DNA-RNA Hybrid Molecule-Based Nanocarriers and Their Preparation Methods and Applications

Abstract

The present invention belongs to the technical field of nanocarriers, and relates to a nanocarrier based on a DNA-RNA hybrid molecule, its preparation method and application. The nanocarrier includes a DNA-RNA hybrid molecule, an AS1411 aptamer and a target RNA. The DNA-RNA hybrid molecule is a connecting bridge between the AS1411 aptamer and the target RNA. By using DNA-RNA hybridization technology, a DNA-RNA hybrid nanocarrier delivery system carrying siRNA / saRNA is constructed. This delivery system has extremely low cytotoxicity and excellent targeting compared with traditional transfection reagents, can successfully target and deliver the target small RNA into tumor cells, and up-regulate or down-regulate the expression of its target gene, inducing apoptosis of tumor cells, thereby playing an anti-tumor role. The construction of this delivery system provides a new idea for the development of small RNA delivery systems.

Description

Technical Field:

[0001] The present invention belongs to the technical field of nanocarriers, and relates to a nanocarrier based on a DNA-RNA hybrid molecule, a preparation method thereof, and an application thereof. Background Art:

[0002] Ovarian cancer, as a gynecological tumor, is one of the common cancers that seriously endanger women's lives and health, and its incidence accounts for 2.7% of the total proportion of female cancers. Ovarian cancer is common in patients aged 50-70, but the number of cases in young and middle-aged patients is also relatively large. The occurrence and development of ovarian cancer are related to various factors such as genetic factors and environmental factors, but are also closely related to the expression of some genes.

[0003] The discovery of RNA interference (RNAi) and RNA activation (RNAa) phenomena has provided new methods for gene expression regulation and can bring new strategies for the development of ovarian cancer treatment. Since the discovery that siRNA, saRNA, miRNA, etc. have an obvious regulatory effect on the target genes of cells, people have started the research and development of RNA drugs. Gradually, RNA drugs have shown the advantages of simplicity, high efficiency, clear targets, and low side effects. However, the development of RNA drugs is restricted by the delivery system. In particular, the delivery systems of commercialized siRNA and saRNA have many problems such as poor targeting, strong cytotoxicity, high immunogenicity, and poor delivery efficiency. Currently, common RNA deliveries include liposome methods, antibody or ligand chemical conjugation methods, etc. Therefore, it is very urgent to develop a new delivery system with strong targeting, low cytotoxicity, low immunogenicity, and low cost.

[0004] As a tumor suppressor gene, p21 is one of the important members of the family of cell cycle-dependent kinase inhibitors. p21 participates in cell cycle regulation, is an important cell cycle regulatory factor, participates in the replication and repair processes of intracellular DNA, and acts together with the p53 gene to form the G1 checkpoint of the cell cycle, preventing the occurrence and development of cancer. Therefore, the p21 gene plays a very important role in cell proliferation, and abnormal changes in its activity are often related to various diseases such as cancer.

[0005] On the surface of T lymphocytes, there are many important functional regulatory molecules, which are of great significance for the activation, proliferation and anti-tumor effect of T cells. Compared with the currently familiar immune checkpoints such as VTCN1 (V-set domain containing T cell activation inhibitor 1), PD-L1 (Programmed cell death 1 ligand 1), CTLA-4 (Cytotoxic T-lymphocyte associated protein 4), LAG-3 (Lymphocyte activating 3), HAVCR2 (Hepatitis A virus cellular receptor 2), CD160, CD244, etc., TIGIT is also a cell surface protein. As a member of the immunoglobulin superfamily, TIGIT has significant immunosuppressive functions. Its full name is T cell immune receptor with Ig and ITIM domains (TIGIT). TIGIT is an inhibitory receptor expressed on a variety of immune cells, including activated CD8+ T cells, CD4+ T cells, NK cells, regulatory T cells (Tregs), etc. In addition, TIGIT is highly expressed on regulatory T cells, inhibitory immune cells, and participates in exercising its immunosuppressive function to help form the tumor immunosuppressive microenvironment.

[0006] Aptamers are oligonucleotide molecules screened by in vitro screening technology, namely Systematic Evolution of Ligands by Exponential Enrichment (SELEX), for targeting target proteins or compounds. Aptamers are functionally similar to antibodies, with specific targeting properties, and are generally oligonucleotide sequences. Aptamers have the advantages of small molecular weight, no immunogenicity, and easy modification, and are often used in the preparation of targeted biological detection or sensors. Among various types of aptamers, the AS1411 aptamer was reported by Bates et al. in 1999 and can effectively target nucleolin (NCL), also known as nucleolar protein or C23 protein. Nucleolin is a 110 kDa phosphorylated protein, and its function is closely related to the vital activities of ribosomes, including the transcriptional regulation of rRNA. In recent years, studies have shown that nucleolin, as a transmembrane protein, is mostly distributed on the cell membranes of tumors with strong proliferative ability or low differentiation, and is highly expressed on the surface of tumor cells. Therefore, the aptamer AS1411 that binds to nucleolin can be widely used in the imaging of tumor cells, etc., and can also be applied to the treatment of tumors. However, there has been no report on a DNA-RNA hybrid nanocarrier using an aptamer as a targeting tool to achieve targeted delivery of nucleic acid drugs. Summary of the Invention:

[0007] The object of the present invention is to overcome the disadvantages existing in the prior art and provide a method for preparing and applying a nanocarrier based on a DNA-RNA hybrid molecule. This carrier is guided by the AS1411 aptamer to target the nucleoprotein on the surface of tumor cells, and is connected to the target small RNA nucleic acid drug. Under the endocytosis of cells, the nanocarrier is taken into the cell to complete the targeted and intracellular delivery of the nucleic acid drug, providing a new low-toxicity scheme for the targeted delivery of traditional small nucleic acid drugs.

[0008] To achieve the above object, the present invention provides a nanocarrier based on a DNA-RNA hybrid molecule, including a DNA-RNA hybrid molecule, an AS1411 aptamer, and a small RNA nucleic acid drug (or target RNA). The DNA-RNA hybrid molecule is the connecting bridge between the AS1411 aptamer and the target RNA; the target RNA molecule is covalently bonded to the RNA-3′ in the DNA-RNA hybrid molecule.

[0009] The 3′ end of the DNA strand of the DNA-RNA hybrid molecule is connected to the 5′ end of the AS1411 aptamer.

[0010] The sequence of the AS1411 aptamer is reported in the reference as shown in SEQ ID NO: 1; the DNA sequence of the DNA-RNA hybrid molecule is shown in SEQ ID NO: 2, and the RNA sequence is the 16 bases at the 5' end of SEQ ID NO: 3.

[0011] The bases U and C of the target RNA are 2'-fluorinated modifications, that is, the -OH at the 2nd position of UTP and CTP is replaced by -F.

[0012] The target RNA is p21 gene saRNA or TIGIT gene siRNA.

[0013] The sequence of the p21 gene saRNA is reported in the reference as the 21 bases at the 3' end of SEQ ID NO: 3 and SEQ ID NO: 4; the sequence of the TIGIT gene siRNA is the 3' end of SEQ ID NO: 5 23 bases and shown in SEQ ID NO: 6.

[0014] The present invention also provides a method for preparing the nanocarrier based on the DNA-RNA hybrid molecule. First, synthesize the required target RNA and the DNA-RNA hybrid molecule - AS1411 aptamer, and perform 2'-fluorinated chemical modification on the CTP and UTP of the target RNA; mix the DNA-RNA hybrid molecule - AS1411 aptamer with the corresponding target RNA in equimolar amounts to form a solution, mix it with an equal volume of hybridization buffer (2xPBS - 2 mM MgCl2), heat it in a thermal cycler at 80°C for 3 min, 50°C for 5 min, 25°C for 15 min, and cool it to 4°C on a PCR instrument for 20 min to complete the preparation of the AS1411 aptamer-DNA-RNA hybrid nanocarrier.

[0015] The present invention also provides the application of the nanocarrier based on the DNA-RNA hybrid molecule in the preparation of nucleic acid drugs.

[0016] Compared with the prior art, the present invention constructs a DNA-RNA hybrid nanocarrier delivery system carrying siRNA / saRNA by using DNA-RNA hybridization technology. This delivery system has extremely low cytotoxicity and excellent targeting compared with traditional transfection reagents, can successfully target the target small RNA into tumor cells, and up-regulate or down-regulate the expression of its target gene, inducing apoptosis of tumor cells, thereby playing an anti-tumor role; the construction of this delivery system provides a new idea for the development of small RNA delivery systems: connecting the target RNA with the aptamer through a DNA-RNA hybrid molecule, which not only does not affect the targeting of the aptamer, but also achieves the effect of delivering RNA drugs. Description of the drawings:

[0017] Figure 1 Schematic diagram of the structural principle and electrophoresis results of preparing DNA-RNA hybrid nanoparticles in Example 1 of the present invention. Among them, A is the composition structure of the entire AS1411-P21 hybrid nanoparticle, where black represents DNA and red represents RNA. The 5' end of the short RNA strand can be labeled with FAM green fluorescence; B is the DNA-RNA hybrid nanoparticle after removing the AS1411 aptamer; C is the schematic diagram of the results of the TBM polyacrylamide gel electrophoresis test for the assembly of DNA-RNA hybrid nanoparticles.

[0018] Figure 2 Schematic diagram of the experimental results of the serum stability of the DNA-RNA hybrid nanoparticle in Example 2 of the present invention. The DNA-RNA hybrid nanoparticle band at the uppermost end is clearly visible after 72 hours, showing little difference from that at 0 hour.

[0019] Figure 3 Schematic diagram of the delivery principle of the DNA-RNA hybrid nanoparticle of the present invention.

[0020] Figure 4 Schematic diagram of the experimental results of detecting the cellular uptake of AS1411-P21 DNA-RNA hybrid nanoparticles by fluorescence microscopy in Example 3 of the present invention. Among them, green represents the FAM-labeled DNA-RNA hybrid nanoparticles, and blue represents the DAPI-stained cell nuclei. P21 NC has little cellular uptake without the guidance of AS1411.

[0021] Figure 5 Confocal laser scanning microscopy images of cells incubated with the DNA-RNA hybrid nanoparticle for 48h in Example 3 of the present invention. Among them, green represents the FAM-labeled DNA-RNA hybrid nanoparticles, and blue represents the DAPI-stained cell nuclei.

[0022] Figure 6 Schematic diagram of the experimental results of the cytotoxicity in Example 4 of the present invention.

[0023] Figure 7 Schematic diagram of the experimental results of detecting the effects of DNA-RNA hybrid nanoparticles on the expression of p21 and TIGIT genes by fluorescence quantitative PCR in Example 5 of the present invention. Among them, (A) is the expression level of p21 gene mRNA in each group after treating tumor cells with AS1411-P21; (B) is the expression level of TIGIT gene mRNA in each group after treating tumor cells with AS1411-TIGIT; (C) is the change in the mRNA level of TIGIT gene after incubating IOSE-80 cells with AS1411-TIGIT; (D) is the change in the mRNA level of p21 gene after incubating IOSE-80 cells with AS1411-P21.

[0024] Figure 8 This is a schematic diagram of the experimental results of detecting the effects of DNA-RNA hybrid nanocarriers on the protein expression of p21, TIGIT, and NCL by Western Blot in Example 6 of the present invention. Among them, A is the Western Blot image of p21 and TIGIT genes in SKOV3 cells treated with hybrid nanocarriers; B is the analysis of the Western Blot gray value of SKOV3 cells after treatment with hybrid nanocarriers; C is the Western Blot image of p21 and TIGIT genes in IOSE-80 cells treated with hybrid nanocarriers; D is the analysis of the Western Blot gray value of IOSE-80 cells after treatment with hybrid nanocarriers; E is the Western Blot image of the NCL protein expression in SKOV3 cells and IOSE-80 cells; F is the analysis of the Western Blot gray value of SKOV3 cells and IOSE-80.

[0025] Figure 9 This is a schematic diagram of the experimental results of detecting the apoptosis of tumor cells by flow cytometry in Example 7 of the present invention. Among them, A is the apoptosis rate of tumor cells in the control group and the experimental groups AS1411-P21 and AS1411-TIGIT after incubating with hybrid nanocarriers; B is the analysis of the apoptosis rate of tumor cells after incubating with hybrid nanocarriers. Specific implementation manners:

[0026] The present invention will be further described below through specific examples in combination with the accompanying drawings.

[0027] Example 1:

[0028] This example relates to a nanocarrier based on a DNA-RNA hybrid molecule, including a DNA-RNA hybrid molecule, a small RNA nucleic acid drug (target RNA), and an AS1411 aptamer. The AS1411 aptamer is covalently bonded to the DNA-3′ of the DNA-RNA hybrid molecule, and the target RNA molecule is covalently bonded to the RNA-3′ of the DNA-RNA hybrid molecule ( Figure 1 A).

[0029] The sequence of the AS1411 aptamer (abbreviated as AS1411) is reported in the reference, as shown in SEQ ID NO: 1; the sequence of the DNA in the DNA-RNA hybrid molecule (abbreviated as b) is shown in SEQ ID NO: 2, and its 3′ end is connected to the 5′ end of the aptamer to form a DNA-RNA hybrid molecule-AS1411 aptamer (abbreviated as AS1411-b), and the sequence is shown in SEQ ID NO: 7. The sequence of the RNA in the DNA-RNA hybrid molecule (abbreviated as a) is the 16 bases at the 5′ end of SEQ ID NO: 3, and the remaining 3′ bases of the SEQ ID NO: 3 sequence are the connected drug RNA molecules.

[0030] The bases U and C of the target RNA are both 2′-fluoro modified, that is, the -OH at the 2nd position of UTP and CTP is replaced by -F.

[0031] The target RNA is p21 gene saRNA (abbreviated as p21 saRNA) or TIGIT gene siRNA (abbreviated as TIGIT siRNA).

[0032] The p21 gene saRNA includes an antisense strand (3′ end of P21-a) and a sense strand (P21-c). The reference reports that its sequence is the 21 bases at the 3′ end of SEQ ID NO: 3 and SEQ ID NO: 4, and the UTP and CTP therein are 2′-fluoro modified; the TIGIT gene siRNA includes an antisense strand (3′ end of TIGIT-a) and a sense strand (TIGIT-c), and its sequence is the 23 bases at the 3′ end of SEQ ID NO: 5 and SEQ ID NO: 6, and the UTP and CTP therein are 2′-fluoro modified. NC-a and NC-c are used as negative controls, as Figure 1 B.

[0033] Table 1. Related DNA and RNA sequences

[0034]

[0035] *Note: The small letter f represents that the nucleotide adjacent to the right of this letter f is a 2'-fluoro modified nucleotide

[0036] The sequences described in Table 1 were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0037] The method for preparing the nanocarrier based on the DNA-RNA hybrid molecule is as follows: Mix AS1411-b with the antisense strand a and the sense strand c of the corresponding target RNA in equimolar amounts, and then mix with an equal volume of hybridization buffer (2xPBS - 2 mM MgCl2). Heat in a thermal cycler at 80 °C for 3 min, 50 °C for 5 min, 25 °C for 15 min, and cool to 4 °C for 20 min on a PCR instrument to complete the preparation of the nanocarrier based on the DNA-RNA hybrid molecule. The prepared hybrid nanocarrier can be stored in a low-temperature environment at 4 °C for standby. The hybrid nanocarrier is preferably stored at 4 °C for no more than 48 h. Taking the assembly of AS1411-P21 as an example: Mix AS1411-b with the corresponding P21-a and P21-c in equimolar amounts to calculate the volume, and then add the same volume of hybridization buffer (2xPBS - 2 mM MgCl2). The 5′ end of P21-c can also be labeled with FAM green fluorescence for convenience in subsequent experiments. The usage amounts of the raw materials in the preparation process are shown in Table 2. When assembling AS1411-TIGIT, AS1411-b is mixed with the corresponding TIGIT-a and TIGIT-c in equimolar amounts.

[0038] Table 2. Dosages of each component of the DNA-RNA hybrid nanocarrier

[0039]

[0040] Verify whether the DNA-RNA hybrid nanocarrier is successfully assembled by TBM gel electrophoresis. The observation of the DNA-RNA hybrid strand is more obvious under a specific buffer system. Prepare a specific TBM electrophoresis buffer and an 8% TBM polyacrylamide gel (Table 3), and perform electrophoresis at a constant voltage of 100 V. Since the TBM gel is relatively fragile and has a large adhesiveness, the gel should be carefully removed after electrophoresis. Soak it in the TBM electrophoresis solution containing ethidium bromide solution or enzyme-free water for 30 min for staining, and observe the assembly situation. Electrophoresis is performed on AS1411-b, P21-a, P21-c, P21-a + P21-c, P21-a + AS1411-b, AS1411-b + P21-c, and P21-a + AS1411-b + P21-c, and the results are as Figure 1 shown in C.

[0041] Table 3. Volumes of each component of the 8% TBM polyacrylamide gel electrophoresis

[0042]

[0043] As can be seen from Figure 1 C, after adding AS1411-b and the corresponding P21-a and P21-c in equimolar amounts, successful assembly is achieved, indicating that the AS1411-P21 hybrid nanocarrier is successfully prepared.

[0044] The preparation methods of P21-a+P21-c, P21-a+AS1411-b, and AS1411-b+P21-c involved in this example are the same as those of AS1411-P21. The specific parameters are shown in Table 2.

[0045] Example 2:

[0046] This example involves the serum stability experiment of DNA-RNA hybrid nanocarriers. The DNA-RNA hybrid nanocarriers are AS1411-P21 prepared in Example 1. The specific steps are as follows: Take 1 nmol of DNA-RNA hybrid nanocarriers and add fetal bovine serum (FBS) to make the whole system contain 10% FBS. Samples are taken at time gradients of 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h respectively. The sampling amount each time is 0.1 nmol. Each sampled sample is immediately stored in a -80 °C refrigerator. After the sampling at 72 h is completed, TBM gel electrophoresis is carried out, and electrophoresis, staining, and photographing are performed as described in Example 1 and recorded. The results are as Figure 2 shown.

[0047] As Figure 2 can be seen, by sampling at different times, it is found that the serum stability of DNA-RNA hybrid nanocarriers is very high after 2'-F modification. Hybrid nanocarriers still exist at 72 h. The bottom band of the TBM gel deepens, indicating that the DNA part has been degraded, but the RNA part still exists intact. This experimental result shows that under the protection of RNA fluorination modification, the ability of DNA-RNA hybrid nanocarriers to resist nuclease degradation is significantly improved.

[0048] Example 3:

[0049] This example involves the experiment of detecting the cellular uptake of DNA-RNA hybrid nanocarriers by fluorescence microscopy and laser confocal microscopy. The specific steps are as follows:

[0050] (1) Prepare a 24-well plate, count the well-grown ovarian cancer cells SKOV3, place cell slides in advance, and inoculate cell suspension (1×10 5 cells / well) in the 24-well plate, and pre-culture in an incubator for 24 h;

[0051] (2) Add the DNA-RNA hybrid nanocarriers (AS1411-P21 carrying FAM green fluorescence label) prepared in Example 1 to the complete DMEM medium containing 10% FBS, and add it to each well in the form of medium replacement, keeping the nanocarrier concentration at 1 μM, and culture for 48 h;

[0052] (3) For the fluorescent DNA-RNA hybrid nanocarriers, after the cells are taken out of the incubator, the operation should be carried out in a dark room. Aspirate and discard the culture medium, and rinse the cells three times repeatedly with phosphate (PBS) buffer solution;

[0053] (4) Fix with 4% paraformaldehyde solution for 10 min;

[0054] (5) Rinse the cells three times with PBS buffer solution;

[0055] (6) Prepare PBS buffer solution containing 0.5% Triton X-100, and use this buffer solution to permeabilize the cells at room temperature for 10 min;

[0056] (7) Rinse the cells three times with PBS buffer solution;

[0057] (8) Take out the stock solution of red fluorescent phalloidin. For a 24-well plate, mix and dilute 1-2 μl of the stock solution of red fluorescently labeled phalloidin with 200 μl of PBS buffer solution for staining one well, and incubate at room temperature for 20 min for staining;

[0058] (9) Rinse the cells three times with PBS buffer solution;

[0059] (10) Take out the ready-to-use DAPI staining solution from the -20 °C refrigerator in advance. After the staining solution is fully melted, take an appropriate amount of the staining solution and incubate with the cells at room temperature for 10 min for nuclear fluorescence staining;

[0060] (11) Rinse the cells three times with PBS buffer solution;

[0061] (12) Carefully take out the coverslip from the 24-well plate with a syringe needle. Drop 5-10 μl of fluorescent anti-quenching mounting medium on the coverslip in advance, and carefully mount the coverslip;

[0062] (13) Observe the binding and internalization of the DNA-RNA hybrid nanocarriers with SKOV3 cells under an ordinary upright fluorescence microscope and a laser confocal microscope; use the naked small RNA and DNA-RNA hybrid molecules carrying only green fluorescent labels as the control group (NC) for parallel experiments. The principle is as Figure 3 and the results are as Figures 4-5 shown.

[0063] It can be found from Figure 4 that after 48 h, the nanocarriers in the experimental group fully bind to the cell surface and a large number of fluorescently labeled carriers enter the cells, while in the control group, the naked small RNA P21 carrying green fluorescent labels without using the AS411-DNA-RNA hybrid nanocarriers cannot be delivered into the cells, indicating that the nanocarriers have a very effective targeting delivery function.

[0064] It can be found from Figure 5It can be seen that the green area has the same morphology as the cells, and the nanocarriers are evenly distributed on the cell surface and near the cytoplasm, with a small amount distributed near the nucleus.

[0065] Example 4:

[0066] This example involves the cytotoxicity experiment of DNA-RNA hybrid nanocarriers on cells, using the CCK-8 assay. The specific steps are as follows:

[0067] (1) Count the cells with a cell counting plate, seed them in a 96-well plate, add human ovarian cancer SKOV3 cells to each well according to the amount specified in the instruction manual of the transfection reagent purchased from Sangon Biotech (Shanghai) Co., Ltd., and pre-culture them in a constant temperature incubator with DMEM complete medium containing 10% FBS for 24 h;

[0068] (2) Synthesize DNA-RNA hybrid nanocarriers in vitro. Divide them into a blank control group (untreated), an NC group (negative control group of nanocarriers without adding AS1411 aptamer), an AS1411-NC (control group carrying negative small RNA (sequence from Sangon Biotech (Shanghai) Co., Ltd.)), a TF-P21 (group transfected with p21 saRNA using the transfection reagent), a TF-TIGIT (group transfected with TIGIT siRNA using the transfection reagent), a TF-NC (transfection reagent control group), an AS1411-P21 group, and an AS1411-TIGIT group; Add the corresponding transfection reagent complexes and DNA-RNA hybrid nanocarriers (final concentration 1 μM) to the wells respectively;

[0069] (3) Mix the CCK-8 reagent and serum-free DMEM medium in a ratio of 1:9 to prepare a 10% CCK-8 dilution;

[0070] (4) Incubate in the incubator, take out the culture plate at time gradients of 0 h, 24 h, 48 h, and 72 h respectively, aspirate the medium, and add the CCK-8 dilution prepared in the previous step in the form of medium replacement;

[0071] (5) Incubate the taken-out 96-well cell culture plate in a 37 °C constant temperature incubator for 2 h, take it out after incubation, and place it on an enzyme-labeled instrument for detection;

[0072] (6) Use an enzyme-labeled instrument to detect the absorbance of each well at 450 nm, record the data and perform statistical analysis, and the results are shown in Table 4 and Figure 6 .

[0073] Table 4 Growth inhibitory effects of hybrid nanocarriers and transfected small RNAs on human ovarian cancer SKOV3 cells (`χ±s, n = 6)

[0074]

[0075] From Table 4 and Figure 6 it can be seen that compared with the blank control group, incubation with AS1411-P21 and AS1411-TIGT inhibited the proliferation of SKOV3 cells, and the difference was statistically significant (*P<0.05, **P<0.01); transfection with p21 saRNA and TIGIT siRNA inhibited the proliferation of SKOV3 cells compared with the blank control group, and the difference was statistically significant ( # P<0.05, ## P<0.01). This indicates that the DNA-RNA hybrid nanocarrier has lower cytotoxicity compared to traditional transfection reagents.

[0076] Example 5:

[0077] This example involves a real-time fluorescence quantitative PCR experiment to detect the expression of p21 and TIGIT genes by DNA-RNA hybrid nanocarriers. The specific steps are as follows:

[0078] 1. Pretreatment of cells

[0079] Count according to the rules using a hemocytometer, and seed human ovarian cancer SKOV3 cells in a 6-well plate and pre-culture them in a constant temperature incubator for 24 h; add the assembled DNA-RNA hybrid nanocarrier to 1 μM, carrying p21 saRNA and TIGIT siRNA respectively as the experimental groups (AS1411-P21 and AS1411-TIGIT); the simple DNA-RNA hybrid nanocarrier is the negative control group (NC, 1 μM), mixed with DMEM complete medium and added in the form of medium change; the simple transfection reagent is the blank control group (Control), and operate according to the transfection reagent instruction manual. Incubate at a constant temperature for 48 h.

[0080] 2. Extraction of total cellular RNA

[0081] Operate according to the Trizol total RNA extraction kit instruction manual.

[0082] 3. Reverse transcription of RNA into cDNA

[0083] (1) According to the reaction system in Table 5, remove the residual genomic DNA in the tumor cell RNA extract;

[0084] Table 5 Reaction system for genomic DNA removal

[0085]

[0086] Put into a PCR instrument

[0087] ↓42°C for 2 min

[0088] 4°C ∞

[0089] (2) Prepare cDNA by performing reverse transcription reaction according to the reaction system in Table 6;

[0090] Table 6 Reverse transcription system

[0091]

[0092] Put it into the PCR instrument

[0093] ↓ 37°C for 15 min, 85°C for 5 s

[0094] 4°C ∞

[0095] (3) Use the obtained cDNA product for qPCR or store it in an environment of -20°C.

[0096] 4. Detection of the expression of p21 and TIGIT in tumor cells by qPCR

[0097] After the reverse transcription operation is completed, using the cDNA as a template, strictly according to the operation instructions, perform real-time fluorescence quantitative PCR (qPCR) detection on the samples to detect the expression of TIGIT and p21 genes in tumor cells. The reaction system is shown in Table 7; the detection results are shown in Figure 8 .

[0098] Table 7 Real-time fluorescence quantitative PCR reaction system

[0099]

[0100] Each sample is subjected to 3 repeated experiments, and the relative mRNA expression level in the sample is represented by 2 -ΔΔct indicated.

[0101] Table 8 Primer sequences

[0102]

[0103] From Figure 7 A and Figure 7 B, the results of real-time fluorescence quantitative PCR show that at the mRNA level, the DNA-RNA hybrid nanocarrier AS1411-P21 carrying p21 saRNA effectively increased the expression of the p21 gene, and the DNA-RNA hybrid nanocarrier AS1411-TIGIT carrying TIGIT siRNA effectively decreased the expression of the TIGIT gene, and the difference was statistically significant (*P < 0.05, **P < 0.01); and the mRNA expression level was significantly higher or lower than that of the control group.

[0104] Treat normal ovarian IOSE-80 cells by the same method as above, and the results are as shown in Figure 7 C and Figure 7As shown in Figure D, the treatments of AS1411-P21 and AS1411-TIGIT had no effect on the expression of p21 and TIGIT genes in normal ovarian IOSE-80 cells, and the differences were not statistically significant.

[0105] Example 6:

[0106] This example involves an experiment on detecting the protein expression of p21, TIGIT, and NCL by Western Blot using DNA-RNA hybrid nanocarriers. The protein expressions of p21 and TIGIT genes in SKOV3 cells and IOSE-80 cells were detected by Western Blot method.

[0107] 1. Extraction of total protein from tumor cells

[0108] (1) Pretreat SKOV3 cells according to the method of Example 5;

[0109] (2) Take out the six-well plate from the incubator, discard the old culture medium, and carefully wash the cells three times with PBS buffer;

[0110] (3) Prepare tissue cell lysate. Mix 1152 μl of RIPA lysate, 12 μl of PMSF, 12 μl of phosphatase inhibitor A solution, 12 μl of phosphatase inhibitor B solution, and 12 μl of Cocktail protease inhibitor (1:100) to make 1.2 ml of cell lysate;

[0111] (4) Add the prepared cell lysate into the six-well plate, 200 μl per well. Carefully scrape the cells with a cell scraper, transfer the scraped cells and lysate to a clean 1.5 ml centrifuge tube, and lyse on ice for 30 min. To ensure complete lysis, the centrifuge tube should be inverted up and down every 10 min;

[0112] (5) Centrifuge at 12000 rpm for 15 min at 4℃ in a low-temperature centrifuge, transfer the supernatant to a new EP tube to obtain a new total protein sample, and store it at -20℃ for later use;

[0113] (6) After lysis, centrifuge the sample at 12,000 rpm for 15 min at 4℃ in a low-temperature centrifuge to obtain a white precipitate and supernatant. Transfer the supernatant in the centrifuge tube to a new 1.5 ml centrifuge tube to obtain the total cell protein sample;

[0114] (7) Add an appropriate amount of Protein loading buffer to the obtained total protein sample and heat it at 100℃ for 10 min in a water bath to fully denature it;

[0115] (8) Prepare protein polyacrylamide gel according to the following formula:

[0116] Table 9 Concentrating Gel (5%)

[0117]

[0118] Table 10 Separating Gel (12%)

[0119]

[0120]

[0121] 2. Protein Immunoblotting (Western Blot) Experiment

[0122] (1) Protein Loading: Install the gel plate in advance according to the electrophoresis instrument manual. Use a pipette to aspirate equal amounts of total protein samples from each group, mix well by pipetting up and down, and add them into the corresponding gel wells. Do not use the wells at the edges of the gel plate;

[0123] (2) Electrophoresis: The liquid level of the Western Blot electrophoresis buffer must cover the upper edge of the gel. First, run the protein at a constant voltage of 80V until the protein runs through the concentrating gel and the protein marker starts to separate, then increase the voltage to 120V for constant voltage electrophoresis until the target protein is completely separated. Then, turn off the electrophoresis instrument switch to stop electrophoresis;

[0124] (3) Transfer: Use the wet transfer method. When electrophoresis is completed, turn off the power supply, carefully remove the gel plate, cut the gel corresponding to the position of the target protein according to the position of the protein marker, and place it in the transfer buffer for 15 minutes of equilibration. Cut a PVDF membrane of the same size as the gel corresponding to the target protein band, mark the direction by cutting a corner, soak the PVDF membrane in analytical pure methanol for 5 minutes of activation, and then place the PVDF membrane in the transfer buffer for 15 minutes of full equilibration; Lay a black sponge flat on the transfer cassette, then lay a layer of transfer filter paper on the sponge, use a test tube to drive out the air bubbles in the sandwich, then carefully place the cut polyacrylamide gel on the filter paper, then tightly attach the PVDF membrane to the gel, cover the filter paper and sponge and drive out the air bubbles in the sandwich again. Close the transfer cassette tightly, install it into the electrotransfer tank according to the corresponding positive and negative poles, with the PVDF membrane on the positive electrode side and the polyacrylamide gel on the negative electrode. Add an appropriate amount of transfer buffer until it covers the transfer cassette, place the entire electrotransfer system in an ice box, set a constant current of 300mA on the power supply for transfer, and the transfer time depends on the size of the corresponding protein;

[0125] (4) Blocking: After transfer is completed, place the labeled PVDF membrane in a 3% BSA blocking solution and block for 2 hours;

[0126] (5) Membrane Washing: Wash the PVDF membrane with TBST buffer on a low-speed shaker for 15 - 20 minutes;

[0127] (6) Incubate with primary antibody: Generally, an antibody incubation box is used for incubating antibodies. According to the instruction manuals of the purchased antibodies (Anti-β-actin antibody: Wuhan Boster Biological Technology Co., Ltd.; p21 antibody, TIGIT antibody: Wuhan Abbkine Biological Technology Co., Ltd.; NCL antibody: Affinity Antibody Company), dilute the antibodies with a universal antibody diluent, then place the corresponding PVDF membrane into the grid containing the corresponding antibody solution, incubate at room temperature on a low-speed shaker for 2 h, and then incubate overnight in a refrigerator at 4 °C;

[0128] (7) Wash the membrane: Wash the PVDF membrane with TBST buffer for 15 - 20 min;

[0129] (8) Incubate with secondary antibody: According to the instruction manual of the purchased antibody, dilute the antibody with a universal antibody diluent, and then incubate with the corresponding PVDF membrane on a low-speed shaker at room temperature for 1 h;

[0130] (9) Wash the membrane: Wash the PVDF membrane with TBST buffer on a low-speed shaker for 15 - 20 min. On the shaker, wash the PVDF membrane 3 times with TBST buffer, 10 min each time;

[0131] (10) Develop the image: Take equal volumes of solution A and solution B of the ECL luminescent solution, mix them evenly to prepare the developing solution, spread it evenly on the front side of the PVDF membrane, incubate for 1 - 2 min, and then place it on the imaging system for developing and photographing analysis; The results are as Figure 8 shown.

[0132] As can be seen from Figure 8 A, 8B, the use of DNA-RNA hybrid nanocarriers can effectively deliver the target small RNA and up-regulate and down-regulate the expression of p21 gene and TIGIT gene at the protein level, respectively, with a relatively significant effect, and the difference in gray value analysis is statistically significant ( Figure 8 A, 8B).

[0133] Treat normal ovarian cells IOSE-80 with the above method, and the results are shown in Figure 8 C, 8D. It can be seen that the hybrid nanocarriers have no obvious effect on the expression of p21 and TIGIT proteins in normal cells. Among them, the expression level of NCL protein in ovarian cancer cells SKOV3 is higher than that in normal ovarian cells IOSE-80, and the difference is statistically significant (*P < 0.05, **P < 0.01) Figure 8 E, 8F.

[0134] Example 7:

[0135] This example involves an experiment on detecting the apoptosis rate of cells by flow cytometry, and the specific steps are as follows:

[0136] (1) Treat ovarian cancer cells SKOV3 cells according to Example 5 and group them; the test samples include cells without fluorescence reagent staining treatment as negative controls, and cells stained with Annexin V-FITC and PI alone for adjustment compensation. The experiment is divided into a control group and an experimental group. The control group is cells not incubated with the hybrid nanocarrier (Control group) and cells of the random sequence group without the AS1411 aptamer (NC group); the experimental group is the AS1411-P21 group and the AS1411-TIGIT group;

[0137] (2) Collect cells: Take the cells out of the incubator, wash the cells with PBS, collect the PBS for each wash and centrifuge at 1000 rpm for 5 min, digest the cells with trypsin without EDTA, carefully collect the cells after digestion, centrifuge at 1000 rpm at 4 °C for 5 min, and discard the supernatant;

[0138] (3) Wash the cells: Prepare pre-cooled PBS buffer at 4 °C in advance, wash the cells twice with it, centrifuge at 1000 rpm at 4 °C for 5 min each time, and discard the supernatant;

[0139] (4) Resuspend the cells: Add 100 μl of 1xBinding Buffer to the cells washed in the previous step, and gently blow to make a single-cell suspension;

[0140] (5) Cell staining: Add 5 μl of Annexin V-FITC and 5 μl of PI Staining Solution, and gently blow to mix; incubate in the dark at room temperature for 10 min; add 400 μl of 1xBinding Buffer and gently mix; after staining, the samples are detected with a flow cytometer within 1 h; the results are as Figure 9 shown.

[0141] It can be seen from Figure 9 that after treatment with AS1411-P21 and AS1411-TIGIT, the apoptosis rate of tumor cells increased significantly, and the difference was statistically significant ( * P < 0.05, ** P < 0.01), and the apoptosis rate of tumor cells treated with AS1411-TIGIT was greater than that of tumor cells treated with AS1411-P21.

Claims

1. A nanocarrier based on DNA-RNA hybrid molecules, characterized in that, It includes a DNA-RNA hybrid molecule, the AS1411 aptamer, and the target RNA. The DNA-RNA hybrid molecule is the connecting bridge between the AS1411 aptamer and the target RNA; the AS1411 aptamer is covalently bonded to DNA-3' in the DNA-RNA hybrid molecule, and the target RNA molecule is covalently bonded to RNA-3' in the DNA-RNA hybrid molecule; The sequence of the DNA-RNA hybrid molecule is shown by the 16 bases at the 5′ end of 5'-GGATCAATCATGGCAA-3' and 5'-UUGCCAUGAUUGAUCC-UACUUGGAGAAUGAGUUGGUU-3'; The bases U and C of the target RNA are 2′-fluoro modified; The target RNA is TIGIT gene siRNA or p21 gene saRNA; The sequence of the TIGIT gene siRNA is shown by the 23 bases at the 3′ end of 5'-UUGCCAUGAUUGAUCC-GGAAUGAUGACAGGCACAAUAUU-3' and 5'-UAUUGUGCCUGUCAUCAUUCCUU-3'; The sequence of the p21 gene saRNA is shown by the 21 bases at the 3′ end of 5'-UUGCCAUGAUUGAUCC-UACUUGGAGAAUGAGUUGGUU-3' and 5'-CCAACUCAUUCUCCAAGUAUU-3'; 2. The preparation method of the nanocarrier based on DNA-RNA hybrid molecules according to claim 1, wherein First, synthesize the required target RNA and DNA-RNA hybrid molecule-AS1411 aptamer, and perform 2′-fluoro chemical modification on the bases U and C of the target RNA; mix the DNA-RNA hybrid molecule-AS1411 aptamer and the corresponding target RNA in equimolar amounts, then mix with an equal volume of hybridization buffer, heat in a thermal cycler at 80 °C for 3 min, 50 °C for 5 min, 25 °C for 15 min, and cool to 4 °C on a PCR instrument for 20 min to complete the preparation of the DNA-RNA hybrid nanocarrier.

3. Use of the nanocarrier based on the DNA-RNA hybrid molecule according to claim 1 in the preparation of nucleic acid drugs.

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