A method for effectively inhibiting the assembly of HIV-1 virus based on synthetic oligonucleotides

Abstract

The present invention discloses a method for effectively inhibiting the assembly of HIV-1 virus based on synthetic oligonucleotides. This method first designs a series of synthetic RNA oligonucleotides to self-assemble into an RNA self-assembled nanomaterial with a more complex spatial structure and multiple stem-loop structures. Then, the RNA oligonucleotides and their self-assembled nanomaterials are used to effectively bind to the HIV-1 structural protein Gag molecule, thereby interfering with virus assembly and effectively inhibiting the release of HIV-1 virus. The present invention realizes for the first time the inhibition of the assembly of HIV-1 virus using synthetic RNA oligonucleotides and self-assembled nanomaterials, expands the application of RNA oligonucleotides in the biomedical field, and provides new ideas for disease treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a method capable of effectively interfering with the assembly of HIV-1 virus in living cells. Background Art

[0002] The assembly and release process of human immunodeficiency virus type 1 (HIV-1) is an important stage in its life cycle. This process is mainly mediated by the Gag protein. Gag polymerizes on the cell membrane with viral RNA molecules as the backbone, assembles into virus particles, and then is released from the cell membrane of the host cell. So far, a large number of research results have proved that the Gag-RNA interaction plays a key role in virus assembly. The Gag molecules in the assembly on the cell membrane can interact with a variety of RNAs, including viral RNA and cellular endogenous RNAs (Selective and nonselective packaging of cellular RNAs in retrovirus particles. (J Virol. 2007;81(12):6623-6631.)) and so on. In vitro aqueous solution experiments show that within a certain RNA length range, the longer the RNA interacting with Gag, the stronger the degree of Gag assembly (Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1 (J Virol. 1995;69(10):6487-6497.)).

[0003] In 2014, Chen et al. proposed the concept of using natural small RNAs to block virus particle assembly. The study found that the microRNA (miRNA) molecules that are ubiquitous in cells can bind to the Gag protein, disrupt the polymerization process of Gag on viral RNA, and cause the failure of virus particle assembly. These Gag proteins with assembly failures will be transported to the cytoplasm by the host cell through the endocytic pathway and degraded in lysosomes (MicroRNA binding to the HIV-1 Gag protein inhibits Gag assembly and virus production (Proc Natl Acad Sci U S A. 2014 Jul 1; 111(26): E2676–E2683.)). In 2017, Qu et al. further revealed the role of autophagy in the process of miRNA-Gag interaction inhibiting virus assembly (Inhibition of retroviral Gag assembly by non-silencing miRNAs promotes autophagic viral degradation (Protein Cell. 2018; 9(7): 640–651.)).

[0004] Despite the above advantages, there are still some problems when miRNAs are used to inhibit virus assembly. For example, experiments on overexpressing miRNAs in cells are required, but it is difficult to control the expression level of miRNAs; the selected miRNAs are limited to the molecular species endogenous to organisms, so the sequence, structure, base composition, molecular size, and chemical modification type are limited.

[0005] The property of base complementarity of RNA molecules can help nucleic acid strands self-assemble into complex nanostructures. As a natural biomaterial, it has the advantages of high modifiability, good thermal stability, and high biosafety. Researchers have confirmed that multiple nucleic acid strands can be designed based on the base complementarity property to self-assemble into RNA nanoparticles with controllable sizes in aqueous solutions, and it has been confirmed that some RNA particles can have biological functions in cells or even in vivo. Currently, using special RNA sequences or structures as self-assembly materials has become a rapidly developing research field (Advancement of the emerging field of RNA nanotechnology (ACS Nano. 2017; 11(2): 1142-1164.)). Summary of the Invention

[0006] In view of the above deficiencies in inhibiting virus assembly with miRNA, the present invention designed a series of artificially synthesized RNA oligonucleotides and found that the Gag protein has a binding preference for stem-loop-structured RNA oligonucleotides on the cell membrane, and this binding can interfere with virus assembly. Introducing this concept into the design of self-assembling RNA elements, RNA self-assembling nanomaterials with more complex spatial structures and multiple stem-loop structures were obtained, further enhancing the inhibitory effect of artificially synthesized RNA oligonucleotides on HIV-1 virus assembly.

[0007] Specifically, the present invention first provides an artificially synthesized stem-loop-structured oligonucleotide that can effectively bind to the HIV-1 structural protein Gag molecule. Such an oligonucleotide can establish RNA self-assembling nanomaterials with more complex spatial structures through the method of controllable self-assembly (Controllable self-assembly of RNAtetrahedrons with precise shape and size for cancer targeting (Adv Mater. 2016; 28(34): 7501-7.)).

[0008] The RNA oligonucleotide that can self-assemble into an RNA self-assembling nanomaterial with multiple stem-loop structures can be an oligonucleotide chain with the following characteristics:

[0009] 1. It has a stem-loop structure, and has an extended single strand at the 5' or 3' end of the stem-loop structure. The length of the extended single strand is preferably 16-20 nt;

[0010] 2. The loop structure in the stem-loop structure consists of 15-25 bases, and the length of the stem structure is at least 4 base pairs; software such as UNAFold can be used to verify whether the designed RNA oligonucleotide has a stem-loop structure;

[0011] 3. Different RNA oligonucleotides are complementary through the single strand located at the 5' or 3' end of the stem-loop structure, and self-assemble to form an RNA self-assembling nanomaterial with a more complex spatial structure; the sequence of the single strand can be freely designed, and based on the principle of base complementary pairing, the assembly between two, three or even more RNA oligonucleotides can be achieved (see Figure 2 );

[0012] 4. The RNA oligonucleotide sequence is a nonsense oligonucleotide sequence, that is, it should have no homology with the human genome and the virus genome.

[0013] Based on the above RNA oligonucleotides, the present invention provides a method for inhibiting HIV-1 virus assembly, including the following steps:

[0014] 1) Design at least two RNA oligonucleotides that can self-assemble into RNA self-assembled nanomaterials with multiple stem-loop structures, and then artificially synthesize the RNA oligonucleotides;

[0015] 2) After mixing the multiple RNA oligonucleotides synthesized in step 1) at equimolar concentration in 1× Tris buffer (100 mM NaCl, 50 mM Tris, pH 8.0), perform high-temperature denaturation - slow cooling annealing. Complementary pairing occurs through the single-stranded regions at the 5' or 3' ends of the stem-loop structures in each RNA oligonucleotide monomer, thereby achieving self-assembly; then purify the product to obtain RNA self-assembled nanomaterials with multiple stem-loop structures;

[0016] 3) Transfect cells infected with HIV-1 virus with the RNA self-assembled nanomaterials to interfere with the assembly of HIV-1 virus in cells.

[0017] The RNA oligonucleotides designed in step 1) should be nonsense oligonucleotide sequences that have no homology with the human genome and the viral genome; have stem-loop structures and have extended single-stranded regions at the 5' or 3' ends of the stem-loop structures; in step 2), each RNA oligonucleotide monomer achieves self-assembly through complementary pairing of the extended single-stranded regions at the 5' or 3' ends of the stem-loop structures. For example, two RNA oligonucleotide monomers self-assemble to form a dumbbell-shaped RNA self-assembled nanomaterial, and three RNA oligonucleotide monomers self-assemble to form a "Y"-shaped RNA self-assembled nanomaterial.

[0018] Preferably, in the stem-loop structure of the RNA oligonucleotides designed in step 1), the loop structure consists of 15 - 25 nt, the length of the stem structure is at least 4 base pairs, and the length of the extended single-stranded region is 16 - 20 nt.

[0019] Furthermore, to resist degradation by nucleases in the cellular environment, the RNA oligonucleotides are oligonucleotides in which all bases are 2'-O-methylated modifications. Other chemical modifications with the ability to resist nuclease degradation can also be used, such as phosphorothioate (PS) modification, locked nucleic acid (LNA) modification, morpholino modification, etc.

[0020] In an embodiment of the present invention, four RNA oligonucleotides shown in SEQ ID No: 1 to SEQ ID No: 4 in the sequence listing are designed. Among them, when the RNA oligonucleotides shown in SEQ ID No: 1 and 2 are mixed in equal amounts and self-assembled, an RNA self-assembled nanomaterial with a dumbbell-shaped structure can be formed; when the RNA oligonucleotides shown in SEQ ID No: 1, 3, and 4 are mixed in equal amounts and self-assembled, an RNA self-assembled nanomaterial with a "Y"-shaped structure can be formed, as Figure 2 shown.

[0021] Preferably, in step 2), a 3000 NMWL cellulose filter column is used to purify the self-assembled product.

[0022] Preferably, in step 3), the RNA self-assembled nanomaterial is transfected into cells by electroporation.

[0023] In an embodiment of the present invention, the feasibility and effectiveness of the technical solution of the present invention are verified by the following experiments:

[0024] 1) Cultivate cells and transfect the viral gene vector pNL43ΔPolΔEnv-Gag to express viral RNA and Gag protein in the cells;

[0025] 2) Electroporate RNA oligonucleotides or their self-assembled nanomaterials, perform Gag-RNA oligonucleotide immunoprecipitation, and measure the interaction between the two, that is, use a specific antibody to extract the Gag protein on the cell membrane, detect the enrichment degree of RNA oligonucleotides in the immunoprecipitate, and analyze the binding ability of different oligonucleotides to Gag molecules on the cell membrane;

[0026] 3) Electroporate RNA oligonucleotides or their self-assembled nanomaterials, and analyze the virus particle release efficiency, that is, measure the release efficiency of HIV-1 virus after being interfered by RNA oligonucleotides or their self-assembled nanomaterials by western blot method to confirm the effectiveness of this method.

[0027] Advantages of the present invention:

[0028] The present invention constructs an RNA self-assembled nanomaterial with a complex spatial structure by a self-assembly method, further improving the ability of artificially synthesized RNA oligonucleotides to inhibit virus assembly. Utilizing the characteristics of easy operation and high controllability of artificially synthesized small molecule RNAs, it effectively inhibits the release of HIV-1 virus and expands the application of RNA oligonucleotides in the biomedical field. This is the first time to achieve the inhibition of HIV-1 virus assembly using artificially synthesized RNA oligonucleotides and self-assembled nanomaterials, providing a new idea for disease treatment. Description of the Drawings

[0029] Figure 1 . (A) Interaction between stem-loop oligonucleotide and HIV-1 virus Gag protein on the cell membrane. (B) Inhibitory effect of stem-loop oligonucleotide on the release of HIV-1 virus. The values in the figure represent the mean ± standard error. Asterisks (*) represent significant differences (independent samples T-test, **P < 0.01, ***P < 0.001).

[0030] Figure 2 . Schematic diagram of self-assembled nanoparticles of stem-loop oligonucleotide. DB is composed of equal amounts of mixed MoD#1 and MoD#2, and TB is composed of equal amounts of mixed MoD#1, MoD#3 and MoD#4.

[0031] Figure 3 . Effects of stem-loop oligonucleotide and self-assembled nanoparticles on the release efficiency of HIV-1 virus. The values in the figure represent the mean ± standard error. Asterisks (*) represent significant differences (one-way ANOVA test, Tukey's post hoc comparison, *P < 0.05, ***P < 0.001).

[0032] Figure 4 . The inhibitory effect of self-assembled nanoparticles on the release of HIV-1 virus is related to the concentration. The values in the figure represent the mean ± standard error. Asterisks (*) represent significant differences (one-way ANOVA test, Tukey's post hoc comparison, **P < 0.01, ***P < 0.001). Detailed implementation manners

[0033] The following examples illustrate the inhibitory effect of the stem-loop oligonucleotide developed by the present invention on the release of HIV-1 virus.

[0034] The interaction between viral RNA and Gag protein plays a key role in virus assembly, mediating the assembly of thousands of Gag proteins on the cell membrane. In this example, by introducing synthetic RNA oligonucleotides, they bind to the Gag protein on the cell membrane, thus competing with viral RNA and interfering with the viral RNA-Gag interaction, ultimately inhibiting the assembly and release of virus particles. The specific implementation manners are as follows.

[0035] 1 Reagents and instruments

[0036] 1.1 Main reagents and materials

[0037] 1) Nonsense oligonucleotide sequence (can be synthesized by Integrated DNA Technologies)

[0038] Table 1

[0039]

[0040] In Table 1, the oligonucleotides Module 1 and Module 2 with stem-loop structures are mixed in equal amounts and self-assembled into dumbbell-shaped nanoparticles, as shown by Dumbbell in Figure 2 ; the oligonucleotides Module 1, Module 3, and Module 4 with stem-loop structures are mixed in equal amounts and self-assembled into branched "Y"-shaped nanoparticles, as shown by Tribell in Figure 2 .

[0041] 2) Plasmid pNL43ΔPolΔEnv-Gag (for expressing Gag protein and viral RNA) (MicroRNA binding to the HIV-1 Gag protein inhibits Gag assembly and virus production (Proc Natl Acad Sci U S A. 2014; 111(26):E2676–E2683.)).

[0042] 3) Plasmid extraction kit (available from Omega Bio-tek).

[0043] 4) Human cervical cancer cell line (HeLa cells).

[0044] 5) DMEM medium containing 10% (vol / vol) fetal bovine serum (available from PAN TM Biotech) and 1×GlutaMAX TM (available from Thermo Fisher) (available from CORNING); 10×PBS (available from CORNING); trypsin (available from Thermo Fisher).

[0045] 6) Transfection reagent 6 (available from Promega).

[0046] 7) Electroporation kit (available from Invitrogen), Minute TM Cell membrane protein isolation kit (available from Invent).

[0047] 8) Reagents required for immunoprecipitation (IP) of Gag-RNA oligonucleotides: IP lysis buffer, Brij buffer, Brij blocking solution (all formulations are shown in the specific experimental procedures).

[0048] 9) Reagents required for the synthesis and purification of RNA self-assembled nanoparticles: 10×Tris buffer, 1×TBM buffer, RNA gel (all formulations are shown in the specific experimental procedures).

[0049] 10) Reagents required for Western Blot to detect virus release efficiency: cell lysis buffer, electrophoresis buffer, transfer buffer, TBST solution, blocking solution (all formulations are shown in the specific experimental procedures).

[0050] 11) Antibodies: HIV-Ig (from NIH), anti-p24 antibody (available from EMD Millipore), anti-chemical dye FAM antibody (available from Abcam).

[0051] 1.2 Main instruments

[0052] 1) Gel electrophoresis apparatus, biochemical incubator, shaker, constant temperature incubator, PCR instrument.

[0053] 2) Cell culture incubator, biological safety cabinet.

[0054] 3) Electroporator, gel imager.

[0055] 2 Experimental methods

[0056] 2.1 Cell transfection

[0057] When the HeLa cells in the T25 culture flask grow to 50%-70% confluence, use according to the instructions 6 Transfect 5 μg of pNL43ΔPolΔEnv-Gag into HeLa cells.

[0058] 2.2 Electroporation of RNA oligonucleotides

[0059] Twenty-four hours after transfection, treat the cultured HeLa cells with trypsin, wash with 1×PBS and resuspend, add 100 μL of electroporation 1×PBS buffer to make the total number of HeLa cells in the solution reach 500,000 (for virus release efficiency analysis) or 2 million (for immunoprecipitation), add 0.1 nmol of RNA oligonucleotide STL1 / UN1, 0.1 nmol of MoD, 0.05 nmol of DB or 0.03 nmol of TB nanoparticles (for virus release efficiency analysis) or 0.5 nmol of FAM-modified STL1 / UN1 (for immunoprecipitation analysis). Use The electroporation system, with parameters set to 1005 V, 35 ms pulse width, and 2 pulses. Add 10 mL of medium to wash the electroporated cells once, centrifuge to obtain the cells, resuspend and culture in a six-well plate.

[0060] 2.3 Immunoprecipitation (IP) of Gag-RNA oligonucleotides

[0061] Reagents required:

[0062] ① IP lysis buffer:

[0063]

[0064] Take 0.5 mL of vanadyl ribonucleoside complexes solution, heat it to 60 °C, and add the above lysis solution. After aliquoting, store it at -20 °C. Add 1× protease inhibitor and SUPERase-In TM RNase inhibitor (to a final concentration of 10 U / μL).

[0065] ② Brij buffer:

[0066] 5× Brij 50 mL

[0067] 10× PBS 50 mL

[0068] Make up to 500 mL with deionized water

[0069] Store at room temperature.

[0070] ③ Brij blocking solution:

[0071] Skim milk powder 1 g

[0072] Make up to 20 mL with Brij buffer

[0073] Use immediately after preparation.

[0074] Experimental procedure: Cells are cultured for 4 hours after electroporating RNA oligonucleotides. Use the Minute TM Cell Membrane Protein Isolation Kit to extract the cell membrane fraction, and add 500 μL of IP lysis solution and mix well. Use nProtein A-Sepharose TM beads (GE Healthcare) for pre-adsorption for 4 hours. Take the supernatant and add 8 μg of HIV Ig antibody (NIH), and culture overnight at 4 °C. Add 20 μL of nProtein A-Sepharose TMMicrobeads (GE Healthcare), cultured at 4 °C for 4 hours. Precipitate the microbeads, wash them 3 times with IP lysis buffer and IP lysis buffer added with 1 M urea respectively. Take one-fifth for Western Blot and detect the protein content with anti-p24 antibody (EMD Millipore). Add 100 μL (0.1% SDS) IP lysis buffer and 30 μg proteinase K to the remaining microbeads, and culture them in a 50 °C reactor for 30 minutes. Add 100 μL phenol-chloroform solvent, mix thoroughly by rapid shaking, centrifuge at 21000×g for 10 minutes, take out the upper colorless transparent solution obtained by extraction, add 100% ice ethanol and sodium acetate, and place it in a -20 °C refrigerator overnight. Centrifuge at 21000×g for 30 minutes, aspirate the supernatant to obtain the RNA precipitate, dissolve it with RNase-free water, and perform dot blot on a nylon membrane. 2 Irradiate for 1 minute with an intensity of 2800 J / cm

[0075] 2.4 Synthesis and characterization of RNA self-assembled nanoparticles

[0076] Reagents required for the synthesis and purification of RNA self-assembled nanoparticles:

[0077] ① 10×Tris buffer:

[0078] 1 M Tris 5 mL

[0079] 5 M NaCl 2 mL

[0080] Make up to 10 mL with deionized water

[0081] Store at room temperature.

[0082] ② 1×TBM buffer:

[0083]

[0084] ③ RNA gel:

[0085]

[0086] Prepare in a 60 °C water bath, quickly add TEMED and drip it into the fixed gel plate, insert a comb, and wait for 30 minutes until the gel solidifies.

[0087] Reagents required for Western Blot - MOPS buffer, transfer buffer, TBST buffer, blocking solution, etc. For the preparation method, please refer to 2.5 "Testing the virus particle release efficiency".

[0088] Experimental procedure: Mix RNA oligonucleotide substrates in 1×Tris buffer (total volume of 50 μL. For the synthesis of Dumbbell, use 5 μM each of Module 1 and Module 2; for the synthesis of Tribell, use 3.3 μM each of Module 1, Module 3, and Module 4). Use a PCR instrument and set the program as follows:

[0089] ① Heat at 95°C for 5 minutes;

[0090] ② Cool down slowly to 25°C at a rate of 1°C per minute;

[0091] ③ Incubate at 25°C for 10 minutes.

[0092] Load the synthesized product onto a 6% native PAGE gel and perform electrophoresis at a constant voltage of 90 V in 1×TBM buffer for one hour. Use a low molecular weight single-stranded RNA marker (ss RNA marker, NEB) as the molecular weight marker. Stain the PAGE gel with 1X Gold (Life Technologies) for 10 minutes. Image the gel using a ChemiDoc XRS+ (Bio-Rad) gel imager. Analyze the band intensity using ImageJ software to calculate the synthesis efficiency of RNA self-assembled nanostructures.

[0093] To purify the obtained product, cut the product band from the RNA PAGE gel and place it in an EP tube. Mash it with a sterile plastic rod. Soak it in 1×Tris-NaCl buffer overnight, transfer the supernatant to a 3000 NMWL cellulose filter column, centrifuge at an acceleration of 14000×g for 30 minutes, and recover the remaining liquid in the filter column to obtain the purified product. The obtained product needs to be verified again by RNA gel electrophoresis, and the steps are as described above.

[0094] 2.5 Testing the virus release efficiency

[0095] Reagents required:

[0096] ① Cell lysis buffer:

[0097]

[0098] Store at room temperature and store at -20°C in the refrigerator after adding 1X protease inhibitor.

[0099] ② Transfer buffer:

[0100] 20× Transfer Buffer 50 mL

[0101] Absolute methanol 200 mL

[0102] Make up to 1 L with deionized water

[0103] Store at 4 °C

[0104] ③ TBST Solution:

[0105] 10× TBS Solution 50 mL

[0106] 10% Tween 2.5 mL

[0107] Make up to 500 mL with deionized water

[0108] Store at room temperature

[0109] ④ Electrophoresis Buffer:

[0110] 20× SDS MOPS 50 mL

[0111] Make up to 1 L with deionized water

[0112] Store at room temperature

[0113] ⑤ Blocking Solution:

[0114] TBST Solution 50 mL

[0115] Non-fat milk powder 2.5 g

[0116] Shake well until dissolved and use immediately after preparation

[0117] Experimental procedure: Cells were cultured for 8 hours after electroporating RNA oligonucleotides, and virus particles and cell samples were collected separately. The culture medium was centrifuged at 1000×g for 10 minutes, and cell debris was removed using a 450 nm needle filter. Add 280 magnetic beads (2 μL / mL), then centrifuge at 100000×g for 50 minutes in an ultracentrifuge, and aspirate the supernatant to obtain virus particles

[0118] Both virus particles and cell samples were lysed in cell lysis buffer containing protease inhibitor (10 μL / mL) at 4 °C for half an hour, centrifuged at 21,000×g for 30 minutes at 4 °C to remove the precipitate. Western Blot experiments were performed on both samples to detect Gag protein using HIV-Ig antibody, and density analysis was carried out using Fiji software to compare the Gag content and the HIV-1 virus release efficiency. Virus release efficiency = Gag content of virus-like particles / (Gag content of virus-like particles + Gag content of cells)

[0119] 2.6 Evaluation of the Effect of This Technology on Inhibiting the Assembly of HIV-1 Virus

[0120] 1) Transfect pNL43ΔPolΔEnv-Gag into HeLa cells. 24 hours after cell transfection, electroporate the stem-loop structured RNA oligonucleotide probe (STL1) and the unstructured RNA oligonucleotide probe (UN1) respectively. 4 hours after electroporation, extract the cell membrane fraction for immunoprecipitation experiment of Gag-RNA oligonucleotide, and calculate the Gag-RNA binding rate (as shown in A of Figure 1 ). The immunoprecipitation results show that Gag mainly binds to STL1 on the membrane. And test its inhibitory effect on the release of HIV-1 virus 8 hours after electroporation (as shown in B of Figure 1 ).

[0121] 2) Take the stem-loop structured RNA oligonucleotide as the basic assembly module, mix them in 1×Tris buffer according to equal molecular weight, then carry out high-temperature denaturation - slow-cooling annealing, and complement through the single-strands at the 3' end of the stem-loop structure in each component unit to achieve self-assembly, and synthesize RNA self-assembled nanoparticles with multiple stem-loop structures (as shown in Figure 2 ), and test its inhibitory effect on the release of HIV-1 virus. 24 hours after HeLa cells are transfected with pNL43ΔPolΔEnv-Gag, perform blank electroporation (MN), electroporate the basic assembly module of RNA oligonucleotide (Module 1, represented by MoD in Figure 3 ), electroporate the RNA self-assembled nanoparticles (Dumbbell or Tribell, represented by DB and TB respectively in Figure 3 ), and detect the release efficiency of HIV-1 virus 8 hours later (as shown in Figure 3 ). The detection results of virus release efficiency show that the basic assembly module of RNA oligonucleotide with stem-loop structure can effectively inhibit the release of HIV-1 virus. Further, the RNA nanoparticles self-assembled from the stem-loop structured RNA oligonucleotides have a more significant improvement in inhibiting the release of HIV-1 virus compared with the single basic assembly module.

[0122] 3) Electroporate different concentration gradients of RNA self-assembled nanoparticles TB by the same method and detect the virus release efficiency, and it can be seen that its inhibitory effect on virus release is related to its working concentration (as shown in Figure 4 ). SEQUENCE LISTING <110> Peking University <120> A Method for Effectively Inhibiting the Assembly of HIV-1 Virus Based on Artificially Synthesized Oligonucleotides <130> WX2021-03-228 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 43 <212> RNA <213> Artificial sequence <400> 1 gacagcguaa gugaugucgu gacugucgag cugcacgcug ccg 43 <210> 2 <211> 43 <212> RNA <213> Artificial sequence <400> 2 gacagcguaa gugaugucgu gacuguccgg cagcgugcag cuc 43 <210> 3 <211> 43 <212> RNA <213> Artificial sequence <400> 3 gacagcguaa gugaugucgu gacuguccgg cagcgacaug agg 43 <210> 4 <211> 43 <212> RNA <213> Artificial sequence <400> 4 gacagcguaa gugaugucgu gacugucccu cauguugcag cuc 43 <210> 5 <211> 28 <212> RNA <213> Artificial sequence <400> 5 gucaccucag cguaagugau gucgugac 28 <210> 6 <211> 20 <212> RNA <213> Artificial sequence <400> 6 cucagcguaa gugaugucgu 20

Claims

1. A method for inhibiting the assembly of HIV-1 virus for non-therapeutic purposes, comprising the following steps: 1) Design at least two RNA oligonucleotides capable of self-assembling into RNA self-assembled nanomaterials with multiple stem-loop structures, and then artificially synthesize the RNA oligonucleotides; wherein, the RNA oligonucleotides are nonsense oligonucleotide sequences, have stem-loop structures, and have extended single strands at the 5' or 3' ends of the stem-loop structures, and are selected from the oligonucleotides shown in SEQ ID No: 1 to SEQ ID No: 4 in the sequence listing; 2) Mix the various RNA oligonucleotides synthesized in step 1) at equimolar concentrations in 1×Tris buffer, perform high-temperature denaturation - slow cooling annealing, and each RNA oligonucleotide monomer is complementary through the extended single strand located at the 5' or 3' end of the stem-loop structure to achieve self-assembly. Among them, the oligonucleotides shown in SEQ ID No: 1 and SEQ ID No: 2 are mixed in equal amounts to self-assemble into a dumbbell-shaped RNA self-assembled nanomaterial, or the oligonucleotides shown in SEQ ID No: 1, SEQ ID No: 3, and SEQ ID No: 4 are mixed in equal amounts to self-assemble into a "Y"-shaped RNA self-assembled nanomaterial; then purify the product to obtain an RNA self-assembled nanomaterial with multiple stem-loop structures; 3) Transfect cells infected with HIV-1 virus with the RNA self-assembled nanomaterial to interfere with the assembly of HIV-1 virus in the cells.

2. The method according to claim 1, wherein The RNA oligonucleotide is an oligonucleotide in which all bases are 2'-O-methylated modified oligonucleotides; or the RNA oligonucleotide has other chemical modifications with the ability to resist nuclease degradation, selected from phosphorothioate modification, locked nucleic acid modification, and morpholino modification.

3. The method according to claim 1, wherein In step 2), a 3000 NMWL cellulose filtration column is used to purify the self-assembled product.

4. The method according to claim 1, wherein In step 3), the RNA self-assembled nanomaterial is transfected into cells by electroporation.

Images