Annular long single-stranded ssDNA, single-stranded DNA assembly and preparation method and application thereof

Abstract

The invention provides annular long single-stranded ssDNA, a single-stranded DNA assembly and a preparation method and application of the annular long single-stranded ssDNA and the single-stranded DNA assembly. The annular long single-stranded ssDNA comprises an aptamer sequence and a DNA assembly skeleton sequence; wherein the aptamer sequence is used for identifying any biomacromolecules or micromolecules; basic groups in molecules of the annular long single-stranded ssDNA are complementarily paired and are self-assembled to form a single-stranded DNA assembly. The method comprises the following steps: preparing long single-stranded ssDNA (single-stranded deoxyribonucleic acid) of milligram to acrylic level by utilizing a biological fermentation method; due to the self-folding characteristic of the annular long single-stranded ssDNA, the aptamer functionalized single-stranded DNA nano assembly with highly accurate valence state and spatial distribution and excellent structural stability can be efficiently constructed. The obtained nano-scale single-stranded DNA assembly has a strong affinity effect on the new coronavirus, and can efficiently block the combination of the new coronavirus and host cells so as to inhibit virus infection. The preparation method has the advantages of low cost, stable product structure and excellent uniformity of products among batches, and is suitable for animal level new coronavirus infection inhibition application research.

Description

Technical Field

[0001] The present application relates to the fields of biochemistry and molecular biology, and specifically to a circular long single-stranded ssDNA, a single-stranded DNA assembly, and a preparation method and application thereof. Background Art

[0002] DNA possesses remarkable programmability and is therefore widely used to construct a variety of precisely controllable DNA nanoassemblies. DNA nanotechnology has achieved rapid development in recent years through the precise control of the size, shape, rigidity, and chemical composition of nanoassemblies. DNA nanoassemblies, with their highly flexible programmable properties, can precisely regulate the spatial arrangement and valence of functional units (such as aptamers and antibodies), effectively mimicking some of the functions of complex biological systems such as the cytoskeleton, transmembrane pores, and viral capsids.

[0003] Although DNA nanotechnology has made significant progress, its application potential in the biomedical field has not yet been fully explored. Among the key factors restricting its application, one of the biggest challenges is the lack of a scalable and sustainable synthesis method that can meet the core requirements of high purity, high yield, low cost and batch consistency. Existing assembly methods based on multi-stranded DNA usually require dozens or even hundreds of short-chain DNA for construction, which not only leads to high synthesis costs and complex operations, but also requires tedious purification steps to remove by-products, low preparation efficiency, and significant differences between different batches, making it difficult to meet the needs of large-scale production.

[0004] In contrast, single-stranded DNA assembly technology (ssDNA assembly) is a more efficient construction method. This technology uses a long single-stranded DNA to form the desired structure through self-folding, significantly reducing the synthesis cost and experimental complexity, while also having the potential to achieve large-scale production. Single-stranded DNA assembly technology is flexible in design and can precisely control the valence state and spatial distribution of nucleic acid functional materials by inserting functional nucleic acid units at specific sequence positions. In particular, the application of bio-fermentation methods in the preparation of long single-stranded DNA (ssDNA) has shown significant advantages, such as low cost and high purity, making it particularly suitable for in vivo research that requires a large amount of nucleic acid material. At the same time, single-stranded DNA assemblies form complete, gap-free structures through self-folding, do not require complex purification steps, and the preparation process has highly consistent structural uniformity, excellent stability, and extremely low batch-to-batch variation. These characteristics make single-stranded DNA assemblies a highly promising nucleic acid functional material for in vivo research. Summary of the Invention

[0005] The purpose of the present invention is to provide a circular long single-stranded ssDNA, a single-stranded DNA assembly, and a preparation method and application thereof, so as to solve the problems existing in the current functionalized DNA assembly system prepared using backbone chains and stapler chains, such as high production cost of nucleic acid raw materials, poor biological stability of the assembly, and insufficient uniformity of the functional units of the assembly itself.

[0006] In order to solve the above technical problems, this application adopts the following technical solutions:

[0007] According to the first aspect of the present application, a circular long single-stranded ssDNA is provided, wherein the circular long single-stranded ssDNA comprises an aptamer sequence and a DNA assembly backbone sequence; wherein the aptamer sequence is used to recognize any biological macromolecule or small molecule;

[0008] The intramolecular bases of the circular long single-stranded ssDNA are complementary and self-assembled to form a single-stranded DNA assembly.

[0009] ssDNA (Single-Stranded DNA), also known as single-stranded DNA, refers to a DNA molecule composed of a single nucleotide chain. Unlike double-stranded DNA (dsDNA), it lacks another complementary chain.

[0010] Aptamer sequences are used to recognize any biomacromolecule or small molecule. They can be specifically designed based on the target binding specificity of any biomacromolecule or small molecule, so that the single-stranded DNA assembly formed by the circular long single-stranded ssDNA can target and recognize the desired biomacromolecule or small molecule, thereby achieving a certain purpose, such as disease treatment; the disease is, for example, tumors. In some embodiments, the aptamer sequence is a segment of DNA that can bind with affinity to the SARS-CoV-2 RBD region.

[0011] The biomacromolecules may be, for example, enzymes and cytokines; the biomicromolecules may be, for example, drugs, toxins, metabolites, and the like.

[0012] Preferably, the length of the circular long single-stranded ssDNA is 500-10000 nt.

[0013] Preferably, the nucleic acid sequence of the circular long single-stranded ssDNA comprises any one or more combinations of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 13.

[0014] Preferably, the nucleic acid sequence of the circular long single-stranded ssDNA is any one or more combinations of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 13.

[0015] Preferably, the biological macromolecules or small molecules that can be recognized by the aptamer sequence are selected from any one or more combinations of the following groups: viruses, proteins, small molecules and metal ions.

[0016] Preferably, the virus is selected from any one or more combinations of the following groups: wild-type new coronavirus, conservative mutants of new coronavirus, human immunodeficiency virus, dengue virus, and norovirus.

[0017] Preferably, the aptamer sequence comprises the sequence shown in SEQ ID NO: 14, or the aptamer sequence is the sequence shown in SEQ ID NO: 14.

[0018] According to a second aspect of the present invention, a single-stranded DNA assembly is provided. The single-stranded DNA assembly is obtained by self-assembly of the above-mentioned circular long single-stranded ssDNA.

[0019] Preferably, the single-stranded DNA assembly is a closed-loop nanostructure.

[0020] More preferably, the single-stranded DNA assembly has no gaps.

[0021] Preferably, after self-assembly, the backbone sequence of the DNA assembly forms the morphological structure of the single-stranded DNA assembly; and the aptamer sequence forms the aptamer functional unit of the single-stranded DNA assembly.

[0022] Preferably, the valence of the aptamer functional unit is monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent.

[0023] It should be noted that the valence of the aptamer functional unit is determined by the biological macromolecule or small molecule targeted by the aptamer functional unit.

[0024] Preferably, the morphological structure of the single-stranded DNA assembly is linear, triangular, quadrilateral or pentagonal.

[0025] More preferably, the aptamer functional units are distributed at the vertices or endpoints, the edges of the framework or the internal cavity region of the single-stranded DNA assembly.

[0026] According to a third aspect of the present invention, a method for preparing the above-mentioned single-stranded DNA assembly is provided, the preparation method comprising the following steps:

[0027] The circular long single-stranded ssDNA is recombined into the vector to obtain a recombinant phagemid;

[0028] transducing the recombinant phagemid and the helper phagemid into a host to obtain a recombinant host;

[0029] culturing the recombinant host and collecting the circular long single-stranded ssDNA;

[0030] The collected circular long single-stranded ssDNA is self-assembled to obtain the single-stranded DNA assembly.

[0031] The helper phagemid may be, for example, HP17_KO7.

[0032] Preferably, the vector is a phagemid; more preferably, it is an M13ori phagemid vector or a flori phagemid vector.

[0033] Preferably, transducing the recombinant vector into the host also includes transducing a helper phagemid into the host; for example, the helper phagemid is HP17_KO7.

[0034] Preferably, the host is Escherichia coli; for example, the Escherichia coli is XL-1 Blue competent Escherichia coli.

[0035] Preferably, the self-assembly comprises the steps of denaturing and renaturing the circular long single-stranded ssDNA.

[0036] More preferably, the temperature for denaturation and annealing of the circular long single-stranded ssDNA is 22-95°C.

[0037] Preferably, the self-assembly 2+ was carried out in a buffer solution.

[0038] More preferably, the Mg 2+ The content is 12.5-20mM.

[0039] According to a fourth aspect of the present invention, a composition is provided, comprising the above-mentioned single-stranded DNA assembly and / or the single-stranded DNA assembly obtained by the above-mentioned preparation method.

[0040] According to the fifth aspect of the present invention, provided is the use of the above-mentioned single-stranded DNA assembly, and / or the single-stranded DNA assembly obtained by the above-mentioned preparation method, and / or the above-mentioned composition in the preparation of kits and / or chips for virus detection and clinical medical diagnosis.

[0041] Beneficial effects

[0042] As described in the background technology section of this application, the present invention has the following advantages over the prior art: 1) In the prior art, when preparing DNA assemblies with multiple short-chain DNAs, the main source of the short-chain DNAs relies on chemical synthesis, and the production cost of the chemical synthesis method is high and the purity is significantly limited by the length of the short-chain DNA; in addition, in the process of assembling assemblies with multiple short-chain DNAs, it is difficult to avoid the problem of defective DNA assembly products or functional unit valences that do not conform to the design, which will make the subsequent purification steps more complicated and difficult to obtain the ideal target product; and the single-stranded DNA assembly proposed in this application is assembled by self-folding of circular long single-stranded DNA, and the obtained single-stranded DNA assembly has no gaps, which is more suitable for physiological environments containing enzymes or metal ions as a biomaterial. 2) The circular long single-stranded DNA provided by the present application through the biological fermentation method has small differences between product batches, high yield and low cost, and is easy to standardize and commercialize, so it is suitable for application in animal experimental systems with large material requirements. 3) The preparation method proposed in this application can customize the morphology and size of the DNA assembly, the type of functional units, their spatial distribution and valence according to different application requirements to improve the neutralization efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a schematic diagram of the biological preparation process of long single-stranded DNA;

[0044] Figure 2 The preparation and characterization results of the two structures, msTri3a and cssTri3a, are shown in Figures a and b. Figures a and b show the agarose gel electrophoresis and atomic force microscopy results of msTri3a and cssTri3a, respectively.

[0045] Figure 3 The yield statistics of cssTri3a and msTri3a are shown in the figure. The upper row is the statistical result of AFM, and the lower row is the statistical result of gel electrophoresis.

[0046] Figure 4 Characterization of the structural stability of cssTri3a and msTri3a; wherein, a is the gel electrophoresis characterization result during the reaction of DNase I with cssTri3a and msTri3a, b is the change in band intensity during the reaction of DNase I with cssTri3a and msTri3a, and c is the AFM characterization result of cssTri3a and msTri3a after storage at 4°C and 23°C for 14 days, respectively;

[0047] Figure 5 The binding curves and dissociation constants (K) of cssTri3a and CoV2-6C3 with wild-type SARS-CoV-2 RBD-magnetic beads, respectively. d ) characterization results;

[0048] Figure 6 It is the fluorescence characterization of the virus neutralization inhibition performance of different types of DNA structures against the new coronavirus pseudovirus;

[0049] Figure 7 It is the characterization of the half-neutralization concentration of cssTri3a against the new coronavirus pseudovirus particles;

[0050] Figure 8 This is a cryo-electron microscopy image of cssTri3a binding to the novel coronavirus pseudovirus particles;

[0051] Figure 9 The synthesis and characterization of 10 single-stranded DNA assemblies with different shapes, sizes and valence states; a is the design drawings of the 10 single-stranded DNA assemblies, b is the gel electrophoresis characterization results of the 10 single-stranded DNA assemblies, and c is the AFM image of the 10 single-stranded DNA assemblies;

[0052] Figure 10 This is a characterization of the virus neutralization inhibition performance of 10 single-stranded DNA assemblies against the new coronavirus pseudovirus, where a is the fluorescence image representation and b is the statistical result of the neutralization efficiency;

[0053] Figure 11 These are the statistical results of immune factor levels in mouse serum after cssTri3a was injected into mice. DETAILED DESCRIPTION

[0054] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0055] Example 1 Preparation of circular single-stranded DNA by bio-amplification

[0056] Helper phagemid method to obtain circular single-stranded DNA.

[0057] Recombinant phagemids were purchased from Jiutian Gene Biotechnology Co., Ltd. (Tianjin), and helper phagemid HP17_KO7 was ordered from Addgene. All other chemical reagents and solvents were purchased from Sigma-Aldrich. All buffer solutions were prepared using 18.2 MΩ·cm ultrapure water (Millipore system).

[0058] The circular long single-stranded DNA sequence of the present application is shown in Table 1 below; it includes cssTri, cssTri3a, cssTri3a-E, cssLTri-6a, cssLTri3a-1, cssLTri3a-2, cssLTri3a-3, cssLTri3a-4, cssLine3a, cssQuad, cssQuad3a, cssQuad4a, and cssCpt3a.

[0059] Table 1

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] The structure of the circular long single-stranded DNA in Table 1 is: DNA assembly backbone sequence + aptamer sequence. Specifically, the DNA assembly backbone sequences in each circular long single-stranded DNA in Table 1 are different, as shown in Table 2. The aptamer sequences attached to each circular long single-stranded DNA are identical, as shown in SEQ ID NO: 14: CGCAGCACCCAAGAACAAGGACTGCTTAGGATTGCGATAGGTTCGG.

[0071] In this embodiment, referring to Figure 1 The method for preparing circular long single-stranded DNA comprises the following steps:

[0072] Get 100 μ L XL-1Blue Escherichia coli competent cells, thaw on ice for 10 minutes. Subsequently, 0.05pmol recombinant phagemid plasmid and 0.05pmol helper phagemid HP17_KO7 are gently added into XL-1Blue Escherichia coli competent cells, then hatched on ice for 30 minutes. The mixture is heat-shocked at 42 ℃ for 60 seconds, and immediately returned to cool on ice for 3 minutes. Next, 900 μ L 2 × YT culture medium (16.0 g / L tryptone, 10.0 g / L yeast extract, 5.0 g / L sodium chloride, pH 7.0) is added, and the mixture is vibrated at 37 ℃ and 200rpm for 1 hour. Subsequently, the sample after 200 μ L conversion is coated on the LB agar plate containing 100 μ g / mL ampicillin and 50 μ g / mL kanamycin, and the plate is inverted, and hatched at 37 ℃ for 12-15 hours.

[0073] For routine ssDNA production in Erlenmeyer flasks, a single colony picked from an agar plate was inoculated into 3-5 mL of 2×YT medium (16.0 g / L tryptone, 10.0 g / L yeast extract, 5.0 g / L sodium chloride, pH 7.0) containing 100 μg / mL ampicillin and 50 μg / mL kanamycin. The culture was incubated in a shaking incubator at 37°C and 220 rpm for 10-12 hours until the OD 600 The pH value was approximately 1.0. Subsequently, 1 mL of the harvested culture was added to 300 mL of 2×YT medium (16.0 g / L tryptone, 10.0 g / L yeast extract, 5.0 g / L sodium chloride, pH 7.0) containing 100 μg / mL ampicillin and 50 μg / mL kanamycin in a 1-liter sterile Erlenmeyer flask. The culture was incubated in a shaking incubator at 37°C and 220 rpm for 10-12 hours.

[0074] The harvested culture was centrifuged at 4,000 g and 4°C for 15 minutes twice. The supernatant was transferred to a clean bottle and PEG 8000 (40 g / L) and NaCl (30 g / L) were added. After vigorous stirring until the powder dissolved, the mixture was incubated on ice for 30 minutes and then centrifuged at 5,000 g and 4°C for 30 minutes. The supernatant was discarded and the precipitated phagemid particles were redissolved in 10 mL of Tris buffer (10 mM, pH 8.5) and centrifuged at 16,000 g and 4°C for 10 minutes to remove any remaining bacterial contaminants. The supernatant was collected, which contained the purified recombinant phagemid particles.

[0075] Extraction of circular long single-stranded DNA in shake flasks: To extract ssDNA plasmids, the protein coating is removed from the phagemid particles. This is achieved by gently mixing 2 volumes of NaOH (0.2 M containing 1% SDS) into the phagemid solution by stirring. The mixture is incubated at room temperature for 3 minutes, and then 1.5 volumes of KOAc (3 M, adjusted to pH 5.5 with glacial acetic acid) are added and mixed gently by inversion. The sample is incubated on ice for 10 minutes and centrifuged at 16,000g and 4°C for 30 minutes. The supernatant is collected and mixed with 2 volumes of absolute ethanol, followed by incubation at -20°C for 30-60 minutes. The mixture is centrifuged at 16,000g and 4°C for 30 minutes. It is then centrifuged again at 16,000g and 4°C for 15 minutes to remove residual salts. The precipitated particles are air-dried for 10 minutes, dissolved in water, and stored at -20°C.

[0076] Extraction of circular long single-stranded DNA in fermenter: A 50 L fermenter (Biotech-JS, Shanghai Baoxing Bioengineering Equipment Co., Ltd., China) was used. 600 mL shake flask culture (OD 600 The OD value was approximately 1.0, which was inoculated into 30 L of 2×YT medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin. Dissolved oxygen levels and pH were continuously monitored, and 25% NH₄OH was used to maintain the fermentation system pH between 6.8 and 7.2. Fermentation was performed at 400 rpm and 37°C. 600 When the OD reached 1.0–1.5, a supplementary nutrient solution containing 141 mg / mL yeast extract and 28% (v / v) glycerol was added at a flow rate of approximately 15 mL / min for 4 hours. 600 The readings reached about 20.

[0077] Results: The gel electrophoresis results of the circular long single-stranded ssDNA obtained in this example are as follows Figure 2 As shown, the lane has a single band, proving that the DNA is of good purity.

[0078] Example 2 Preparation and characterization of functionalized triangular single-chain assemblies

[0079] All oligonucleotides were purchased from Sangon (Shanghai, China) and purified by HPLC.

[0080] The main structure of the single-stranded DNA assembly designed in this embodiment is designed based on the PX structural concept. The aptamer sequence is matched and placed in the middle of the vertex or edge of the triangular assembly, but the design ensures that the introduction of the aptamer sequence will not affect the base pairing of the DNA assembly backbone sequence. The DNA assembly backbone sequence strictly follows the Watson-Crick base complementary pairing principle. Therefore, the target circular long single-stranded ssDNA not only contains the necessary sequence for folding to form the main structure of the single-stranded DNA assembly, but also contains the aptamer sequence, so that it has functional characteristics.

[0081] Preparation of functionalized triangular single-stranded assemblies: Nanodrop was used to quantify the circular long single-stranded DNA. 1-10 pmol of circular long single-stranded ssDNA was added to 1×TAE / MgCl2 solution. 2+ Annealing was performed in a buffer (40 mM Tris, 2 mM EDTA, 12.5 mM MgCl2, pH 8.0) with the following annealing schedule: 90°C for 5 min, 75°C for 10 min, 60°C for 10 min, 45°C for 10 min, and 22°C for 10 min.

[0082] Similarly, all single-stranded DNAs assembled into the multi-stranded DNA assembly msTri and the multi-stranded DNA assembly msTri3a were quantified using UV-visible absorption spectrometer, and the concentration of all DNA single strands was quantified to 100 μM. 2+ The final concentration of each of the ten single-stranded DNA strands in the buffer was 1 μM, and denaturation and assembly were performed. Annealing procedure: 90°C for 5 min, 65°C for 20 min, 45°C for 20 min, 37°C for 30 min, and 20°C for 30 min.

[0083] The sequences used to synthesize multi-stranded DNA assemblies are shown in Table 2. Among them, MS3-1 to MS3-10 are used to synthesize msTri, and these structures do not contain aptamers. TMS-1 to TMS-3, which replace MS3-1, MS3-2, and MS3-3, respectively, are used to synthesize msTri3a. Part of the sequences in these three chains, TMS-1 to TMS-3, are aptamer sequences, while part of the sequences participate in backbone formation, and these structures contain aptamers.

[0084] Table 2

[0085]

[0086]

[0087] Gel electrophoresis characterization: cssTri3a single-stranded DNA assembly was characterized by 1% agarose gel electrophoresis at 100 V for 2 h; msTri3a multi-stranded DNA assembly was characterized by 6% PAGE gel electrophoresis under the same conditions.

[0088] Atomic force microscopy characterization: 5 μL of DNA sample with a concentration of 2–5 nM was added to the freshly torn mica surface, allowed to stand for 3 minutes, and then 30 μL of fixation buffer (25 μL of 1×TAE / Mg 2+ and 5 μL of 100 mM NiCl2). AFM images were acquired in liquid mode using a ScanAsyst-Fluid+ probe and analyzed using NanoScope Analysis 1.7 software.

[0089] Yield analysis was performed on the gel electrophoresis and AFM results using Image J. For gel electrophoresis, product yield = (target band intensity / total lane intensity) × 100%. For AFM statistical analysis, product yield = (target sample single particle number / (target sample single particle number + byproduct single particle number)) × 100%.

[0090] Reference Figure 2 The agarose electrophoresis showed that the annealed single-stranded DNA lane had a clear single target band, indicating that the circular single-stranded DNA provided by the present invention can efficiently self-fold into the designed single-stranded DNA assembly, and the product purity is excellent; the multi-stranded DNA assembly product lane had multiple bands, indicating the presence of by-products. Figure 2 a is the result of a multi-stranded DNA assembly; and Figure 2 b is the result of single-stranded DNA self-assembly.

[0091] Figure 2 The atomic force microscopy characterization in b shows that the single-stranded DNA assembly has good monodispersity and structural integrity, indicating that the circular single strand provided by the biological preparation method provided by the present invention can be used to prepare functionalized single-stranded DNA assemblies.

[0092] Figure 3 Yield analysis showed that the yield of cssTri3a single-stranded DNA assembly was close to 100%, while the yield of msTri3a multi-stranded DNA assembly was about 50%, which indicates that the single-stranded DNA assembly provided by the invention does not require purification in subsequent applications.

[0093] Example 3 Comparative analysis of the stability of single-stranded DNA assemblies and multi-stranded DNA assemblies

[0094] Enzyme cleavage resistance assay: DNase I at a final concentration of 0.06 U / mL and 50 nM cssTri3a single-stranded DNA assemblies and msTri3a multi-stranded DNA assemblies were mixed with 25 μL of the supplier's DNase I (purchased from NBE) reaction solution and incubated at 37°C for the specified time points (t = 0, 0.5, 1, 2, 4, and 6 h). The reactions were then analyzed by electrophoresis on a gelRed pre-stained agarose gel in 1% 1× TAE / Mg buffer at 90 V for 1 h. The agarose gel was then imaged on a GelDoc XR+ system (Biorad), and band intensity was analyzed using Quantum One software. Stability analysis at different storage temperatures: The annealed cssTri3a single-stranded DNA assemblies and msTri3a multi-stranded DNA assemblies were stored at 4°C and 25°C for 14 days, and 2-5 nM samples were taken for AFM morphology characterization. The characterization steps are as described in Example 2.

[0095] Figure 4 a and Figure 4 The gel band intensity analysis in b showed that the band intensity of the cssTri3a single-stranded DNA assembly decreased by up to 90% after incubation with DNase I for up to 6 h, while the band intensity of the msTri3a multi-stranded DNA assembly decreased by ∼40%. Figure 4 AFM characterization of c showed that after 14 days at 4°C and 25°C, respectively, the cssTri3a ssDNA assembly maintained structural integrity and monodispersity, while the msTri3a MMDA exhibited significant aggregation. This demonstrates the excellent stability of the long ssDNA assemblies designed and prepared in this invention.

[0096] Example 4 Analysis of the inhibitory performance of the single-stranded DNA assembly cssTri3a at the pseudovirus level

[0097] Analysis of the inhibitory performance of cssTri3a ssDNA assemblies at the pseudovirus level: HEK293T cells overexpressing ACE2 were cultured overnight in 384-well plates. CoV2-6C3 (synthesized by Sangon Biotech (Shanghai) Co., Ltd.; sequence: 5'-CGCAGCACCCAAGAACAAGGACTGCTTAGGATTGCGATAGGTTCGG–3', SEQ ID NO:28) was added. Samples of msTri3a ssDNA, cssTri ssDNA, cssTri1a ssDNA, and cssTri3a ssDNA were incubated with SARS-CoV-2 pseudovirus (purchased from Fubo Bio (Suzhou) Biopharmaceutical Technology Co., Ltd.) at 37°C for 1 hour and then co-cultured with ACE2-transfected HEK293T cells. Six hours after infection, the supernatant was removed and replaced with fresh medium, and the cells were cultured for an additional 48 hours. Immunofluorescence (GFP) expression in infected cells was detected using a Nikon Eclipse Ti microscope. Each sample was tested in triplicate.

[0098] Figure 5 Flow cytometry confirmed that the dissociation constant (K d ) was 0.14 nM, which was lower than that of the single CoV2-6C3 aptamer (K d =42.7nM) is more than 300 times more potent. Figure 6 The results show that compared with the free CoV2-6C3 nucleic acid aptamer, the neutralization efficiency of the monovalent cssTri1a single-stranded DNA assembly increased by 5.4 times, and the neutralization efficiency of the trivalent cssTri3a single-stranded DNA assembly was as high as 90.49%. The neutralization efficiency of the msTri3a multi-stranded DNA assembly was 55.97%, which was significantly lower than that of the cssTri3a single-stranded DNA assembly. The half-maximal inhibitory concentration (IC50) of the cssTri3a single-stranded DNA assembly against SARS-CoV-2 pseudovirus was 1. 50 ) is 34pM( Figure 7 ).at the same time, Figure 8 Cryo-electron microscopy directly observed cssTri3a single-stranded DNA assemblies aggregated and bound to the surface of pseudovirions; cryo-electron microscopy also visually demonstrated that cssTri3a single-stranded DNA assemblies bound to the surface of SARS-CoV-2 pseudovirions. This demonstrates that the single-stranded DNA assemblies provided by the present invention have excellent pseudovirion neutralization performance, and the valence of the nucleic acid aptamer units on the single-stranded assemblies is one of the key factors in effectively improving the efficiency of neutralizing SARS-CoV-2 pseudovirions.

[0099] Example 5 Preparation of single-stranded DNA assemblies with different morphologies and valencies

[0100] Assembly of single-stranded DNA assemblies with different morphologies and valences: Nanodrop was used to quantify the required long single-stranded DNA, and a certain volume of long single-stranded DNA was placed in 1×TAE / Mg 2+ Annealing and assembly were performed in a buffer (40 mM Tris, 2 mM EDTA, 12.5 mM MgCl2, pH 8.0) with a final concentration of 10 nM. Annealing program: 90°C for 5 min, 75°C for 10 min, 60°C for 10 min, 45°C for 10 min, and 22°C for 10 min.

[0101] Gel electrophoresis characterization: Characterization was performed using 1% agarose gel electrophoresis at 100 V for 2 h.

[0102] Atomic force microscopy characterization: 10 μL of 2-5 nM single-stranded DNA assembly was dripped onto freshly ripped mica for 3 minutes. Then, 25 μL of 1× TAE Mg buffer mixed with 100 mM NiCl₂ (5 μL) was dripped onto the mica for 1 minute. Scanning images were taken using an atomic force microscope (AFM) with the SCANASYST-FLUID+ probe and PeakForce QNM inFluid imaging mode.

[0103] Figure 9 Agarose electrophoresis in b shows that the annealed single-stranded DNA lane has a clear single target band, which indicates that the method provided by the present invention can prepare functionalized single-stranded DNA assemblies with different morphologies and valencies, and the product purity is excellent.

[0104] Figure 9 The atomic force microscopy characterization of c showed that the single-stranded DNA assembly had good monodispersity and structural integrity, which indicates that the circular long single chain provided by the biological preparation method provided by the present invention can be used to customize the preparation of functionalized single-stranded DNA assemblies.

[0105] Example 6 Analysis of the inhibitory performance of single-stranded DNA assemblies with different morphologies and valencies at the pseudovirus level

[0106] Analysis of the inhibitory performance of single-stranded DNA assemblies of different morphologies and valencies at the pseudovirus level: HEK293T cells overexpressing ACE2 were cultured overnight in 384-well plates. Triangular assemblies, linear assemblies, square assemblies, and solid triangle assemblies were incubated with SARS-CoV-2 pseudovirus at a final concentration of 20 nM at 37°C for 1 hour. The mixture was then added to ACE2-transfected 293T cells. After 6 hours of infection, a portion of the supernatant was removed and replaced with fresh medium, and the cells were cultured for an additional 48 hours. GFP-expressing infected cells were imaged using a fluorescence microscope (Nikon Eclipse Ti). Each sample was tested in triplicate.

[0107] like Figure 10 As shown in Figure 2, the neutralization performance of DNA assemblies with different morphologies on pseudoviruses is significantly different, and its efficiency is closely related to the spatial distribution and valence of the aptamers on the assembly and the DNA density of the assembly itself (in Figure 10 Among them, the single-stranded DNA assemblies cssTri3a-E, cssCpt3a, cssLine3a, cssQuad3a, cssLTri3a-1, css LTri3a-2, css LTri3a-3, and css LTri3a-4 are trivalent; cssQuad4a is tetravalent; and css LTri6a is hexavalent).

[0108] Example 7 Analysis of the immune stress level performance of cssTri3a in the C57BL / 6 mouse system

[0109] cssTri3a's immune response in mice: PBS and 40 nM cssTri3a were injected into C57BL / 6 nude mice (female, 5-7 weeks old) via tail vein injection. Serum was collected 6 hours, 24 hours, and 7 days after injection. Elasa kits (Solarbio, SEKM-0034, SEKM-0007) were used to quantitatively measure IL-6 and TNF-α levels in serum at different time points after injection.

[0110] like Figure 11 As shown, when C57BL / 6 mice were injected with cssTri3a, serum levels of TNF-α and IL-6, two immune cytokines, did not differ significantly from the control group at 6 hours, 24 hours, and 7 days. These results demonstrate that cssTri3a does not induce chronic or acute immune stress responses in mice, making it suitable for animal-based viral suppression studies.

[0111] Unless otherwise specified, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments can have different values.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A circular long single-stranded ssDNA, characterized in that The circular long single-stranded ssDNA comprises an aptamer sequence and a DNA assembly backbone sequence; wherein the aptamer sequence is used to recognize any biological macromolecule or small molecule; The intramolecular bases of the circular long single-stranded ssDNA are complementary and self-assembled to form a single-stranded DNA assembly; Preferably, the length of the circular long single-stranded ssDNA is 500-10000 nt.

2. The circular long single-stranded ssDNA according to claim 1, characterized in that The nucleic acid sequence of the circular long single-stranded ssDNA comprises any one or more combinations of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 13; Preferably, the nucleic acid sequence of the circular long single-stranded ssDNA is any one or more combinations of the sequences shown in SEQ ID NO: 1 to SEQ ID NO:

13.

3. The circular long single-stranded ssDNA according to claim 1, characterized in that The biological macromolecules or small molecules that the aptamer sequence can recognize are selected from any one or more combinations of the following groups: viruses, proteins, small molecules and metal ions; Preferably, the virus is selected from any one or more combinations of the following groups: wild-type new coronavirus, other new coronavirus variants, human immunodeficiency virus, dengue virus, and norovirus.

4. The circular long single-stranded ssDNA according to claim 1, characterized in that The aptamer sequence comprises the sequence shown in SEQ ID NO: 27, or the aptamer sequence is the sequence shown in SEQ ID NO:

27.

5. A single-stranded DNA assembly, characterized in that The single-stranded DNA assembly is obtained by self-assembly of the circular long single-stranded ssDNA according to any one of claims 1 to 4; Preferably, the single-stranded DNA assembly is a closed-loop nanostructure; More preferably, the single-stranded DNA assembly has no gaps.

6. The single-stranded DNA assembly according to claim 5, characterized in that After self-assembly, the backbone sequence of the DNA assembly forms the morphological structure of the single-stranded DNA assembly; the aptamer sequence forms the aptamer functional unit of the single-stranded DNA assembly; Preferably, the morphological structure of the single-stranded DNA assembly is linear, triangular, quadrilateral or pentagonal; More preferably, the aptamer functional units are distributed at the vertices or endpoints, the edges of the framework or the internal cavity region of the single-stranded DNA assembly.

7. The method for preparing a single-stranded DNA assembly according to claim 5 or 6, characterized in that: The preparation method comprises the following steps: The circular long single-stranded ssDNA is recombined into the vector to obtain a recombinant phagemid; transducing the recombinant phagemid and the helper phagemid into a host to obtain a recombinant host; culturing the recombinant host and collecting the circular long single-stranded ssDNA; self-assembling the collected circular long single-stranded ssDNA to obtain the single-stranded DNA assembly; Preferably, the vector is a phagemid; more preferably, it is an M13ori phagemid vector or a flori phagemid vector; Preferably, the host is Escherichia coli.

8. The method for preparing a single-stranded DNA assembly according to claim 7, wherein the self-assembly comprises the steps of denaturing and renaturing the circular long single-stranded ssDNA; Preferably, the temperature for denaturation and annealing of the circular long single-stranded ssDNA is 22-95°C; Preferably, the self-assembly 2+ More preferably, the Mg 2+ The content is 12.5-20mM.

9. A composition, characterized in that Comprising the single-stranded DNA assembly according to claim 5 or 6 and / or the single-stranded DNA assembly obtained by the preparation method according to claim 7 or 8.

10. Use of the single-stranded DNA assembly according to claim 5 or 6, and / or the single-stranded DNA assembly obtained by the preparation method according to claim 7 or 8, and / or the composition according to claim 9 in the preparation of kits and / or chips for virus detection and clinical medical diagnosis.

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