM TCEP in the dark for 1 hour to alkylate cysteine thiols, preventing disulfide formation (e.g., forming Ac-*CRRY-NH2SEQ ID NO: 8, where AC-*C is an N-acetyl cysteine-acetamide moiety). This mixture was then used in co-assembly under standard conditions: 4.5 nM M13mp18, 30-fold excess of staple strands, 70 mM NaCI, 0.25 mM TCEP, 0.25 mM AC-*CRRY-NH2, 2.5 mM IAA, and 10% DMSO, incubated at 37 °C for 60 hours before AGE analysis.

[0035] DETAILED DESCRIPTION

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0037] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0038] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of’’ excludes any element, step, or ingredient not specified in the claim.

[0039] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0040] As used herein, the term “or” can be conjunctive or disjunctive.

[0041] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0042] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0043] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0044] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0045] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15-30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure.

[0046] Described herein is a new pathway for folding DNA nanostructures using peptides. This approach employs biomolecules to trigger the dynamic folding of DNA / RNA nanostructures under physiological conditions. The technology utilizes a positively charged peptide instead of metal ions to stabilize Watson-Crick base pairs during both thermal and isothermal annealing processes, thereby enabling the folding of DNA nanostructures under physiological conditions. This concept could potentially extend to other organic molecules, offering novel controls over dynamic DNA assembly.

[0047] Beyond thermal annealing, limited methods can be explored to improve the folding efficiency of DNA (RNA) structures at physiological environment.

[0048] A positive-rich tetrapeptide with 3 arginine groups at the N-terminal and 1 tyrosine group at the C-terminal was designed and synthesized. The N terminal was capped with -Ac group and C terminal was capped with -NH2group. Each arginine group contained 1 positive charge to form polyvalent metal ion analogue with three positive charges. Multiple positive charges enabled the DNA nanostructures to be folded at low peptide concentration.

[0049] One embodiment described herein is a method of forming a nucleic acid nanostructure using peptide-assisted assembly, the method comprising: contacting a nucleic acid with one or more charged peptides to form a combination; incubating the combination for a period of time; and forming the nucleic acid nanostructure. In one aspect, the nucleic acid comprises DNA, RNA, or a combination thereof. In another aspect, the nucleic acid comprises a single stranded or a double stranded nucleic acid. In another aspect, the nucleic acid comprises a single-stranded circular DNA scaffold. In another aspect, the nucleic acid comprises an M13mp18 scaffold. In another aspect, the combination further comprises a plurality of nucleic acid staple strands. In another aspect, the combination is incubated under physiological conditions for about 1 hour to about 60 hours. In another aspect, the combination is incubated at about 37 °C to about 40 °C for about 3 hours to about 24 hours. In another aspect, the combination is incubated in a buffer comprising Tris-acetate and NaCI without Mg2+. In another aspect, a concentration of the NaCI in the combination ranges from about 1 mM to about 200 mM. In another aspect, the charged peptides comprise positively charged peptides. In another aspect, the charged peptides comprise at least one arginine residue, tyrosine residue, cysteine residue, or combinations thereof. In another aspect, the charged peptides comprise an N-terminal acetyl group. In another aspect, the charged peptides comprise at least one phosphate group. In another aspect, the charged peptides comprise an arginine-to-phosphate ratio of about 0.1 :1 to about 2:1. In another aspect, the charged peptides comprise one or more peptides having the amino acid sequence of any one of SEQ ID NO: 1-7. In another aspect, a concentration of the charged peptides in the combination ranges from about 5 pM to about 75 pM. In another aspect, the combination further comprises tris(2-carboxyethyl)phosphine (TCEP), iodoacetamide (IAA), alkaline phosphatase (ALP), dimethylsulfoxide (DMSO), or combinations thereof. In another aspect, the nucleic acid nanostructure is a 1-dimensional (1 D), 2-dimensional (2D), or 3-dimensional (3D) nanostructure. In another aspect, the nucleic acid nanostructure is a 2D or 3D DNA origami nanostructure.

[0050] Another embodiment described herein is a kit for forming a nucleic acid nanostructure using peptide-assisted assembly, the kit comprising: a nucleic acid scaffold; one or more charged peptides; a buffer comprising Tris-acetate and NaCI; and a plurality of nucleic acid staple strands.

[0051] Another embodiment described herein are peptide and DNA origami structures disclosed herein.

[0052] Another embodiment described herein is a method for forming DNA-peptide origami structures described herein.

[0053] Another embodiment described herein is a method for isothermal folding of ssRNA nanostructure using positive-charged peptide.

[0054] Another embodiment described herein is a method for programming DNA assembly with charge-switching peptides.

[0055] Another embodiment described herein is a method for using enzymes to program DNA with charge-switching peptides.

[0056] Another embodiment described herein is a method for crystallizing peptide and DNA origami using the hanging drop method.

[0057] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0058] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0059] Clause 1 . A method of forming a nucleic acid nanostructure using peptide-assisted assembly, the method comprising: contacting a nucleic acid with one or more charged peptides to form a combination; incubating the combination for a period of time; and forming the nucleic acid nanostructure.

[0060] Clause 2. The method of clause 1, wherein the nucleic acid comprises DNA, RNA, or a combination thereof.

[0061] Clause 3. The method of clause 1 or 2, wherein the nucleic acid comprises a single stranded or a double stranded nucleic acid.

[0062] Clause 4. The method of any one of clauses 1-3, wherein the nucleic acid comprises a single-stranded circular DNA scaffold.

[0063] Clause 5. The method of any one of clauses 1-4, wherein the nucleic acid comprises an M13mp18 scaffold.

[0064] Clause 6. The method of any one of clauses 1-5, wherein the combination further comprises a plurality of nucleic acid staple strands.

[0065] Clause 7. The method of any one of clauses 1-6, wherein the combination is incubated under physiological conditions for about 1 hour to about 60 hours.

[0066] Clause 8. The method of any one of clauses 1-7, wherein the combination is incubated at about 37 °C to about 40 °C for about 3 hours to about 24 hours.

[0067] Clause 9. The method of any one of clauses 1-, wherein the combination is incubated in a buffer comprising Tris-acetate and NaCI without Mg2+.

[0068] Clause 10. The method of any one of clauses 1-9, wherein a concentration of the NaCI in the combination ranges from about 1 mM to about 200 mM.

[0069] Clause 11. The method of any one of clauses 1-10, wherein the charged peptides comprise positively charged peptides. Clause 12. The method of any one of clauses 1-11 , wherein the charged peptides comprise at least one arginine residue, tyrosine residue, cysteine residue, or combinations thereof. Clause 13. The method of any one of clauses 1-12, wherein the charged peptides comprise an N-terminal acetyl group.

[0070] Clause 14. The method of any one of clauses 1-13, wherein the charged peptides comprise at least one phosphate group.

[0071] Clause 15. The method of any one of clauses 1-14, wherein the charged peptides comprise an arginine-to-phosphate ratio of about 0.1 :1 to about 2:1 .

[0072] Clause 16. The method of any one of clauses 1-15, wherein the charged peptides comprise one or more peptides having the amino acid sequence of any one of SEQ ID NO: 1-7.

[0073] Clause 17. The method of any one of clauses 1-16, wherein a concentration of the charged peptides in the combination ranges from about 5 pM to about 75 pM.

[0074] Clause 18. The method of any one of clauses 1-17, wherein the combination further comprises tris(2-carboxyethyl)phosphine (TCEP), iodoacetamide (IAA), alkaline phosphatase (ALP), dimethylsulfoxide (DMSO), or combinations thereof.

[0075] Clause 19. The method of any one of clauses 1-18, wherein the nucleic acid nanostructure is a 1 -dimensional (1 D), 2-dimensional (2D), or 3-dimensional (3D) nanostructure.

[0076] Clause 20. The method of any one of clauses 1-19, wherein the nucleic acid nanostructure is a 2D or 3D DNA origami nanostructure.

[0077] Clause 21. A kit for forming a nucleic acid nanostructure using peptide-assisted assembly, the kit comprising: a nucleic acid scaffold; one or more charged peptides; a buffer comprising Tris-acetate and NaCI; and a plurality of nucleic acid staple strands.

[0078] EXAMPLES

[0079] Materials

[0080] For all experiments except otherwise specified, single-stranded M13mp18 template DNA (500 pg, in DI H2O), abbreviated M13MP18, was purchased from tilibit. Oligonucleotide staple strands were obtained from IDT as 200 pM water solutions and used without further purification. Magnesium chloride anhydrous (> 98%) (MgCI2), sodipM Chloride (> 98%), tris(hydroxymethyl)aminomethane (> 99.9%) (Tris), acetic acid (> 99.8%) were obtained from Sigma-Aldrich. Agarose was purchased form Bio-Rad. Mica used inAFM imaging was purchased from Ted Pella. Deionized MilliQ water (resistivity 18 MO cm) was used for all experiments.

[0081] TA Buffer For all experiments except otherwise specified, a TA buffer was used with the following final composition of tris(hydroxymethyl)aminomethane (40 mmol L-1). The pH value was adjusted to 8.2 with acetic acid.

[0082] Peptides

[0083] Peptide SEQ ID NO

[0084] Ac-RRRY-NH21

[0085] Ac-RRRRY-NH22

[0086] Ac-RRRRRY-NH23

[0087] AC-RRRYP-NH24

[0088] AC-RRRRYP-NH25

[0089] Ac-CRRY-NH26

[0090] NH2-YRRCAc-Ac-CRRY-NH27

[0091] Ac-*CRRY-NH28

[0092] Ac- refers to an N-acetyl moiety; -NH2 refers to an amine moiety on the carboxyl terminus; Yprefers to an phosphate moiety added to the tyrosine sidechain (phosphotyrosine); NH2-YRRCAc-AcCRRY-NH2is a dipeptide covalently linked by a disulfide bond (-S-S-) between the terminal acetyl-cysteine residues (forming cystines); Ac-*C refers to an N-acetyl cysteine-acetamide moiety.

[0093] Peptide Structures and SEQ ID NO

[0094]

[0095] Peptide concentrations were determined via the absorbance at 280 nm through UV-vis using the following equation: c (M) = 71280 / 1280 M ciiT'

[0096] Thermal Annealing of 2D and 3D DNA Origamis

[0097] For triangular 2D DNA origami, 2 nM M13MP18 scaffold and 10-time excess of staple strands (207 strands) were mixed in 1x TAE buffer with 12.5 mM Mg(OAc)2. The samples were heated and kept at 70 °C for 5 min and then subjected to 3h thermal ramp from 70 °C to 20 °C. For square-lattice 3D DNA origami, 4.5 nM M13MP18 scaffold and 30-time excess of staple stands (164 strands) were mixed in 1 * TAE buffer with 12.5 mM Mg(OAc)2. The samples were heated and kept at 70 °C for 5 min and subjected to 12h thermal ramp from 70 °C to 20 °C. DNA origami nanostructures were characterized with AGE and AFM without staple purification.

[0098] Isothermal Co-Assembly of P3-2D DNA Origami

[0099] Isothermal co-assembly of triangular 2D DNA origami were conducted through incubation of M13MP18 scaffold, 10-time excess staple strands and peptide (Ac-RRRY-NH2; SEQ ID NO: 1). For a typical synthesis, 2 nM M13mp18, 20 nM each staple (207 strands) and 50 pM (Ac- RRRY-NH2; SEQ ID NO: 1) were mixed and incubated at 37 °C for 3 h in TANa buffer ([NaCI] = 2 mM). The product was characterized with AGE and AFM without staple purification.

[0100] Isothermal Co-Assembly of P3-3D DNA Origami

[0101] Isothermal co-assembly of P3-3D DNA origami were conducted through incubation of M13MP18 scaffold, 30-time excess of staples and peptide (AC-RRRY-NH2; SEQ ID NO: 1). For a typical synthesis, 4.5 nM M13MP18 scaffold, 135 nM each staple (164 strands) and 330 pM (Ac-RRRY-NH2; SEQ ID NO: 1) were mixed and incubated at 37 °C for 60 h in TANa buffer ([NaCI] = 20 mM). The product was characterized with AGE and AFM without staple purification.

[0102] Isothermal Co-Assembly of P3-DNA Tile, P3-bKL 8b RNA Tile, P3-RNA Origami

[0103] Isothermal co-assembly of DNA / RNA nanostructures was performed by incubating the components with 50 pM (Ac-RRRY-NH2; SEQ ID NO: 1) peptide in TANa buffer ([NaCI] = 2 mM) at 37 °C for 12 hours. The final concentrations of P3-DNA tile, P3-bKL 8b RNA tile, P3-RNA ssOrigami were 250 nM (4 strands), 20 ng / pL (1 strand), 20 ng / pL, respectively. The product was characterized with AFM without staple purification.

[0104] Isothermal Co-Assembly of P4-3D DNA Origami

[0105] Isothermal co-assembly of P4-3D DNA origami were conducted through incubation of M13mp18 scaffold, 30-time excess staple and (Ac-RRRRY-NH2; SEQ ID NO: 2). For a typical synthesis, 4.5 nM M13mp18, 135 nM each staple (164 strands) and 125 pM(Ac-RRRRY-NH2; SEQ ID NO: 2) were mixed and incubated at 37 °C for 60 h in TANa buffer ([NaCI] = 100 mM). The product was characterized with AGE and AFM without staple purification.

[0106] Isothermal Co-Assembly of P5-3D DNA Origami

[0107] Isothermal co-assembly of P5-3D DNA origami were conducted through incubation of M13mp18 scaffold, 30-time excess staple and (Ac-RRRRRY-NH2; SEQ ID NO: 3). For a typical synthesis, 4.5 nM M13mp18, 135 nM each staple (164 strands) and 50 pM (Ac-RRRRRY-NH2; SEQ ID NO: 3) were mixed and incubated at 37°C for 60 h in TANa buffer ([NaCI] = 100 mM). The product was characterized with AGE and AFM without staple purification.

[0108] Turbidity Measurements

[0109] Samples for turbidity measurement were measured on an automated microplate reader (BioTek). 2 pL sample was loaded on a TAKE3 plate for each measurement. Absorbance at 500 nm was collected for indicating of turbidity. Raw data was redacted to 1 cm-1using the following equation:

[0110] 0.5 mm

[0111] A = Aox - - — x 20 a where A is the absorbance reduced to 1 cm-1, Ao is the data collected, and d is the depth of each well on TAKE3 plate. Sigmoidal Equation

[0112] The turbidity change of peptide-DNA coacervation in response to NaCI was fitted with the following sigmoidal equation: where Aminis smallest absorbance and Amaxwas the highest absorbance, IC50 is the half-maximal inhibitory concentration, and Hill Slope is the simulated curve factor.

[0113] P3-3D DNA Origami Co-Assembly and P3-DNA Phase Separation Diagram

[0114] As co-assembly of P3-3D DNA origami was only successful within a narrow NaCI range, the co-assembly map of charge-unbalanced [R]:[P] ratios was plotted at variable NaCI concentrations. The NaCI concentration at which turbidity dropped to 5% of the initial value was defined as the critical point for phase transition. Phenomenological P3-DNA LLPS binodal curve was then fitted based on Voorn-Overbeek (VO) theory.1 To illustrate that the co-assembly was only successful near the binodal curve, the co-assembly diagram was overlaid with the P3-DNA phase diagram. In both cases, the phosphate concentration was maintained at 1 mM, using 4.5 nM M13mp18 and 135 nM of each staple strand (164 strands). For construction of the P3-DNA phase diagram, mixtures of P3 (AC-RRRY-NH2; SEQ ID NO: 1) and DNA were prepared at varying [R]:[P] ratios (0.25:1, 0.5:1 , 1 :1 , 2:1 , 3:1 , and 5:1), and each condition was tested across a range of NaCI concentrations (0, 20, 50, 70, 100, 150, and 200 mM). Similarly, for the P3-3D DNA origami co-assembly diagram, the same DNA components and [R]:[P] ratios were used, with samples incubated at 37 °C for 60 hours across NaCI concentrations of 0, 20, 50, 70, 100, and 200 mM before analysis via agarose gel electrophoresis.

[0115] P4-3D DNA Origami Co-Assembly and p4-DNA Phase Separation Diagram

[0116] To determine the P4-3D DNA origami co-assembly conditions under charge-unbalanced states, the turbidity changes of charge unbalanced DNA / peptide mixture was monitored in response to varying NaCI concentrations. The NaCI concentration at which turbidity dropped to 5% of the initial value was defined as the critical point for phase transition. The co-assembly behavior of P4-3D DNA origami under these conditions mirrored that observed in charge- unbalanced P4-DNA liquid-liquid phase separation (LLPS), with 5% remaining turbidity marking the critical NaCI concentration. Mixtures of P4 (Ac-RRRRY-NFL; SEQ ID NO: 2) and DNA were prepared at varying [R]:[P] ratios (0.25:1 , 0.5:1 , 1 :1 , 2:1 and 5:1), and each condition was tested across a range of NaCI concentrations (0, 50, 100, 150, 200, and 250 mM). Because of the strong interaction between P4 and DNA strands, high P4 concentration caused the aggregation immediately when mixing and hinder the co-assembly near the phase transition curve.

[0117] To solve this problem and construct the P4-3D DNA origami co-assembly diagram, ALP- triggered charge-switching of the (Ac-RRRRpY-NH2; SEQ ID NO: 5) peptide (P4p) was employed to investigate the conditions required for co-assembly. By slowly increasing the total positive charge from +2 to +4 to the binodal curve, this method prevented the aggregation pf P4-DNA. Similarly, the same DNA components and [R]:[P] ratios were used, with samples incubated at 37 °C for 24 hours across NaCI concentrations of 0, 50, 100, 150, 200 and 250 mM before agarose gel electrophoresis analysis.

[0118] Alkaline Phosphatase (ALP)-Responsive Assembly of 2D DNA Origami

[0119] Isothermal co-assembly of triangular 2D DNA origami were conducted through incubation of M13MP18 scaffold, 10-time excess of staples (207 strands) and (Ac-RRRpY-NH2; SEQ ID NO: 4) (tyrosine residue is modified with phosphate). For a typical synthesis, 2 nM M13mp18, 20 nM each staple (207 strands) and 50 pM (Ac-RRRpY-NH2; SEQ ID NO: 4) were mixed and incubated at 37 °C for 12h in TANa buffer ([NaCI] = 2 mM). 2 U / mL ALP enzyme were added into the mixture to cut the phosphate on the peptide C-terminus, thereby triggering the assembly of triangular 2D DNA origami. The product was characterized with AGE and AFM without staple purification.

[0120] Alkaline Phosphatase (ALP)-Responsive Co-Assembly of 3D DNA Origami with P3p

[0121] Alkaline phosphatase (ALP)-responsive co-assembly of P3-3D DNA origami (square lattice) were conducted through incubation of M13MP18 scaffold, 30-time excess of staples and (Ac-RRRpY-NH2; SEQ ID NO: 4) in a TANa buffer. For a typical synthesis, 4.5 nM M13mp18, 135 nM each staple (164 strands) and 330 pM (Ac-RRRpY-NH2; SEQ ID NO: 4) were mixed and incubated at 37 °C for 60 h in TA buffer. 2 U / mL ALP enzyme were added into the mixture to cut the phosphate on the peptide C-terminus, thereby triggering the co-assembly of square-lattice DNA origami. The product was characterized with AGE and AFM without staple purification.

[0122] Alkaline Phosphatase (ALP)-Responsive Assembly of 3D DNA Origami with P4p

[0123] Alkaline phosphatase (ALP)-responsive co-assembly of P4-3D DNA origami (square lattice) were conducted through incubation of M13MP18 scaffold, 30-time excess of staples and (Ac-RRRRpY-NH2; SEQ ID NO: 5). For a typical synthesis, 4.5 nM M13mp18, 135 nM each staple (164 strands) and 50 pM (Ac-RRRRpY-NH2; SEQ ID NO: 4) were mixed and incubated at 37 °C for 60 h in TANa buffer ([NaCI] = 100 mM). 2 U / mL ALP enzyme were added into the mixture to cut the phosphate on the peptide C-terminus, thereby triggering the assembly of square-lattice DNA origami. The product was characterized with AGE and AFM without staple purification.

[0124] ALP Responsive Assembly of ssRNA Origami P3p

[0125] Isothermal co-assembly of triangular ssRNA origami were conducted through incubation of ssRNA and (Ac-RRRpY-NH2; SEQ ID NO: 4). For a typical synthesis, 20 ng / pL ssRNA and 50 pM (Ac-RRRpY-NH2; SEQ ID NO: 4) were mixed and incubated at 37 °C for 12 h in TANa buffer ([NaCI] - 2 mM). 2 U / mL ALP enzyme were added into the mixture to cut the phosphate on the peptide C-terminus, thereby triggering the assembly of triangular DNA origami. The product was characterized with AGE and AFM without staple purification.

[0126] Rapid Co-Assembly of 2D and 3D DNA Origami with ALP Responsive P3p

[0127] As the isothermal peptide-DNA origami co-assembly only presented highest yield near the peptide-DNA binodal curve and rapidly decrease with higher or lower NaCI, especially for 3D DNA origami. It was found that peptide-3D DNA origami co-assembly at the binodal curve presented very slow kinetics and required as long as 60 hours. The AGE results demonstrated that insufficient incubation would cause stuck in the agarose gel which indicate the aggregated DNA nanostructures. The aggregates transferred slowly into DNA origamis through incubation. Because of the narrow co-assembly range, aggregation is inevitable if the peptide was directly mix with DNA strands, which caused the aggregation and slowed down the co-assembly. To prevent the aggregation during sample preparation and accelerate the co-assembly kinetics, a charge switching peptide P3p was used with the tyrosine at C-terminal attached to a phosphate group. The designed P3p (Ac-RRRpY-NH2; SEQ ID NO: 4) with a negative charge of +1 was not able to trigger the co-assembly due to single phase of peptide-DNA mixture. However, the total charge increased to +3 which locates near the peptide-DNA bimodal curve, thereby triggering the co-assembly without causing aggregation.

[0128] For rapid 2D DNA origami co-assembly, 2 nM M13mp18 and 10-time staples (207 strands) was mixed with 400 pM P3p (Ac-RRRpY-NH2; SEQ ID NO: 4) in 1x TA (2 mM NaCI) and 0 U / mL, 1 U / mL, 2 U / mL, 4U / mL, 6U ALP, 8U / mL, 10 U / mL ALP were added respectively. After 1-hour incubation at 37 °C, the samples were tested with AGE. Triangular 2D DNA origami was successfully assembled with 6U / mL ALP isothermal incubation at 37 °C within 1 h. For rapid 3D DNA origami co-assembly, 4.5 nM M13mp18 and 30-time excess of staples (164 strands) was mixed with 330 M P3p in 1 * TA (20 mM NaCI) and 0 U / mL, 2U / ml_, 4 U / mL, 6 U / mL, 6U ALP and 8U / mL ALP were added respectively. After 3-hour incubation at 37 °C, the samples were tested with AGE. 3D DNA origami was successfully assembled with 4U / mL-8U / mL ALP through isothermal incubation at 37 °C within 3 h.

[0129] DMSO Triggered Charge-Switching Peptide for 3D DNA Origami Co-Assembly

[0130] For DMSO-triggered 3D DNA origami co-assembly, a DMSO-responsive, chargeswitching peptide with an N-terminal cysteine was designed. Upon oxidation, the cysteine residues formed covalent disulfide bonds (-S-S-), converting the peptide from P2 to P4. Although the total number of charges remained unchanged, the charge density increased from +2 to +4, resulting in stronger electrostatic interactions for co-assembly. For DMSO triggered 3D DNA origami co-assembly, Ac-CRRY-NH? (SEQ ID NO: 6) was mixed with TCEP to prevent the disulfide bonds under the oxidation of air (Scheme 1). For a typical co-assembly, 4.5 nM M13MP18 and 30-time excess of staples (164 strands) were mixed with 0.25 mM CRRY and 0.25 mM TCEP in 1* TA buffer with varied NaCI. 10% DMSO was then added into the mixture to trigger the co-assembly. To confirm that DNA origami was formed due to the formation of disulfide bonds, iodoacetamide (IAA) was added to block the cysteine group, which would inhabit the formation of disulfide bonds with the existence of DMSO (Scheme 2). For a typical synthesis, 10 mM IAA was first incubated with 1 mM CRRY and 1 mM TCEP in dark for 1h to block the cysteine group. Afterwards, the mixture was transferred to 3D DNA origami co-assembly conditions. The final concentration of each component was 4.5 nM M13MP18 and 30-time excess of staples, 70 mM NaCI, 0.25 mM TCEP, 0.25 mM CRRY, 2.5 mM IAA and 10% DMSO. The mixture was incubated at 37 °C for 60 h before AGE.

[0131] Scheme 1

[0132]

[0133] Agarose Gel Electrophoresis

[0134] 2% agarose gel were prepared in 1 * TAE buffer (45 mM Tris pH 8.0, 45 mM boric acid, 1 mM EDTA) supplemented with 12.5 mM MgCL. 10 pL sample was mixed with 2 pL loading dye and 1.2 pL 100 mM MgCI2. The annealed products were subjected to native agarose gel electrophoresis at 100 V in an ice-water bath. The agarose gel was post stained with 3* GelRed before imaging.

[0135] AFM Imaging in Air Mode

[0136] AFM images were obtained by the dry mode due to its convenience and the smaller amount of damage caused by the probe tips. However, liquid AFM provides better resolution and was used. Sample preparation for dry AFM was as follows: (1) the annealing mixtures were diluted ten times with 1x TAE-Mg buffer, (2) deposited 5 pL onto a freshly cleaved mica (Ted Pella) to adsorb for 1 min, (3) dried the specimen with compressed air, (4) rinsed it with 30 pL of 3 mM Mg(OAc)2and (5) dried the specimen with compressed air again. Images were acquired on a Multimode 8 AFM in the ‘ScanAsystAir in Air’ mode using ScanAsystAir probes (Bruker). For liquid AFM, sample preparation was like that of dry AFM except that, after sample deposition, 30 pL of imaging buffer (10 mM NiCh and 10 mM Tris HCI, pH 8.0) was added onto the mica. Images were obtained in liquid tapping mode using tipAof SNL-10 probes (Bruker). Based on the stability study of a model heterodimer system, all structures were expected to be stable at room temperature, at which AFM sample preparation and imaging were performed. Images were processed and prepared with Gwyddion.

[0137] DNA Nanostructure Design

[0138] Triangular DNA origami (2D): Triangular DNA origami was designed.

[0139] Nucleic acid nanostructures design and sequences.

[0140]

[0141]

[0142] Isothermal folding of ssRNA origamis using positively-charged peptides was performed using similar strategies as described herein.

[0143] Programming DNA Assembly with Charge-Switching Peptides:

[0144] By applying enzyme to the r-R-r-pY, the total charge of the tetrapeptide can be increased to trigger the assembly of DNA origami. Peptide-DNA Co-Crystallization

[0145] Crystals were prepared from a hanging drop method.

Claims

CLAIMSWhat is claimed:

1. A method of forming a nucleic acid nanostructure using peptide-assisted assembly, the method comprising: contacting a nucleic acid with one or more charged peptides to form a combination; incubating the combination for a period of time; and forming the nucleic acid nanostructure.

2. The method of claim 1 , wherein the nucleic acid comprises DNA, RNA, or a combination thereof.

3. The method of claim 1 , wherein the nucleic acid comprises a single stranded or a double stranded nucleic acid.

4. The method of claim 1 , wherein the nucleic acid comprises a single-stranded circular DNA scaffold.

5. The method of claim 4, wherein the nucleic acid comprises an M13mp18 scaffold.

6. The method of claim 1 , wherein the combination further comprises a plurality of nucleic acid staple strands.

7. The method of claim 1, wherein the combination is incubated under physiological conditions for about 1 hour to about 60 hours.

8. The method of claim 7, wherein the combination is incubated at about 37 °C to about 40 °C for about 3 hours to about 24 hours.

9. The method of claim 1 , wherein the combination is incubated in a buffer comprising Trisacetate and NaCI without Mg2+.

10. The method of claim 9, wherein a concentration of the NaCI in the combination ranges from about 1 mM to about 200 mlVI.

11. The method of claim 1 , wherein the charged peptides comprise positively charged peptides.

12. The method of claim 1 , wherein the charged peptides comprise at least one arginine residue, tyrosine residue, cysteine residue, or combinations thereof.

13. The method of claim 1 , wherein the charged peptides comprise an N-terminal acetyl group.

14. The method of claim 1 , wherein the charged peptides comprise at least one phosphate group.

15. The method of claim 1 , wherein the charged peptides comprise an arginine-to-phosphate ratio of about 0.1 :1 to about 2:1.

16. The method of claim 1, wherein the charged peptides comprise one or more peptides having the amino acid sequence of any one of SEQ ID NO: 1-7.

17. The method of claim 1 , wherein a concentration of the charged peptides in the combination ranges from about 5 pM to about 75 pM.

18. The method of claim 1 , wherein the combination further comprises tris(2- carboxyethyl)phosphine (TCEP), iodoacetamide (IAA), alkaline phosphatase (ALP), dimethylsulfoxide (DMSO), or combinations thereof.

19. The method of claim 1 , wherein the nucleic acid nanostructure is a 1-dimensional (1 D), 2- dimensional (2D), or 3-dimensional (3D) nanostructure.

20. The method of claim 19, wherein the nucleic acid nanostructure is a 2D or 3D DNA origami nanostructure.

21. A kit for forming a nucleic acid nanostructure using peptide-assisted assembly, the kit comprising:a nucleic acid scaffold; one or more charged peptides; a buffer comprising Tris-acetate and NaCI; and a plurality of nucleic acid staple strands.

Images