Nucleic acid tetrahedron nanostructure and preparation method and application thereof

Abstract

The invention discloses a nucleic acid tetrahedron nanostructure as well as a preparation method and application thereof. The nucleic acid tetrahedron nanostructure is composed of circRNA for coding an antigen, an ISD chain and two specific single-stranded DNA sequences, is simple and easy to prepare, can enter cells through cell membrane microfossa mediated endocytosis without the help of a transfection reagent, and is positioned to lysosome. The nanostructure is split to release circRNA coding antigen protein, and the circRNA coding antigen protein is delivered to APC and ISD chains in drainage lymph nodes (dLN) to activate cGAS-STING pathways, so that antigen presentation and immune activation are synergistically enhanced, T cells are efficiently and continuously activated, and the effect of tumor immunotherapy is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a nucleic acid tetrahedron nanostructure and a preparation method and application thereof. Background Art

[0002] Tumor metastasis and recurrence are the hottest topics in contemporary cancer treatment research. Effective anti-tumor treatment of solid tumors requires the activation of strong cellular and humoral immunity to achieve clinical efficacy.

[0003] mRNA has the ability to express any protein and has been widely used in recent years in many fields, including vaccine development, protein replacement therapy, and gene editing. Multiple preclinical and clinical trials of mRNA-based cancer vaccines (e.g., ClinicalTrials.gov identifier: NC03313778, NCT02410733, NCT03948763, NCT03953235, and NCT04161755) have demonstrated positive therapeutic effects against melanoma, non-small cell lung cancer, cancer, colorectal tumors, and pancreatic cancer. Despite these significant advances, the sensitivity of mRNA to enzymes, its autoimmunogenicity, and the delivery efficiency, efficacy, and safety of mRNA vaccines still need to be further improved to achieve better anti-tumor effects.

[0004] Inspired by the reverse splicing of pre-mRNAs with various biological functions, which are connected to 3' acceptor splice sites via a 5' donor splice site and form a 3'-5' phosphodiester bond at the junction site to generate natural circular RNA (circRNA), recent attempts to develop synthetic circRNA therapies and vaccines have shown better safety, simplified synthesis, and greater scalability than their linear mRNA counterparts, highlighting their potential as cancer therapeutic vaccines. However, delivery of antigen-encoding mRNA alone to antigen-presenting cells (APCs) may not fully activate anti-tumor immune responses and often requires co-stimulation with immune adjuvants.

[0005] Therefore, there is an urgent need for a circRNA vaccine vector that can simultaneously achieve antigen presentation and immune activation to achieve tumor immunotherapy. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a nucleic acid tetrahedral nanostructure (circRNA-TDN ISD ), can enter cells without the help of transfection reagents, localize to lysosomes, synergistically enhance antigen presentation and immune activation, and achieve the effect of tumor immunotherapy.

[0007] A second object of the present invention is to provide a method for preparing the nucleic acid tetrahedral nanostructure.

[0008] A third object of the present invention is to provide an application of the nucleic acid tetrahedral nanostructure.

[0009] The present invention provides a nucleic acid tetrahedron nanostructure, which consists of a circular RNA (circRNA) S3, an interferon-stimulated gene DNA chain (ISD) S4, and two complementary DNA chains S1 and S2.

[0010] Furthermore, the circular RNA is circRNA-1a or circRNA-1b; when the circular RNA is circRNA-1a, the obtained nucleic acid tetrahedral nanostructure is circRNA-1a-TDN ISD When the circular RNA is circRNA-1b, the resulting nucleic acid tetrahedral nanostructure is circRNA-1b-TDN ISD ;

[0011] Among them, circRNA-1a is complementary to the tetrahedral skeleton, while the coding region of circRNA-1b is independent.

[0012] circRNA-1a-TDN ISD The S1 nucleotide sequence is shown in SEQ ID NO: 1.

[0013] circRNA-1a-TDN ISD The S2 nucleotide sequence is shown in SEQ ID NO: 2.

[0014] The nucleotide sequence of circRNA-1a is shown in SEQ ID NO: 3.

[0015] circRNA-1a-TDN ISD The nucleotide sequence of S4 (ISD) is shown in SEQ ID NO: 4.

[0016] circRNA-1b-TDN ISD The S1 nucleotide sequence is shown in SEQ ID NO:5.

[0017] circRNA-1b-TDN ISD The nucleotide sequence of S2 is shown in SEQ ID NO:6.

[0018] The nucleotide sequence of circRNA-1b is shown in SEQ ID NO: 7.

[0019] circRNA-1b-TDN ISD The nucleotide sequence of S4 (ISD) is shown in SEQ ID NO:8.

[0020] SEQ ID NO: 1 to 8 sequences are shown in the following table:

[0021] Table 1. Oligonucleotide chain sequences

[0022]

[0023]

[0024] The present invention also provides a method for preparing the nucleic acid tetrahedral nanostructure, comprising the following steps:

[0025] Preparation of S11. IVT Template: Genetically engineer a recombinant plasmid containing the circular RNA S3 sequence. Transform the plasmid into E. coli and screen using a culture medium containing a pre-selected antibiotic. The screened strain is inoculated into E. coli culture medium and cultured with shaking at 37°C and 200 rpm for 8–12 hours. The resulting E. coli is then lysed to obtain a plasmid product. The plasmid product is then digested with a restriction endonuclease to obtain a linearized plasmid product. This linearized plasmid product is then sequenced and verified, and the verified product is used as the IVT template.

[0026] S12. Using the IVT template obtained in step S11, in vitro transcription is performed to obtain a transcription product; deoxyribonuclease is then added to remove the IVT template, LiCl is added for precipitation, and the precipitated product is washed with 70% ethanol to obtain linear RNA;

[0027] S13. The linear RNA obtained in step S12 is heated, then placed on ice, GTP is added, and T4 RNA ligase reaction buffer is added for reaction. After the reaction is complete, the product is purified to obtain circular RNA;

[0028] S14. The circular RNA obtained in step S13 is mixed with the interferon-stimulated gene DNA chain (ISD) S4 and two complementary DNA chains S1 and S2 in TM buffer, and the solution is quenched to obtain the nucleic acid tetrahedral nanostructure.

[0029] Furthermore, in step S11, the recombinant plasmid sequence at least includes a T7 promoter, homology arms, elements of a replacement intron-exon (PIE) construct, a spacer, an IRES, and a coding sequence.

[0030] Furthermore, in step S12, the in vitro transcription is performed using a T7 high-yield RNA transcription kit (NEB); the mass volume ratio of DNA: RNA polymerase in the transcription system is 1:2 μg / μL, the total volume of the transcription system is 20-50 μL, and the transcription time is 8 to 12 hours; the volume mass ratio of the deoxyribonuclease added after transcription to the DNA in the system is 1:1 μL / μg, and after adding the deoxyribonuclease, the reaction is carried out at 37°C for 15 minutes; 1 / 2 volume of 5M LiCl at a concentration of the transcription system is added, vortexed, and placed in -40°C for precipitation for 2-12 hours, centrifuged at 13300 rpm at 4°C for 1 hour, the supernatant is aspirated, and the precipitate is washed with 70% ethanol at 4°C.

[0031] Furthermore, in step S13, the specific process conditions for heating the linear RNA obtained in step S12 are as follows: heating at 65°C for 3 min; adding 100 μg of the linear RNA obtained in step S12 to a 40 μL reaction system, adding 1.2 μL of a 100 mM GTP stock solution to a final concentration of 3 mM; then adding 4 μL of 10×T4 RNA ligase reaction buffer (NEB), vortexing the system, and setting the reaction temperature to 55°C for 15 min.

[0032] The purification was carried out by liquid chromatography; a 7.8×300mM column with a particle size of 5 μm and a pore size of The RNA was purified by liquid chromatography using an exclusion column. During the purification process, the volume ratio of RNA:Binding Buffer:anhydrous ethanol was 1:2:3. The liquid was transferred to the adsorption column and placed in a collection tube. The Wash buffer in the kit was diluted with four times the volume of anhydrous ethanol. The adsorption column was then washed twice with 500-1000 μL of the diluted Wash buffer. The column was centrifuged at 13,300 rpm for 2 minutes at 4°C to completely remove the Wash buffer. 50-100 μL of DEPC water was added to the adsorption column to dissolve the RNA on the adsorption column. The column was allowed to stand for 5-15 minutes to fully dissolve the RNA. The RNA solution was then centrifuged at 13,300 rpm for 2 minutes at 4°C.

[0033] Furthermore, in step S14, the molar ratio of the four nucleic acid sequences of circular RNA, interferon-stimulated gene DNA chain (ISD) S4, DNA chain S1, and DNA chain S2 is 1:1:1:1, and the final concentration of any one nucleic acid sequence in the reaction system is 3 μM; the MgCl2 concentration in the TM buffer used is 10 mM and the pH is 7.4.

[0034] The quenching is to heat the reaction system at 95° C. for 10 minutes and then at 4° C. for 30 minutes to allow the bases to be complementary and paired. The reaction is carried out in a PCR thermal cycler.

[0035] The present invention also provides an application of the nucleic acid tetrahedron nanostructure in anti-tumor treatment.

[0036] The nucleic acid tetrahedral nanostructure of the present invention is a simple and typical pyramid structure. The tetrahedral nanostructure does not require transfection reagents and can effectively enter mammalian cells. It has the advantages of natural biocompatibility, structural stability, programmability and easy internalization.

[0037] At the same time, interferon-stimulating gene DNA chains were incorporated into the nucleic acid tetrahedral nanostructure. The addition of ISD activated the cGAS-STING pathway. This signaling pathway is crucial for sensing abnormal DNA accumulation in the cytoplasm. When DNA damage or excessive DNA accumulation occurs within the cell, cGAS binds to it, generating second messenger cyclic dinucleotides. Cyclic dinucleotides activate downstream STING, thereby inducing the expression of inflammatory cytokines such as type I interferon, ultimately initiating an immune response. ISD can directly interact with the STING protein and has the highest cGAS-STING pathway activation effect, significantly enhancing DC maturation and antigen presentation, leading to a strong SIINFEKL-specific cytotoxic T lymphocyte (CTL) response and greatly enhancing the immune response.

[0038] In addition, after the nucleic acid tetrahedral nanomaterials are broken down in the lysosomes, the circular RNA therein is more stable than ordinary linear RNA and can stably translate antigens, greatly improving the efficiency of nucleic acid vaccines.

[0039] The nucleic acid tetrahedron nanomaterial provided by the present invention induces less acute systemic inflammation and has higher safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the process for preparing circular RNA in Example 1;

[0041] Figure 2 This is the gel electrophoresis diagram obtained by gel electrophoresis verification in Example 1;

[0042] Figure 3 This is the gel electrophoresis diagram of the agarose gel electrophoresis verification of the self-assembled TDN in Example 2;

[0043] Figure 4 This is a graph showing the particle sizes of circRNA and TDN measured using dynamic light scattering in Example 2;

[0044] Figure 5 This is a fluorescence localization image of the tetrahedral structure taken up by cells using a laser confocal scanning microscope in Example 4;

[0045] Figure 6In Example 5, flow cytometry was used to measure the expression of circRNA-lipo3000 and circRNA-TDN at different concentrations. ISD Antigen expression effect diagram;

[0046] Figure 7 This is the western blot image obtained by the chemical imager in Example 6;

[0047] Figure 8 This is a diagram showing the effects of mouse T cell immune activation for different durations obtained by flow cytometry analysis in the T cell activation experiment in Example 7;

[0048] Figure 9 This is a graph showing the weight changes of mice at different time points after administration in Example 8;

[0049] Figure 10 This is a graph showing the changes in tumor volume in the E.G7-OVA lymphoma xenograft mouse model after administration of the drug in Example 9;

[0050] Figure 11 This is a graph showing the changes in tumor volume after administration of E.G7-OVA in the tumor recurrence model in Example 9;

[0051] Figure 12 This is a graph showing the enzyme levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice after administration in Example 10. DETAILED DESCRIPTION

[0052] Example 1 Preparation of circular RNA

[0053] Schematic diagram of the preparation method of circular RNA Figure 1 As shown, the following steps are included:

[0054] For the circRNA backbone, the T7 promoter, homology arms, elements of the replacement intron-exon (PIE) construct, spacer, IRES, and coding sequence were synthesized by GenScript and cloned into the puc19 plasmid. The puc19 plasmid was transformed into Escherichia coli and screened using a medium containing preset antibiotics. The screened strain was inoculated into Escherichia coli culture medium and cultured with shaking at 37°C and 200 rpm for 8-12 hours. The obtained E. coli was then lysed using an endotoxin-free plasmid extraction kit (TIANGEN) to obtain a plasmid product. The plasmid product was digested with XbaI restriction endonuclease and purified using DNA extraction solution, chloroform, anhydrous ethanol, and sodium acetate to obtain a linearized plasmid product. The linearized plasmid product was then sequenced and verified, and the verified product was used as an IVT template.

[0055] The DNA extraction solution is a mixture of phenol, chloroform and water, wherein phenol: chloroform: water = 25:24:1.

[0056] The obtained IVT template was used to perform in vitro transcription using a T7 high-yield RNA transcription kit (NEB), with DNA:RNA polymerase = 1 μg:2 μl, a transcription system of 20 μl, and a transcription time of 8-12 hours to obtain a transcription product; deoxyribonuclease DNaseI was then added to remove the IVT template, and LiCl was added for precipitation. The precipitated product was washed with 70% ethanol to obtain linear RNA;

[0057] The obtained linear RNA was heated at 65°C for 3 minutes, then placed on ice, GTP was added to a final concentration of 3 mM, and then T4 RNA ligase reaction buffer was added to react for 15 minutes.

[0058] After the reaction, the product was purified by liquid chromatography; a 7.8×300mM column with a particle size of 5 μm and a pore size of The RNA was purified by liquid chromatography using an exclusion column. During the purification process, the volume ratio of RNA:Binding Buffer:anhydrous ethanol was 1:2:3. The liquid was transferred to an adsorption column, and the adsorption column was placed in a collection tube. The adsorption column was washed twice with 500 μL of Wash buffer and centrifuged at 13,300 rpm for 2 minutes at 4°C to completely remove the Wash buffer. 50-100 μL of DEPC water (depending on the amount of RNA) was added to dissolve the RNA on the adsorption column. The RNA solution was obtained by centrifugation at 13,300 rpm for 2 minutes at 4°C.

[0059] The resulting RNA solution was subjected to an enzyme digestion reaction to remove unconnected linear RNA in the circularization reaction. RNaseR (Biyuntian, 20U / μL) was used in an amount of 1-3μL. The total enzyme digestion system was 60ul. 6μL of 10×RNase R Reaction Buffer was added and digested at 37°C for 15 minutes. The results were verified by agarose gel electrophoresis. The resulting gel electrophoresis pattern is shown below. Figure 2 shown.

[0060] Example 2 Preparation and Characterization of Nucleic Acid Tetrahedron Nanostructures

[0061] The nucleic acid tetrahedral nanostructure consists of a circRNA, an interferon-stimulated gene DNA chain (ISD), and two complementary DNA chains. The three oligonucleotides S1, S2, and S4-ISD were prepared by Shanghai Sangon Biotechnology Co., Ltd. The four nucleic acid sequences were mixed in a molar ratio of 1:1:1:1 in TM buffer (10 mM MgCl2, 1×TAE, pH = 7.4) to a final concentration of 3 μM for each single chain, and the solution was quenched.

[0062] The quenching is to heat the reaction system at 95° C. for 10 minutes and then at 4° C. for 30 minutes to allow the bases to be complementary and paired. The reaction is carried out in a PCR thermal cycler.

[0063] Agarose gel electrophoresis was used to verify the formation of self-assembled TDN. A 1.5% native agarose gel was prepared in 1× TAE buffer supplemented with 10 mM MgCl2 and an appropriate amount of nucleic acid dye. The samples were then loaded and the gel was run in an ice-water bath at 70 V for 1 hour. The bands were visualized and photographed using a UVP bioimaging system. The resulting gel electrophoresis pattern is shown in Figure 2. Figure 3 shown.

[0064] Dynamic light scattering (DLS) was used to measure the particle size of circRNA and TDN. Figure 4 shown.

[0065] exist Figure 3 Because circular RNAs migrate more slowly than their actual size, their molecular weight appears larger. The slower migration of circular RNAs compared to their larger linear precursors likely reflects differences in their topological structures. Linear RNA circularization efficiency does not reach 100%, resulting in a much lower concentration of circRNA in band 3 than in band 2. Band 3 contains both successfully circularized circRNAs and short fragments of the linear precursor that cleave at the circularization temperature. After enzyme digestion, the uncircularized linear RNA band disappears.

[0066] from Figure 4 The measurement results show that the particle size of circRNA is about 8-9nm, and the particle size of TDN is about 40-50nm. This shows that the quenching operation of oligonucleotide sequences in TM buffer can efficiently produce base complementary pairing and form DNA tetrahedral structures.

[0067] Example 3 Preparation of circRNA or ISD chain and transfection reagent complex

[0068] 3.1 Preparation of lipo3000-circRNA and lipo3000-ISD chains

[0069] Lipofectamine TM 3000 is a new transfection reagent developed to improve nucleic acid delivery, which can be used to transfect nucleic acids into various eukaryotic cells that are difficult to transfect. TM Preparation of DNA-Lipofectamine in Reduced Serum Medium TMAfter the 3000 complex is formed, it can be directly added to cells containing cell culture medium without being affected by serum and antibiotics. There is no need to remove the transfection complex, replace or add culture medium after transfection.

[0070] In the experiment, circRNA was mixed with the transfection reagent Lipofectamine TM 3000 were diluted in Opti-MEM at a ratio of 1 nmol:2 μL. TM In reduced serum medium, the amount of medium used for each 1 nmol of circRNA is 250 μl. TM 3000 and P3000 were mixed and diluted in Opti-MEM at a ratio of 1 nmol:2 μL:2 μL. TM Vortex the mixture to increase the speed of complex formation between the nucleic acid chain and the transfection reagent, and let it stand at room temperature for 10 minutes before transfection into the cells.

[0071] 3.2 Preparation of LNP-circRNA

[0072] To prepare 1 mL of 2 mM SM102 lipid stock, the following ingredients are needed: 47.34 μL 2 mM SM102, 63.21 μL 400 μM DSPC, 119.09 μL 1.54 mM cholesterol, 75.27 μL 60 μM DMG-PEG2000, and 695.09 μL anhydrous ethanol. Add all reagents, vortex to mix, and store in a refrigerator at 4°C until ready to use.

[0073] The required volume of SM102 was calculated as follows: n(SM102) = 6 * n(nucleic acid sequence) * number of bases in the nucleic acid sequence, and V(SM102) = n(SM102) / c(SM102). The amount of 100 mM sodium citrate buffer (SCB, pH = 4) was adjusted according to the volume of circRNA in the experiment, so that the final concentration of SCB in the system was 20 mM. Anhydrous ethanol was added to the SM102 lipid stock solution so that V(SM102) + V(C2H5OH) = 3[V(circRNA) + V(SCB)]. All reagents were added to the EP tube in proportion and vortexed to mix. The mixed reagents were transferred to the dialysis tube, which was placed in a bottle containing DPBS solution and placed in a shaker at 40 rpm at 4°C overnight. The purpose of dialysis was to stabilize the complex particles and reduce the effect of anhydrous ethanol on the experimental animals. At room temperature, the dialyzed LNP-circRNA solution was centrifuged at 5000 x g using a 10 kb ultrafiltration tube to the required volume.

[0074] Example 4 Cellular Uptake and Antigen Expression of Tetrahedral Nucleic Acid Nanostructures

[0075] The glass bottom of the confocal dish was treated with polylysine (PLL) at 37°C for 30 min, rinsed with DPBS, and placed in a 37°C cell culture incubator overnight. The next day, DC2.4 cells were cultured at a density of 2×10 4 cells / dish were seeded on the bottom of a glass dish, and then 400 μl of culture medium was added after stabilization in a 37°C incubator for 2 h. After 24 h, cy5-circRNA-1a-TDN prepared in Example 2 was added. ISD The incubation concentration was 0.4 μM. After 1 hour, the cells were aspirated and washed with DPBS. Then, they were incubated with the nuclear dye Hoechst33342 for 45 minutes and the lysosomal dye Lyso-TrackerGreen (1 μM) for 15 minutes. The excess dye was removed by washing with DPBS, and 500 μl of DPBS was added. The uptake and localization of the fluorophore-labeled tetrahedral structure in DC2.4 cells were evaluated by laser confocal scanning microscopy. The results are shown in Figure 2. Figure 5 shown.

[0076] It can be clearly seen from the figure that when cy5-circRNA-1a-TDN prepared in Example 2 was added to DC2.4 cells ISD After incubation at 37 °C for 1 h, the tetrahedron labeled with cy5 fluorescent group was located at the same position as the lysosomal dye Lyso-TrackerGreen, indicating that cy5-circRNA-1a-TDN ISD It can reach the lysosomes well without the help of drug transfection reagents, and unwind the base complementary pairing structure in the lysosomes, releasing circRNA-1a expressing SIINFEKL antigen and interferon-stimulated gene DNA chain, thereby delivering antigens to antigen-presenting cells and activating the cGAS-STING pathway, forming an efficient and sustained activation of specific CD8+ T cell effector immunity.

[0077] Example 5 circRNA antigen expression

[0078] This embodiment is provided with an experimental group and a control group. The experimental group is the nucleic acid tetrahedral nanostructure prepared in Example 2, which enters the cell via the endocytosis pathway mediated by caveolin, is degraded by lysosomes in the cytoplasm, and releases circRNA and ISD chains. The control group is prepared in Example 3, in which lipo3000 is used as a transfection reagent to carry circRNA expressing SIINKFEL antigen information, and enters the cell in the form of recombinant nucleic acid. CircRNA binds to ribosome-transcribed antigen protein in the cytoplasm, and after APC treatment, presents SIINKFEL antigen on its surface. After binding to the added SIINKFEL fluorescent antibody, the fluorescence intensity is measured by flow cytometry, and the antigen expression effect of the two different delivery methods can be measured.

[0079] DC2.4 with 5×10 5 The cells were seeded at a density of 10 cells / well in a 12-well plate. After 24 hours, the serum-containing medium was aspirated and rinsed with DPBS. An appropriate amount of Opti-MEM serum-free medium was added. The circRNA-1a / 1b-TDN prepared in Example 2 was added to the 12-well plate at concentrations of 0.05 μM, 0.1 μM, 0.2 μM, and 0.4 μM, respectively. ISD and circRNA-1a / 1b-lipo3000 prepared in Example 3. After incubation for 6 h, the medium was replaced with serum-containing medium and incubated for another 18 h. DC2.4 was gently scraped from the 12-well plate with a cell scraper, centrifuged, and rinsed twice with DPBS. The cells were incubated with fluorescent antibodies at 4°C for 30 min and washed, and the fluorescence intensity was measured by flow cytometry.

[0080] like Figure 6 As shown, whether circRNA-1a or circRNA-1b, circRNA-TDN ISD Both circRNA-lipo3000 and circRNA-lipo3000 showed the highest antigen expression effect at the incubation concentration of 0.4 μM, so this concentration was selected as the incubation concentration in subsequent experiments. In the tetrahedral nucleic acid nanostructure, the antigen expression effects of circRNA-1a and circRNA-1b were almost the same.

[0081] Example 6 Western blotting experiment

[0082] Phosphorylated IRF-3 (p-IRF3), phosphorylated TBK-1 (p-TBK1), and phosphorylated STING (p-STING) are markers of cGAS-STING pathway activation. The activation level of the cGAS-STING pathway was investigated by measuring the expression of these phosphorylated proteins.

[0083] 4T1 cells were cultured at 5×10 5 The cells were seeded into 6-well plates at a density of 10 cells / well and cultured at 37°C for 16 h. ISD(500nM) and ISD-lipo3000 (500nM) prepared in Example 3 were added to the wells and incubated for 24h or 48h, and only cell culture medium was added to the blank control group. After gently scraping the 4T1 cells with a cell scraper, RIPA lysis buffer containing protease and phosphatase inhibitors was added, and the cells were placed in an ice box on a shaker for 20min. After lysis, the cells were centrifuged at 2000rpm for 5min and the supernatant was separated to extract the protein, and the protein content was quantified using a BCA protein assay kit. The protein was denatured at 100℃ for 5min, and SDS-PAGE electrophoresis was performed using a gel electrophoresis apparatus (Bio-Radmini, USA). The protein was transferred to a PVDF membrane, blocked with 5% BSA solution, and the corresponding antibody was added and incubated on a shaker at 4℃ overnight. The PVDF membrane was washed 3 times with 1‰ PBST detergent, incubated with HRP-conjugated antibody at room temperature for 1 hour, washed again, and then quantified according to 1ml / 10cm 2 Add ultrasensitive ECL chemiluminescent reagent to the PVDF membrane in a certain proportion, and obtain Western blot images using a chemiluminescent imager. The image results are as follows: Figure 7 shown.

[0084] The experimental results show that circRNA-1a-TDN ISD The expression of cGAS-STING pathway phosphorylation proteins (48h) was significantly greater than that of circRNA-1a-TDN ISD (24h), which indicates that increasing the incubation time can increase the activation effect of the cGAS-STING pathway. ISD (24h) There is no obvious gap, which indicates that the tetrahedral structure can unravel and release the ISD chain after entering the cell.

[0085] Example 7: T cell activation experiment of nucleic acid tetrahedron nanostructures

[0086] In order to verify the nucleic acid tetrahedral nanostructure (circRNA-TDN ISD ) on T cell activation effect, C57BL / 6 female mice (n=5) were subcutaneously injected with ① DPBS (50 μl), ② LNP-circRNA-1a (10 μM, 50 μl) prepared in Example 3, and ③ circRNA-1a-TDN prepared in Example 2 on days 0, 14, and 28, respectively. ISD (10μM, 50μl) and ④circRNA-1b-TDN ISDOn days 21, 35, 49, and 63, blood was collected from the mouse orbits and stained for CD8+ tetramers. The immune response was studied by flow cytometry. The results are shown in Table 2. Figure 8 shown.

[0087] Before the completion of three doses of repeated administration, in the T cell activation effect test on the 21st and 35th days, LNP-circRNA-1a (10 μM, 50 μl) prepared in Example 3 and ③ prepared in Example 2

[0088] Both circRNA-1a-TDNISD (10 μM, 50 μl) and circRNA-1b-TDNISD (10 μM, 50 μl) showed an increasing trend in SIINFEKL-specific CD8+ T lymphocyte activation effect in peripheral blood mononuclear cells, with the highest activation effect on day 35 and a decreasing trend after cessation of administration, indicating that these vaccines showed dose-dependent SIINFEKL+CD8+T cell responses.

[0089] LNP-circRNA-1a triggered the largest proportion of SIINFEKL+CD8+T cell activation among all PBMC CD8+T cells. This may be because lipid nanoparticles have been more mature in drug delivery applications, while DNA tetrahedron is a less studied drug delivery system. circRNA-1a-TDN ISD Similar levels of effect were shown on days 21 and 63, indicating that DNA tetrahedron as the framework of circRNA has good development prospects as a new type of nucleic acid vaccine.

[0090] LNP-circRNA-1a and circRNA-1a-TDN ISD showed similar effector memory T cells (T em ) retained the effect and was consistent with the results of eliciting SIINFEKL-specific CD8+ T cells in all PBMC CD8+ T cells.

[0091] Example 8 Effect of drug injection on mouse body weight

[0092] Before the first two doses (day 0 and day 7) and at 12h, 24h, 36h, 48h and 72h after the two doses, the weight changes of the mice were recorded using an electronic scale, and the circRNA-1a-TDN prepared in Example 2 was calculated according to [measured weight / weight before the dose]. ISD The body weight change rate of mice in the , LNP-circRNA-1a and blank groups was compared to observe the effects of the drugs on the health status of the mice.

[0093] like Figure 9 As shown in the figure, if the weight of mice decreases after drug administration, it means that the drug has side effects, such as decreased appetite, abnormal liver and kidney metabolic function caused by the drug, etc., which affect the weight of mice.

[0094] From the experimental results, it can be seen that within 12 hours after the two administrations, the circRNA-1a-TDN prepared in Example 2 ISD The average weight of both the LNP-circRNA-1a group and the LNP-circRNA-1a group prepared in Example 3 decreased, among which the decrease in the LNP-circRNA-1a group prepared in Example 3 was more significant, which indicates that compared with the nucleic acid transfection reagent LNP containing ethanol, the tetrahedral structure composed of DNA avoids the effects of additional transfection reagents on the organism and has less effect on liver metabolic function; 24 hours after the two doses, the weight of the mice began to increase again and returned to the pre-dose level within 72 hours. The short-term fluctuation in weight indicates that the drug can be rapidly metabolized after exerting its effect, avoiding accumulation in the organism and thus producing toxicity. Combined with the small change in weight in the blank control group, this indicates that acute stress can affect experimental animals, such as environmental changes and dosing operations.

[0095] Example 9 Experimental Study on Tumor Inhibition and Immune Effects of Nucleic Acid Tetrahedron Nanostructures

[0096] E.G7-OVA cells (1×10 6 ) E.G7-OVA lymphoma xenograft mouse model was established in the right hip of C57BL / 6 female mice. One week later, the average tumor volume of the mice reached approximately 100 mm 3 .

[0097] On days 7, 13, and 19, mice with established tumor models were subcutaneously injected with ① LNP-circRNA-1a (10 μM, 50 μl) prepared in Example 3 and ② circRNA-1a-TDN prepared in Example 2. ISD (10μM, 50μl), ③ DPBS (50μl), five mice per group, the size of the mouse tumor was measured every three days starting from the 7th day, and the tumor volume was calculated according to the formula: length (mm) * width (mm) * width (mm) / 2. ISD Whether the vaccine has an inhibitory effect on tumor growth.

[0098] Depend on Figure 10As shown, the tumors in the DPBS group grew rapidly, and circRNA-1a-TDN ISD The tumor growth rate of the group was between that of the DPBS group and the LNP-circRNA-1a group, showing a significant inhibitory effect; while the tumor growth in the LNP-circRNA-1a group was slower.

[0099] At the next tumor volume measurement (day 10, day 16, day 22) at each dosing time point (day 7, day 13, day 19), the tumor growth rate slowed significantly.

[0100] The method for verifying the nucleic acid tetrahedral nanostructure (circRNA-TDN) in Example 5 was used. ISD ) on the activation effect of T cells in mice, the E.G7-OVA tumor recurrence model was established on the seventh day after the fourth blood draw (day 70), and the tumor volume of the mice was measured every three days starting from the seventh day (day 77) after tumor cell implantation to observe the activation effect of circRNA-TDN ISD Whether the vaccine has an inhibitory effect on tumor recurrence or metastasis.

[0101] Depend on Figure 11 It can be seen that the tumor began to grow on the 7th day after the tumor recurrence model was established; from the 7th day to the 16th day, the changes in tumor volume of the four groups of mice were not much different; from the 16th day onwards, the tumor in the DPBS group grew rapidly, and circRNA-1a-TDN ISD and circRNA-1b-TDN ISD The tumors in the LNP-circRNA-1a group also began to grow, but their growth rate was significantly inhibited compared to the DPBS group; while the tumors in the LNP-circRNA-1a group grew more slowly. This is consistent with the tumor growth inhibition trend in Example 9, where a tumor model was first established and then administered to mice. This indicates that the tumor growth inhibition effect of the LNP-circRNA-1a prepared in Example 3 is better than that of the circRNA-1a-TDN prepared in Example 2. ISD This may be because lipid nanoparticles have been developed more maturely in drug delivery applications, and nucleic acid tetrahedron nanomedicines still have a lot of room for improvement compared to this mature delivery system, and this is consistent with the activation effect of SIINFEKL-specific T lymphocytes in PMBC in Example 7. The results of Example 8 show that circRNA-1a-TDN prepared in Example 2 ISD The effect of LNP-circRNA-1a on liver metabolic function was smaller than that of LNP-circRNA-1a prepared in Example 3. In the two experiments of primary tumor treatment and immunosuppression of recurrent and metastatic tumors in Example 9, circRNA-TDN was found to be ISDWhen the tumor volume of the group was measured for the last time, its size was only 1 / 2 of that of the DPBS group. These data also show that the attempts of nucleic acid tetrahedron nanomedicine and circRNA vaccine therapy have achieved antigen presentation and immune activation, providing a new and promising method for tumor immunotherapy.

[0102] Example 10 Biochemical indicators of liver and kidney function in mice

[0103] Three groups of 5 C57BL / 6 female mice were set up, and each group was administered with circRNA-1a-TDN prepared in Example 2. ISD , LNP-circRNA-1a prepared in Example 3 and a blank control group were administered twice to the DNA tetrahedron group and the LNP group on day 0 and day 14, respectively, at a dose of 1 nmol / mouse / time. On the second day after the second administration, i.e., on the 15th day, blood was taken by removing the mouse eyeballs. In order to avoid hemolysis, the mouse beard should be shaved before blood collection, and the blood should be directly suspended and dripped into a clean EP tube to avoid contact with the tube wall. The blood was marked and left to stand at room temperature for 2 hours, then centrifuged at 3000 rpm for 15 minutes at 4 ° C, and the upper serum was carefully sucked out into a new EP tube to measure the concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) liver function indicators in the serum.

[0104] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are two enzymes in the liver. When the liver is damaged, the levels of these two enzymes will increase. Figure 12 As shown in the results of serum biochemical tests of the three groups of mice, the LNP-circRNA-1a prepared in Example 3 was significantly better than the circRNA-1a-TDN prepared in Example 2. ISD Compared with the blank control group, the levels of ALT and AST were significantly increased, indicating that there was obvious liver damage; while the circRNA-1a-TDN prepared in Example 2 ISD The ALT and AST levels in the control group were slightly increased compared with those in the blank control group, but not as high as those in the LNP-circRNA-1a prepared in Example 3. This indicates that the DNA tetrahedral structure circRNA-1a-TDN prepared in Example 2 ISD Compared with the lipid nanoparticle delivery of circRNA in Example 3, the effect on liver function is smaller and the biosafety of the drug is higher.

Claims

1. A nucleic acid tetrahedral nanostructure, characterized in that: The nucleic acid tetrahedron nanostructure consists of a circular RNA S3, an interferon-stimulated gene DNA chain S4, and two complementary DNA chains S1 and S2.

2. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that The circular RNA is circRNA-1a or circRNA-1b; when the circular RNA is circRNA-1a, the obtained nucleic acid tetrahedral nanostructure is circRNA-1a-TDN ISD When the circular RNA is circRNA-1b, the resulting nucleic acid tetrahedral nanostructure is circRNA-1b-TDN ISD .

3. The nucleic acid tetrahedral nanostructure according to claim 2, characterized in that: circRNA-1a complementarily pairs with the tetrahedral backbone, while the coding region of circRNA-1b is independent.

4. The nucleic acid tetrahedral nanostructure according to claim 2, characterized in that Nucleic acid tetrahedral nanostructure circRNA-1a-TDN ISD The nucleotide sequence of the DNA chain S1 is shown in SEQ ID NO: 1, the nucleotide sequence of the DNA chain S2 is shown in SEQ ID NO: 2, the nucleotide sequence of the circular RNA S3 is shown in SEQ ID NO: 3, and the nucleotide sequence of the ISD S4 is shown in SEQ ID NO:

4.

5. The nucleic acid tetrahedral nanostructure according to claim 2, characterized in that Nucleic acid tetrahedral nanostructure circRNA-1b-TDN ISD The DNA chain S1 nucleotide sequence is shown in SEQ ID NO: 5, the DNA chain S2 nucleotide sequence is shown in SEQ ID NO: 6, the circular RNA S3 nucleotide sequence is shown in SEQ ID NO: 7, and the ISD S4 nucleotide sequence is shown in SEQ ID NO:

8.

6. A method for preparing a nucleic acid tetrahedral nanostructure according to any one of claims 1 to 5, characterized in that: The following steps are involved: Preparation of S11. IVT Template: Genetically engineer a recombinant plasmid containing the circular RNA S3 sequence. Transform the plasmid into E. coli and screen using a culture medium containing a pre-selected antibiotic. The screened strain is inoculated into E. coli culture medium and cultured with shaking at 37°C and 200 rpm for 8–12 hours. The resulting E. coli is then lysed to obtain a plasmid product. The plasmid product is then digested with a restriction endonuclease to obtain a linearized plasmid product. This linearized plasmid product is then sequenced and verified, and the verified product is used as the IVT template. S12. Using the IVT template obtained in step S11, in vitro transcription is performed to obtain a transcription product; deoxyribonuclease is then added to remove the IVT template, LiCl is added for precipitation, and the precipitated product is washed with 70% ethanol to obtain linear RNA; S13. The linear RNA obtained in step S12 is heated, then placed on ice, GTP is added, and then T4 RNA ligase reaction buffer is added for reaction. After the reaction is complete, the product is purified and enriched by precipitation with anhydrous ethanol to obtain circular RNA; S14. The circular RNA obtained in step S13 is mixed with the interferon-stimulated gene DNA chain S4 and two complementary DNA chains S1 and S2 in TM buffer, and the solution is quenched to obtain the nucleic acid tetrahedral nanostructure.

7. The preparation method according to claim 6, characterized in that In step S11, the recombinant plasmid sequence at least includes a T7 promoter, homology arms, elements of a replacement intron-exon construct, a spacer, an IRES, and a coding sequence; In step S12, the in vitro transcription is performed using a T7 high-yield RNA transcription kit; the mass volume ratio of DNA: RNA polymerase in the transcription system is 1:2 μg / μL, the total volume of the transcription system is 20-50 μL, and the transcription time is 8-12 hours; the volume mass ratio of the deoxyribonuclease added after transcription to the DNA in the system is 1:1 μL / μg, and after adding the deoxyribonuclease, the reaction is incubated at 37°C for 15 minutes; 1 / 2 volume of 5M LiCl at a concentration of the transcription system is added, vortexed, and placed in -40°C for precipitation for 2-12 hours, centrifuged at 13300 rpm at 4°C for 1 hour, the supernatant is aspirated, and the precipitate is washed with 70% ethanol at 4°C.

8. The preparation method according to claim 6, characterized in that In step S13, the specific process conditions for heating the linear RNA obtained in step S12 are as follows: heating at 65°C for 3 minutes; adding 100 μg of the linear RNA obtained in step S12 to a 40 μL reaction system, and adding 1.2 μL of a 100 mM GTP stock solution to a final concentration of 3 mM; then adding 4 μL of 10×T4 RNA ligase reaction buffer, vortexing the system, and the reaction temperature is 55°C for 15 minutes. The product was purified by liquid chromatography using a 7.8×300mM column with a particle size of 5 μm and a pore size of The product was purified by liquid chromatography using a size exclusion column; During the purification process, the volume ratio of RNA:BindingBuffer:anhydrous ethanol is 1:2:3; transfer the liquid to the adsorption column and place the adsorption column in a collection tube. Use four times the volume of anhydrous ethanol to dilute the Washbuffer in the kit, then take 500-1000μL of the diluted Washbuffer to wash the adsorption column twice. Centrifuge at 13300rpm for 2 minutes at 4°C to completely remove the Washbuffer. Add 50-100μL of DEPC water to dissolve the RNA on the adsorption column. Let it stand for 5-15 minutes to fully dissolve. Centrifuge at 13300rpm for 2 minutes at 4°C to obtain the RNA solution.

9. The preparation method according to claim 6, characterized in that In step S14, the molar ratio of the four nucleic acid sequences of circular RNA, interferon-stimulated gene DNA chain S4, DNA chain S1, and DNA chain S2 is 1:1:1:1, and the final concentration of any nucleic acid sequence in the reaction system is 3 μM; The TM buffer used had a MgCl2 concentration of 10 mM and a pH of 7.4; The quenching is to heat the reaction system at 95° C. for 10 minutes and then at 4° C. for 30 minutes to allow the bases to be complementary and paired. The reaction is carried out in a PCR thermal cycler.

10. Use of the nucleic acid tetrahedral nanostructure according to any one of claims 1 to 5 or the nucleic acid tetrahedral nanostructure prepared by the preparation method according to any one of claims 6 to 9 in anti-tumor treatment.

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