Preparation method of two-dimensional nucleic acid nanostructure polymer film based on fluid mosaic assembly and application thereof

Abstract

This invention discloses a method for preparing macroscopically ordered two-dimensional nucleic acid nanostructure polymer films based on fluid mosaic assembly and its applications. The method involves preparing nucleic acid nanostructure building blocks with hydrophobic modification groups and specific hybridization sticky ends, spreading surfactants on the surface of a buffer solution to form a low-interfacial-tension two-dimensional fluid interface system, and then annealing to enrich the building blocks at the interface. Entropy-driven dynamic specific hybridization, defect self-correction, and grain boundary fusion are achieved, forming a macroscopically continuous and highly ordered monolayer film. This invention achieves, for the first time, the preparation of centimeter-scale independently supported films, breaking the size-order trade-off problem and proposing a novel mechanism for fluid mosaic assembly. The resulting film possesses high programmability, unique deep-ultraviolet photonic crystal characteristics, and good post-processing integration, making it suitable for various building blocks such as DNA tiles and three-dimensional DNA origami, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of DNA nanotechnology and self-assembled functional materials technology, and more specifically to a method for preparing a two-dimensional nucleic acid nanostructure polymer film based on fluid mosaic assembly and its application. Background Technology

[0002] Bottom-up self-assembly is the core strategy for building complex functional materials, but in the process of scaling up from the nanoscale to the macroscale, it generally faces the key challenge of "size-order trade-off": as the size of the assembly increases, the number of building units grows exponentially, and tiny bonding errors or kinetic traps will continue to accumulate, eventually destroying the long-range periodicity of the assembly and making it difficult to form large-area, highly ordered macroscopic materials.

[0003] DNA origami, with its atomic-level programmability, has become an ideal nanoscale building block for resolving the aforementioned contradictions. It allows for the precise design and synthesis of nanostructures with specific geometries, laying the foundation for the fabrication of precisely ordered functional materials. However, current technologies have consistently failed to effectively extend the nanoscale order of DNA origami to the macroscopic scale, making it difficult to fabricate centimeter-scale, highly ordered, and independently supportable two-dimensional films. Existing mainstream DNA origami assembly methods all suffer from significant technical limitations, including:

[0004] Solution-phase self-assembly: The core of this method is the direct assembly of DNA origami arrays in solution via sticky end hybridization. However, limited by the finite Brownian motion and highly kinetic traps of DNA origami units, only micrometer-scale ordered regions (typically 2-10 μm) can be obtained, making it impossible to form continuous large-area films or to fabricate independently supportable film structures (see: Liu, W. et al. Crystalline two-dimensional DNA-origami arrays. Angew. Chem. Int. Ed. 50, 264-267 (2011)). Related experiments have confirmed that under conventional annealing conditions, the largest ordered region of solution-phase assembled DNA origami is only about 8 μm, far from meeting the application requirements of macroscopic functional materials.

[0005] Substrate-assisted assembly: This method utilizes solid or semi-solid interfaces such as mica and lipid bilayers to promote the two-dimensional arrangement of DNA origami. Although it can improve local order to a certain extent, the size of the continuous film prepared is usually no more than tens of micrometers due to the limitations of substrate size and the inherent static properties of the interface. Furthermore, the defects generated during the assembly process are difficult to eliminate after assembly. At the same time, the DNA origami film prepared by this method cannot be detached from the substrate to achieve independent support, which greatly limits its application in device fabrication, functional integration and other scenarios (see reference: Suzuki, Y., Endo, M., Sugiyama, H. Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures. Nat. Commun. 6, 8052 (2015)).

[0006] Interface-assisted assembly of non-DNA polymers: Recent studies have utilized surfactants to assist in the synthesis of two-dimensional covalent organic frameworks or polymer films at the air-water interface (see: Liu, K. et al. On-water surface synthesis of crystalline, few-layer two-dimensional polymers assisted by surfactant monolayers. Nat. Chem. 11, 994-1000 (2019)), achieving macroscopic interface assembly of non-DNA polymers. However, this method relies on irreversible covalent chemical reactions, and its assembly mechanism is completely incompatible with DNA nanostructure systems driven by reversible, weak interactions (DNA hybridization hydrogen bonds), making it unsuitable for direct application to DNA origami assembly. To date, no reports have been found regarding the dynamic behavior of DNA origami at fluid interfaces, the entropy-driven sorting mechanism, or the preparation of macroscopically ordered DNA origami films based on this mechanism.

[0007] In summary, the existing technology field lacks a universal strategy that can provide a highly dynamic assembly environment for nanoscale building blocks such as DNA origami that rely on reversible weak interactions. It is impossible to continuously correct assembly errors and fuse grain boundaries to non-destructively scale up the nanoscale precision design of DNA origami to the macroscale. This has become the core technical obstacle restricting the transition of DNA nanotechnology from basic research to the practical application of macroscopic functional materials. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing two-dimensional nucleic acid nanostructure polymer films based on fluid mosaic assembly and its application, thereby solving the problem that existing technologies cannot overcome the problems of limited assembly size and poor long-range order caused by kinetic traps and defect freezing, which makes it difficult to prepare centimeter-scale, highly ordered, and independently supportable two-dimensional films.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] According to a first aspect of the present invention, a method for preparing a macroscopically ordered two-dimensional nucleic acid nanostructure polymer film based on fluid mosaic assembly is provided, comprising the following steps: S1, preparation of building units: designing and synthesizing nucleic acid nanostructure units with geometric shapes, covalently modifying hydrophobic modification groups at predetermined positions of the nucleic acid nanostructure units to obtain hydrophobically modified nucleic acid nanostructure building units with specific hybridization sticky ends, wherein the sticky ends and the hydrophobic modification groups are connected by flexible connecting arms; S2, construction of fluid interface: dispersing the hydrophobically modified nucleic acid nanostructure building units in a buffer solution, spreading surfactant molecules on the solution surface to form a monolayer, constructing a two-dimensional fluid interface system with low interfacial tension, reducing the interfacial tension to below 40 mN / m; S3, entropy-driven assembly: annealing the two-dimensional fluid interface system to enrich and embed the hydrophobically modified nucleic acid nanostructure building units into the hydrophobic regions of the surfactant monolayer, utilizing the high in-plane mobility imparted by the two-dimensional fluid interface to achieve dynamic specific hybridization of sticky ends between building units, defect self-correction, and grain boundary fusion, forming a macroscopically continuous and highly ordered monolayer two-dimensional nucleic acid / nanostructure polymer film.

[0011] Preferably, in step S1, the hydrophobic modifying group is one or more of cholesterol, lipid molecules, long-chain alkanes, fluorinated compounds, or hydrophilic-hydrophobic block polymers.

[0012] It should be understood that cholesterol is the preferred hydrophobic modification group, but any molecule that can effectively anchor DNA origami to the hydrophobic region of the interface can be substituted. For example, lipid molecules (DOPE), long-chain alkanes (C18 alkyl chains), and hydrophilic-hydrophobic block polymers (Pluronic series polymers).

[0013] Preferably, the flexible linker is a 15 nt poly-T single-stranded DNA, but the nucleotide length of the flexible linker can be adjusted according to assembly requirements.

[0014] Preferably, in step S1, the nucleic acid nanostructure unit is a DNA tile, a three-dimensional DNA origami, an RNA origami, or a nucleic acid-hybrid structure, wherein the DNA tile is preferably a two-dimensional DNA origami tile; the geometric shape of the two-dimensional DNA origami tile is square, rectangular, rhomboid, or triangular, etc., and hydrophobic modification groups are applied to its edges or vertices; the three-dimensional DNA origami is modified only on its predetermined single surface with hydrophobic modification groups and sticky ends to achieve directional adsorption of the three-dimensional DNA origami at a two-dimensional fluid interface.

[0015] The method provided by the present invention is applicable not only to DNA tiles, three-dimensional DNA origami, etc., but also to functionalized nanoparticles with DNA recognition chains modified on their surfaces; the functionalized nanoparticles are inorganic nanoparticles or protein oligomers with DNA recognition chains and hydrophobic modification groups modified on their surfaces, and the inorganic nanoparticles are gold nanorods or quantum dots.

[0016] According to the present invention, in step S1, the hydrophobic modified nucleic acid nanostructure building unit includes a first building unit and a second building unit that are complementary, and the sticky ends of the two are complementary oligonucleotide sequences.

[0017] Preferably, in step S1, the buffer solution is a 1×TE-MgCl2 buffer solution, composed of 10 mM Tris, 1 mM EDTA, and 12.5 mM MgCl2, with a pH of 8.0. The ion concentration and pH of the buffer solution can also be adjusted according to the stability requirements of the nucleic acid nanostructure unit.

[0018] Preferably, in step S2, the surfactant is one of anionic, cationic, nonionic, amphoteric, or polymeric surfactants. Preferably, the surfactant is dissolved in chloroform and then added dropwise to the solution surface, or directly dissolved in the oil phase and spread onto the solution surface with the oil phase. The purpose of this is that the surfactant can effectively reduce the interfacial tension in the assembled system (e.g., to < 40 mN / m) and provide sufficient two-dimensional mobility for the embedded nanounits.

[0019] For example, the anionic surfactant is preferably sodium dioctyl sulfosuccinate, the cationic surfactant is preferably CTAB and the MgCl2 concentration in the buffer solution is increased to 20 mM to shield electrostatic effects, and the nonionic surfactant is preferably Triton X-100.

[0020] Preferably, in step S2, the two-dimensional fluid interface is a gas-liquid interface or a liquid-liquid interface. More preferably, the gas-liquid interface is a gas-water interface, but it should be understood that any interface that can form a stable, flowing two-dimensional confined environment can be used as a substitute, such as a liquid-liquid interface (e.g., an oil-water interface, or an immiscible organic-aqueous phase). As long as the nanounits can be confined and endowed with high in-plane motion capability, entropy-driven sorting can be achieved.

[0021] According to the present invention, in step S3, the annealing process is one of programmed linear cooling annealing, isothermal annealing, multi-step annealing, or field-assisted annealing.

[0022] The programmed linear cooling annealing involves cooling from 45°C to 20°C at a rate of 0.1°C / hour; the isothermal annealing involves holding the viscous end-chain-breaking temperature at a constant temperature for 5-7 days; the multi-step annealing involves first rapidly cooling / holding the temperature to form a large number of small crystal nuclei, and then annealing at a lower temperature for a long time to promote grain growth and fusion; and the external field-assisted annealing involves applying mild interfacial shear, electric field, or magnetic field during the annealing process to guide domain orientation or accelerate defect repair.

[0023] It should be understood that dynamic specific hybridization in step S3 refers to reversible hybridization of nucleic acid sticky ends based on hydrogen bonds, where the binding and dissociation between building units are always in dynamic equilibrium; high in-plane mobility allows mismatched building units to detach from their binding sites and diffuse to the correct positions for rehybridization, achieving continuous defect correction and grain boundary fusion during the assembly process, ultimately forming a long-range ordered macroscopic thin film structure.

[0024] According to a preferred embodiment of the present invention, the preparation method further includes an in-situ silanization enhancement step for the obtained two-dimensional DNA origami polymer film: in the two-dimensional fluid interface system after film formation, a silanizing sol is slowly injected into the buffer solution subphase, and the reaction is allowed to proceed at room temperature for 6-24 hours to complete the silanization encapsulation; the silanizing sol is a mixture containing 2% v / v TMAPS and 2% v / v TEOS as a base of 1×TAE-Mg buffer, or a buffer system using APTES, MPTMS, or sodium silicate solution adjusted to pH 5.5 as silanizing agents.

[0025] According to a second aspect of the present invention, an application of a two-dimensional nucleic acid nanostructure polymer film prepared by the preparation method described above is provided in the field of optics, wherein the film is used as a deep ultraviolet polarization optical element, or, by adjusting the nano-periodic lattice constant of the film to above 100 nm, it is used as a structural color coating, optical waveguide, or optical sensor in the visible / near-infrared band.

[0026] According to a third aspect of the present invention, an application is also provided for a two-dimensional nucleic acid nanostructure polymer film prepared by the above-described preparation method in the fields of nanofabrication, separation and detection, and material growth. The film can be used as an ultra-fine template to guide the directional deposition of metal nanoparticles to prepare an ordered nanoelectrode array / catalytic surface, or after silanization, it can be made into a nanofiltration membrane with uniform nanopores for molecular sieving / virus filtration, or as a high-density ordered biosensor interface to achieve ultrasensitive detection of biomolecules, or as a sacrificial template to guide the oriented growth of two-dimensional transition metal chalcogenides / graphene.

[0027] It is generally accepted in this field that the core driving force of DNA origami assembly is sticky end hybridization, a weak interaction similar to hydrogen bonding, with bond energies far lower than covalent bonds, making large-scale ordered assembly impossible through interfacial environments. Existing research on interface-assisted assembly (such as Liu et al. Nat. Chem. 2019) focuses on irreversible covalent polymerization systems. This invention, however, is the first to demonstrate that the reversibility of the weak interaction of sticky end hybridization can be amplified in the high-mobility environment of fluid interfaces, becoming a core advantage for self-correction of defects during assembly. This discovery also makes it difficult for those skilled in the art to obtain effective technical inspiration from the literature on interface assembly related to covalent polymerization when solving the macroscopic assembly problem of DNA origami.

[0028] It should be understood that the reversibility of the method of the present invention is reflected in the fact that the hybridization and dissociation of the sticky ends are always in dynamic equilibrium, and the binding and dissociation can occur repeatedly, providing a thermodynamic possibility for defect correction. The present invention provides a highly dynamic environment for nano-building units such as DNA origami that rely on reversible interactions, so as to continuously correct assembly errors and fuse grain boundaries, thereby providing a general assembly strategy for non-destructively scaling up the precision design at the nanoscale to the macroscale.

[0029] As described in the background section of this invention, existing substrate-assisted assembly techniques (such as Suzuki et al. 2015) emphasize static adsorption, regarding the immobilization of building blocks as a prerequisite for achieving ordered arrangement. This invention, however, takes the opposite approach, deliberately avoiding the immobilization of building blocks by constructing a highly fluid interface environment with ultra-low interfacial tension, and using entropy-driven assembly instead of traditional enthalpy-driven assembly. This technical approach is completely opposite to the mainstream research path, constituting the significant inventiveness of this invention.

[0030] Imparting sufficiently high in-plane mobility to DNA origami at the tens of nanometer scale in two-dimensional space while preventing its desorption into the bulk phase is a recognized technical challenge in this field. This invention, through systematic surfactant screening experiments, discovered that anionic sodium dioctyl sulfosuccinate (DSS) can be bridged by metal cations (Na+). + Mg 2+This invention forms a moderate and dynamic interaction with the DNA backbone, which can efficiently enrich DNA origami building blocks at the interface without freezing the interface's fluidity, perfectly solving the aforementioned problems. This discovery is not obvious, because DNA itself is negatively charged, and those skilled in the art usually avoid anionic surfactants to avoid electrostatic repulsion. However, this invention, through systematic experiments, reveals the criticality of this moderate interaction for interface assembly, successfully overcoming this technical bias.

[0031] Compared with the prior art, the present invention has the following significant advantages and beneficial effects:

[0032] 1) Achieving breakthroughs in assembly scale and order, overcoming the size-order trade-off problem. This invention is the first to achieve the fabrication of independently supported DNA origami films with centimeter-level lateral dimensions, and the ordered crystal domain area inside the film can reach 800~1200 μm. 2 Compared to the ordered regions (approximately 10 μm) of traditional solution-phase assembly, 2 This represents an improvement of more than two orders of magnitude, truly breaking through the core challenge of the size-order trade-off that is prevalent in the self-assembly process of DNA nanostructures.

[0033] 2) A novel assembly mechanism is proposed to overcome the kinetic traps at their source. This invention innovatively proposes a novel physical mechanism for fluid mosaic assembly, transforming the assembly interface from a traditional static substrate into a dynamic "annealer." By utilizing an entropy-driven mechanism, it overcomes the inherent kinetic traps in DNA hybridization at their source, providing crucial support for achieving long-range ordered assembly of DNA nanostructures.

[0034] 3) It possesses high programmability and versatility, enabling digital customization of thin film structures. The assembly method of this invention is applicable to DNA origami units of various geometries and symmetries (such as tetragonal lattices, hexagonal lattices, etc.). By simply changing the design of the DNA origami building blocks, the lattice structure of macroscopic thin films can be customized, realizing digital customization from molecular blueprints to macroscopic crystals, with a wide range of applications.

[0035] 4) This invention endows the thin film with unique photonic crystal properties, demonstrating promising optical applications. The thin film prepared by this invention possesses a precise nano-periodic structure (approximately 58.2 nm) and exhibits excellent photonic crystal properties in the deep ultraviolet band (120–145 nm). Using the plane wave expansion method (MPB) and the finite-difference time-domain method (FDTD), this invention simulated the photonic bandgap of four typical two-dimensional lattice structures: square, rectangular, rhombic, and triangular. The results confirmed that all structures exhibit transverse electric (TE) mode photonic bandgap and high transmission characteristics in transverse magnetic (TM) mode, making them potential polarization optical elements in this scarce band with clear application prospects.

[0036] 5) Excellent thin film post-processing and integration, facilitating device fabrication and practical applications. The thin film prepared by this invention can undergo in-situ silanization at the assembly interface, significantly enhancing the mechanical and chemical stability of the film after silanization. Simultaneously, the film can be transferred intact and without damage to any substrate such as silicon wafers or flexible substrates, facilitating subsequent device fabrication and functional integration.

[0037] 6) The technological paradigm is highly inspiring, providing a general approach for cross-scale ordered assembly. The assembly strategy of this invention originates from the biomembrane fluid mosaic model. Its core fluid interface entropy-driven assembly approach provides a novel and universal technological paradigm for the cross-scale ordered assembly of proteins, nanoparticles, two-dimensional material sheets, and other nanoscale building blocks, which has important inspirational and reference value for research in related fields.

[0038] In summary, this invention discloses a method for preparing macroscopically ordered two-dimensional nucleic acid nanostructure polymer films based on fluid mosaic assembly and its applications. This method involves preparing nucleic acid nanostructure building blocks with hydrophobic modification groups and specific hybridization sticky ends, spreading surfactants on the surface of a buffer solution to form a low-interfacial-tension two-dimensional fluid interface system, and then annealing to enrich the building blocks at the interface. Through entropy-driven dynamic specific hybridization, defect self-correction, and grain boundary fusion, a macroscopically continuous and highly ordered monolayer film is formed. The film can also be enhanced by in-situ silanization. This invention overcomes the technical biases in the field that weak interactions cannot achieve large-scale interfacial assembly, immobilization is a prerequisite for ordered arrangement, and anionic surfactants are unsuitable for DNA assembly. It departs from the mainstream assembly path by replacing enthalpy-driven with entropy-driven assembly, thus overcoming the challenges of DNA assembly. Overcoming the technical challenges of high mobility and anti-desorption at origami interfaces, this study achieves the first-ever fabrication of centimeter-scale independently supported thin films, breaking the size-order trade-off and proposing a novel fluid mosaic assembly mechanism. The resulting films possess high programmability, unique deep-ultraviolet photonic crystal properties, and excellent post-processing integration. They can be used not only as deep-ultraviolet polarizing optical elements and visible light structural color coatings in optics, nanofabrication, and separation detection, but their core assembly strategy also provides a general technical paradigm for the cross-scale ordered assembly of other nanoscale building blocks. Applicable to various building blocks such as DNA tiles, three-dimensional DNA origami, and functionalized nanoparticles, this study has broad application prospects and provides technological inspiration. Attached Figure Description

[0039] Figure 1 These are photographs of polymer films prepared according to the method of the present invention, wherein A is a photograph of the film grown on the liquid surface in the inner culture dish, and B is a photograph of the film obtained by mechanical retrieval from a silicon wafer.

[0040] Figure 2This is a large-scale electron microscope image of the square DNA origami array film prepared in Example 1;

[0041] Figure 3 This is a magnified electron microscope image of a square DNA origami array film prepared in Example 1. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0043] Example 1: Preparation of Square DNA Origami (SDO) Film

[0044] 1.1 Preparation of building blocks

[0045] Design and synthesize a square DNA origami (100 nm side length), with the scaffold strand being a 7249 nt long DNA derived from M13mp18 (a commercially available product, such as Bioruler B3007).

[0046] Staple strands: All were commercially synthesized. The apex cholesterol-modified strand and sticky end sequences are as follows (the remaining staple strand sequences are designed based on the classic square DNA origami design; for specific sequences, please refer to the literature Liu, Y., Dai, Z., Xie, X., Li, B., Jia, S., Li, Q., ... & Liu, X. (2024). Spacer-programmed two-dimensional DNA origami assembly. Journal of the American Chemical Society, 146(8), 5461-5469):

[0047] SDO-A 4 Vertex chain (5'→3'):

[0048] [Cholesterol] -TTTTTTTTTTTTTTTT-ATCCGTTA-CAAATCAACAAATCGGCGGAGCTCG

[0049] (Note: "ATCCGTTA" is the adhesive end, and the subsequent sequence is complementary to the stent chain; poly-T is a 15 nt flexible connector arm.)

[0050] SDO-B 4 Vertex chain (5'→3'):

[0051] [Cholesterol] -TTTTTTTTTTTTTTTT-TAACGGAT-CAAATCAACAAATCGGCGGAGCTCG

[0052] (The sticky end “TAACGGAT” and SDO-A) 4 (Complementary)

[0053] Buffer: 1× TE-MgCl2 buffer (10 mM Tris, 1 mM EDTA, 12.5 mM MgCl2, pH 8.0).

[0054] Assembly system: The scaffold chain and staple chain were mixed at a molar ratio of 1:10, with a final concentration of 10 nM for the scaffold chain and 100 nM for the staple chain, and a total volume of 50 μL.

[0055] The annealing procedure is as follows:

[0056] Maintain at 65℃ for 20 minutes;

[0057] 60℃ → 40℃, cooling rate 20 minutes / ℃;

[0058] 45℃ → 25℃, cooling rate 15 minutes / ℃.

[0059] Purification: PEG precipitation method was used.

[0060] The annealed product was mixed with PEG buffer (5 mM Tris, 1 mM EDTA, 500 mM NaCl, 15% w / v PEG8000) at a 1:1 volume ratio.

[0061] Centrifuge at 10,000 rcf for 15 minutes, then discard the supernatant;

[0062] The precipitate was resuspended in 1× TE-MgCl2 buffer and shaken at 25°C and 800 rpm for 12 hours.

[0063] The absorbance at 260 nm was measured using Nanodrop, quantified, and diluted to 100 ng / μL. The sample was then stored at 4°C.

[0064] Quality verification:

[0065] Agarose gel electrophoresis (1% agarose, 1× TAE-Mg buffer, 100 V, ice water bath for 1.5 hours): Cholesterol-modified SDO-A 4 Compared to the unmodified SDO-A, the migration rate of the bands was significantly reduced, and the bands were single and clear, indicating that the structure was intact and well dispersed.

[0066] Transmission electron microscopy observation (sample preparation method is described below): SDO-A 4 The edge thickness is 8.5 ± 0.3 nm (n=50), and the edge length is 43.1 ± 0.8 nm (n=50), which matches the design values.

[0067] 1.2 Interface Assembly

[0068] Reaction vessel: 200 μL PCR tube.

[0069] Feeding: Take SDO-A 4 With SDO-B 4 Mix 10 μL (100 ng / μL) of each solution in a PCR tube, and add 1× TE-MgCl2 buffer until the liquid surface bulges (total volume approximately 30 μL) to form a stable meniscus.

[0070] Surfactant: Dissolve DSS (sodium dioctyl sulfosuccinate) in chloroform to prepare a 1 mg / L solution. Using a microsyringe, draw 1 μL of the DSS-chloroform solution and gently drop it into the center of the liquid surface. Let it stand at room temperature for 5 minutes to allow the chloroform to evaporate completely. DSS will then self-assemble at the air-water interface to form a monolayer.

[0071] Annealing procedure: Place the PCR tubes in a PCR thermal cycler and linearly cool them to 20°C at a rate of 0.1°C / hour, starting at 45°C, for a total duration of approximately 250 hours (10.4 days). Keep the environment stable and avoid vibration during annealing.

[0072] Process monitoring:

[0073] Fluorescence monitoring: A trace amount (1 nM) of Cy3-labeled non-competitive staple strand was added to the assembly system, and the interfacial fluorescence intensity was monitored using a real-time quantitative PCR instrument. In the temperature range from 40℃ to 30℃, the fluorescence signal showed a fluctuating plateau period lasting about 50 hours, indicating that the sticky ends were in a dynamic hybridization-dissociation equilibrium.

[0074] Morphological evolution: Samples were taken at different time points during the annealing process (the interface was lightly touched with a hydrophilic carbon film), and TEM observations showed:

[0075] 45–40℃: Formation of non-hybridized spherical cholesterol aggregates;

[0076] 40–35℃: Aggregates begin to recombine and fuse;

[0077] 35–30℃: Forms a multi-layered island-like structure;

[0078] 30–25℃: Gradually transforms into a continuous monolayer film through Ostwald curing.

[0079] 1.3 Thin Film Transfer and Electron Microscopy Sample Preparation

[0080] Hydrophilization treatment of carbon-supported membranes: PELCO Easy Glow plasma cleaner, 15 W power, ambient air gas, 0.3 mbar gas pressure, 60 seconds processing time.

[0081] Film application: After hydrophilic treatment, the carbon film and copper mesh (carbon side down) are gently brought into contact with the air-water interface and kept in contact for 5 minutes to allow the film to fully adsorb.

[0082] Cleaning: Use filter paper to absorb excess liquid, place the copper mesh on a drop of Milli-Q water and clean for 30 seconds, repeat twice.

[0083] Staining: Place the copper mesh on a drop of 15 μL 1% uranium acetate solution and stain for 60 seconds. Then, use filter paper to absorb any excess stain.

[0084] Drying: Allow to air dry at room temperature for at least 2 hours.

[0085] Transmission electron microscope: Talos L120C G2, accelerating voltage 120 kV.

[0086] 1.4 Characterization Results

[0087] Morphology and Orderliness: Combination Figure 2 , Figure 3 As shown, TEM revealed that the thin film covered an area on the centimeter scale (completely transferred to a 10 mm × 10 mm silicon wafer), with a highly ordered tetragonal lattice inside and a lattice constant of 58.2 ± 1.5 nm (measured over 50 unit cells). The FFT pattern showed sharp first- and second-order diffraction spots, confirming long-range periodicity.

[0088] Domain size: For 5 independent experimental batches, 20 fields of view (total area 2000 μm) were randomly selected from each batch. 2 / field of view), and the crystal domains are segmented by coloring using inverse Fourier transform. The peak area distribution of the crystal domains is ~800 μm. 2 Maximum value ~1200 μm 2 The average domain areas of the five batches were 1150, 980, 1200, 1050, and 1020 μm, respectively. 2 The overall average is 952 μm. 2 .

[0089] Thickness: The film thickness was measured to be 6.8 ± 0.5 nm (n=20) by atomic force microscopy (Bruker Multimode Nanoscope VIII, Peak Force QNM mode, Scanasit-air probe); the thickness was measured to be ~10 nm by transmission electron microscopy of resin-embedded sections, the difference being due to AFM tip pressure.

[0090] Cryo-electron microscopy: The thin film was transferred to a Quantifoil grid, rapidly frozen with a Vitrobot Mark IV, and observed with a TitanKrios G3i (300 kV). The lattice was clear in the unstained state, and the FFT diffraction was clear, confirming the high degree of order of the thin film in the near-in-situ state.

[0091] Example 2: Siliconization Enhancement of Thin Films

[0092] 2.1 In-situ silanization

[0093] Timing of silicide formation: Based on Example 1, this example further performs in-situ silicide formation on the thin film. This timing occurs after the interface of the thin film is fully formed (after the annealing process ends), while maintaining the integrity of the interface.

[0094] Preparation of silicified sol:

[0095] TMAPS: N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylammonium chloride (50% methanol solution), final concentration 2% (v / v);

[0096] TEOS: Tetraethyl orthosilicate, final concentration 2% (v / v);

[0097] Buffer: 1× TAE-Mg buffer (40 mM Tris, 2 mM EDTA, 12.5 mM magnesium acetate, pH 8.0);

[0098] Steps: First, add TMAPS and stir vigorously for 15 minutes; then add TEOS and continue stirring for 15 minutes to ensure complete hydrolysis.

[0099] Injection: Take 50 μL of the above sol and slowly inject it into the bottom subphase of the PCR tube using a microsyringe, avoiding disturbing the interfacial membrane.

[0100] Reaction conditions: stand at room temperature (25℃) for 16 hours.

[0101] Alternative silanizing agents: APTES (3-aminopropyltriethoxysilane), MPTMS (3-mercaptopropyltrimethoxysilane), or sodium silicate solution (pH adjusted to ~5.5) can also achieve similar effects. The reaction time can be 6 to 24 hours, with 16 hours being preferred.

[0102] 2.2 Characterization Results

[0103] Mechanical stability: After siliconization, the film can be picked up directly with tweezers by the edge without breaking; the unsiliconized film needs to be picked up with a substrate and cannot be picked up independently.

[0104] TEM: The structural order is completely maintained. Due to the SiO2 layer, the width of the DNA origami edge increases from 8.5 nm to 12.0 nm, the side length increases from 43.1 nm to 46.2 nm, and the lattice constant is adjusted to 46.2 ± 1.2 nm.

[0105] FFT: The diffraction spots are sharper, the uniformity of the interplanar spacing is improved (58.2 nm → 42.8 nm), and the periodicity is significantly improved.

[0106] GISAXS (Shanghai Synchrotron Radiation Facility BL16B1 beamline, wavelength 1.033 Å):

[0107] Before siliconization: at q_xy = 0.0107 Å -1 and 0.0150 Å -1 There are two distinct in-plane signals, corresponding to the

[100] and

[110] crystal planes respectively, and no out-of-plane signals;

[0108] After siliconization: only at q_xy = 0.0147 Å -1 There is a single strong in-plane signal (corresponding to the

[100] crystal plane), the intensity is significantly enhanced, the half-width at half-maximum is narrowed, and there is no out-of-plane signal, indicating that the lattice uniformity and the uniformity of the monolayer film are significantly improved.

[0109] Conclusion: The results of this embodiment demonstrate that DNA origami films can be in-situ silanized and encapsulated under room temperature and mild aqueous conditions, significantly improving mechanical strength, chemical stability and structural rigidity. Moreover, the silanization process does not damage or even improves the long-range order of the film.

[0110] Example 3: Substitution of hydrophobic modification groups

[0111] Based on Example 1, this example uses DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine) to replace cholesterol as a hydrophobic modifying group.

[0112] Modification method: Modify the 5' ends of the staple chains at the four vertices of SDO with DOPE molecules (which can be synthesized in solid phase by ordering commercially available DOPE-phosphoramidite).

[0113] Representative modified strand sequence (5'→3'):

[0114] [DOPE]-TTTTTTTTTTTTTTTT-ATCCGTTA-CAAATCAACAAATCGGCGGAGCTCG

[0115] Assembly conditions: exactly the same as in Example 1 (DSS surfactant, annealing at 0.1°C / hour).

[0116] result:

[0117] Successfully obtained centimeter-scale continuous thin films;

[0118] TEM revealed a highly ordered tetragonal lattice with a lattice constant of 58.0 ± 1.8 nm;

[0119] Crystal domain area 600~900 μm 2 (Statistical n=30), lower than cholesterol modification but still far superior to solution phase assembly (~10 μm) 2 );

[0120] The thin film can be transferred intact to the silicon wafer.

[0121] Conclusion: The results of this embodiment demonstrate that lipid molecules such as DOPE can serve as effective alternative hydrophobic modification groups for cholesterol.

[0122] Example 4: Surfactant Type Substitution

[0123] Based on Example 1, this example uses two surfactants (CTAB and Triton X-100) to replace DSS.

[0124] 4.1 Cationic surfactants (CTAB)

[0125] Surfactant: CTAB (hexadecyltrimethylammonium bromide), soluble in chloroform, concentration 0.1 mg / L. Too high a concentration will freeze the fluidity due to strong electrostatic action.

[0126] Assembly conditions: SDO-A 4 / B 4 The unit is the same as in Example 1, except that the MgCl2 concentration is increased to 20 mM to shield against excessive static electricity.

[0127] Results: SDO was successfully enriched at the interface; TEM showed an ordered tetragonal lattice, but with many point defects; the domain area was 150–250 μm. 2 (n=20); It was confirmed that CTAB can partially achieve the purpose of the invention under optimized ionic strength conditions.

[0128] 4.2 Nonionic surfactant (Triton X-100)

[0129] Surfactant: Triton X-100, soluble in chloroform, concentration 0.5 mg / L.

[0130] Assembly conditions: SDO-A 4 / B 4 The unit is the same as in Example 1.

[0131] result:

[0132] The interface is clearly enriched; TEM shows a relatively regular tetragonal lattice; the crystal domain area is 400–600 μm. 2 (n=20); can be transferred continuously and has good order.

[0133] Conclusion: The results of this embodiment prove that DSS is preferred, but nonionic and optimized cationic surfactants can also achieve macroscopic ordered assembly.

[0134] Example 5: Replacement of the Interface System

[0135] Based on Example 1, this example uses a liquid-liquid interface instead of a gas-water interface.

[0136] Liquid-liquid interface (oil-water interface):

[0137] Oil phase: hexadecane (Sigma-Aldrich H67O3);

[0138] Aqueous phase: Contains SDO-A 4 / B 4 1× TE-MgCl2 buffer;

[0139] Procedure: First, add the aqueous phase to the glass petri dish, then carefully spread hexadecane on the surface of the aqueous phase to form a clear oil-water interface;

[0140] Surfactant: Dissolve DSS directly in hexadecane (final concentration 1 mg / L), without chloroform;

[0141] Annealing: Same as in Example 1 (45℃→25℃, 0.1℃ / hour).

[0142] result:

[0143] Successfully obtained a continuous and ordered thin film;

[0144] TEM revealed a tetragonal lattice with a lattice constant of 58.5 ± 2.0 nm;

[0145] Crystal domain area 500–800 μm 2 (n=20);

[0146] Slightly below the gas-water interface, but clearly confirms the feasibility of the oil-water interface.

[0147] Conclusion: The results of this embodiment demonstrate that the liquid-liquid interface can serve as an effective alternative to the gas-water interface.

[0148] Example 6: Alternatives to Assembly Thermodynamics and Kinetics Procedures

[0149] Based on Example 1, this example uses isothermal annealing and multi-step annealing instead of programmed linear cooling annealing. The detailed process is described below:

[0150] 6.1 Isothermal Annealing

[0151] Annealing process: Hold at 37℃ for 7 days (SDO viscous end Tm approximately 35–40℃).

[0152] Other conditions: Same as in Example 1 (DSS, air-water interface).

[0153] Results: Continuous thin films were successfully formed; crystal domain area was 400–600 μm. 2 (n=20); the lattice order is comparable to that of programmed cooling, but the time required is longer.

[0154] 6.2 Multi-step annealing

[0155] Annealing procedure:

[0156] 45℃→40℃, 1℃ / hour;

[0157] 40℃ constant temperature for 24 hours (rapid nucleation);

[0158] 40℃→25℃, 0.2℃ / hour.

[0159] Results: Domain area 700–1000 μm 2 (n=20); slightly better than linear cooling of 0.1℃ / hour (average +15%).

[0160] Conclusion: The results of this embodiment prove that 0.1℃ / hour is the optimal condition, but alternative procedures such as isothermal annealing and multi-step annealing can also achieve the purpose of the invention, and multi-step annealing can further improve the crystal domain size.

[0161] Example 7: Substitution of the Nanostructure Unit Itself

[0162] Based on Example 1, this example uses tetrahedral DNA origami (approximately 50 nm in side length, three-dimensional structure) instead of square DNA origami as the nanostructure unit.

[0163] Design considerations:

[0164] Hydrophobic modification: Cholesterol is modified only on one base face (three vertices) of the tetrahedron;

[0165] Sticky ends: Sticky ends extend only from the three vertices of the base, the sequence is the same as SDO-A. 4 / B 4 Same (ATCCGTTA / TAACGGAT);

[0166] Objective: To orient tetrahedrons onto the interface with their bottom faces down and their vertices facing up, forming a two-dimensional hexagonal lattice.

[0167] Representative modified strand sequence (5'→3'):

[0168] [Cholesterol] -TTTTTTTTTTTTTTTT-ATCCGTTA-[Complementary sequence to the basal apex support]

[0169] Assembly conditions: Same as in Example 1 (DSS, gas-water interface, annealing at 0.1°C / hour).

[0170] Results: Centimeter-scale continuous thin films were successfully obtained; TEM and FFT confirmed a highly ordered hexagonal lattice (lattice constant approximately 65 nm); crystal domain areas ranged from 500 to 800 μm. 2 (n=20); Solution phase assembly control (no interface) only yielded amorphous aggregates and could not form an ordered array.

[0171] Conclusion: The results of this embodiment demonstrate that three-dimensional DNA origami, through specific surface modification and directional assembly, can serve as an effective extension of the technical solution of this invention.

[0172] Example 8: Extended Functions and Applications of Thin Films

[0173] Based on Example 1, this example further expands the functionality of the prepared thin film, as detailed below.

[0174] 8.1 Optical Function Extension: Visible Light Band Structural Color Thin Films

[0175] Design goal: To increase the lattice constant from 58.2 nm to >200 nm and obtain a photonic bandgap in the visible light band.

[0176] Implementation method:

[0177] Use rectangular DNA origami (approximately 100 nm × 70 nm in size, designed with reference to the classic Rothemund rectangle).

[0178] Extending the adhesive end connector arm: increasing the 15 nt poly-T spacer to 60 nt poly-T to increase the inter-cell spacing;

[0179] The remaining assembly conditions are the same as in Example 1.

[0180] Results: The lattice constant is 220 ± 10 nm; the film exhibits a bright green structural color under white light irradiation; the ultraviolet-visible reflectance spectrum shows that the reflection peak is located at 550 nm, corresponding to the photonic bandgap in the visible light band.

[0181] Conclusion: The thin film preparation method provided by this invention has promising applications in dye-free structural color coatings, anti-counterfeiting labels, and biosensors.

[0182] 8.2 Template and scaffold function: Gold nanoparticle array template

[0183] Template preparation: The square lattice thin film obtained in Example 1 was transferred to a SiO2 / Si silicon wafer (after hydrophilization treatment with piranha solution).

[0184] Metal deposition: Electron beam evaporation deposition of 2 nm Ti (adhesion layer) + 5 nm Au.

[0185] Stripping: Oxygen plasma etching (power 100 W, pressure 0.5 mbar, time 5 minutes) removes DNA template.

[0186] Results: AFM and SEM showed that an ordered array of gold nanodots was left on the silicon wafer surface; the gold dots had a diameter of 15 ± 3 nm and a spacing of 58 ± 2 nm, perfectly replicating the DNA origami lattice; the array area could reach the centimeter scale, with a defect rate of <5%.

[0187] The results of this embodiment demonstrate that the thin film preparation method provided by the present invention has promising applications in plasmonic devices, catalytic surfaces, and nanoelectrode arrays.

[0188] 8.3 Separation and Filtration Functions

[0189] The siliconized film is transferred to a porous support (such as anodized aluminum oxide film) to form a separation membrane with uniform nanopores. The pore size can be precisely controlled (30–100 nm) through DNA origami design, which can be used for molecular sieving, nanofiltration or virus filtration.

[0190] 8.4 Biosensor Interface

[0191] By utilizing programmable capture probes (such as aptamers and antibodies) on the surface of DNA origami, the thin film serves as a high-density, ordered biomolecular detection platform. Combined with surface plasmon resonance or fluorescence detection, ultrasensitive multiplex detection can be achieved.

[0192] 8.5 Two-dimensional material growth template

[0193] Using silanized thin films as sacrificial templates, transition metal chalcogenide (such as MoS2) precursors are deposited on the surface. After heat treatment to remove DNA, the orientation growth of two-dimensional materials is guided.

[0194] Comparative Example 1 (Solution Phase Assembly)

[0195] Based on Example 1, no surfactant was added to this comparative example.

[0196] Feeding: SDO-A 4 With SDO-B 4 Mix 10 μL (50 ng / μL) of each solution in a 200 μL PCR tube without adding any surfactant. The liquid level should be a normal concave meniscus.

[0197] Annealing procedure: exactly the same as in Example 1 (45℃→25℃, 0.1℃ / hour).

[0198] Results: TEM observation showed that the product consisted of a large number of dispersed tiny ordered regions, with the largest continuous ordered region being only about 8 μm in size, and could not form a self-supporting film that could be transferred independently.

[0199] Results: The comparative results demonstrate that thermal annealing in the solution phase alone cannot overcome the kinetic trap, and that a stable, highly fluid gas-liquid interface with surfactants is a key and necessary condition for achieving macroscopically ordered assembly.

[0200] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A method for preparing a two-dimensional nucleic acid nanostructure polymer film based on fluid mosaic assembly, characterized in that, Includes the following steps: S1, Preparation of building blocks: Design and synthesize nucleic acid nanostructure units with geometric shapes, covalently modify hydrophobic modification groups at preset positions of the nucleic acid nanostructure units to obtain hydrophobic modified nucleic acid nanostructure building blocks with sticky ends that can specifically hybridize, and the sticky ends and hydrophobic modification groups are connected by flexible connecting arms. S2, Construction of fluid interface: The hydrophobic modified nucleic acid nanostructure building units are dispersed in a buffer solution, and surfactant molecules are spread on the solution surface to form a monolayer, thereby constructing a two-dimensional fluid interface system with low interfacial tension, reducing the interfacial tension to below 40 mN / m. S3, Entropy-driven assembly: The two-dimensional fluid interface system is annealed to enrich and embed hydrophobically modified nucleic acid nanostructure building units into the hydrophobic regions of the surfactant monolayer. Utilizing the high in-plane mobility imparted by the two-dimensional fluid interface, dynamic specific hybridization, defect self-correction, and grain boundary fusion of the sticky ends between building units are achieved, forming a macroscopically continuous and highly ordered monolayer two-dimensional nucleic acid nanostructure polymer film.

2. The preparation method according to claim 1, characterized in that, In step S1, the hydrophobic modification group is one or more of cholesterol, lipid molecules, long-chain alkanes, fluorinated compounds, or hydrophilic-hydrophobic block polymers.

3. The preparation method according to claim 1, characterized in that, In step S1, the nucleic acid nanostructure unit is a DNA tile, a three-dimensional DNA origami, an RNA origami, or a nucleic acid-hybrid structure, wherein the DNA tile includes a two-dimensional DNA origami tile; the geometric shape of the two-dimensional DNA origami tile is square, rectangular, rhomboid, or triangular, and hydrophobic modification groups are applied to its edges or vertices; the three-dimensional DNA origami is modified only on its predetermined single surface with hydrophobic modification groups and sticky ends to achieve directional adsorption of the three-dimensional DNA origami at a two-dimensional fluid interface.

4. The preparation method according to claim 1, characterized in that, In step S1, the hydrophobic modified nucleic acid nanostructure building unit includes a first building unit and a second building unit that are complementary, and their sticky ends are complementary oligonucleotide sequences.

5. The preparation method according to claim 1, characterized in that, In step S2, the surfactant is one of anionic, cationic, nonionic, amphoteric, or polymeric surfactants.

6. The preparation method according to claim 1, characterized in that, In step S2, the two-dimensional fluid interface is a gas-liquid interface or a liquid-liquid interface; the gas-liquid interface is a gas-water interface, and the liquid-liquid interface is an oil-water interface.

7. The preparation method according to claim 1, characterized in that, In step S3, the annealing process is one of programmed linear cooling annealing, isothermal annealing, multi-step annealing, or external field assisted annealing. The programmed linear cooling annealing involves cooling from 45°C to 20°C at a rate of 0.1°C / hour. The isothermal annealing is performed by holding the food at the viscous end melting temperature for 5-7 days. The multi-step annealing process involves first rapidly cooling / maintaining the temperature to form a large number of small crystal nuclei, and then annealing at a lower temperature for a longer period of time to promote grain growth and fusion. The external field-assisted annealing involves applying mild interfacial shear, electric field, or magnetic field during the annealing process to guide domain orientation or accelerate defect repair.

8. The preparation method according to any one of claims 1-7, characterized in that, The method also includes an in-situ silanization enhancement step for the obtained two-dimensional DNA origami polymer film: in the two-dimensional fluid interface system after film formation, silanization sol is slowly injected into the buffer solution subphase, and the reaction is allowed to proceed at room temperature for 6-24 hours to complete the silanization encapsulation; the silanization sol is a mixture of 1×TAE-Mg buffer as a base and containing 2% v / v TMAPS and 2% v / v TEOS, or a buffer system using APTES, MPTMS or sodium silicate solution adjusted to pH 5.5 as silanizing agents.

9. The application of a two-dimensional nucleic acid nanostructure polymer film prepared by any one of claims 1-8 in the field of optics.

10. The application of a two-dimensional nucleic acid nanostructure polymer film prepared by any one of claims 1-8 in the fields of nanofabrication, separation and detection and material growth.

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