A copolymer of RA16-A and DNA tetrahedral carrier and its preparation method and application

Abstract

The present invention discloses a copolymer of RA16-A and a DNA tetrahedron carrier, wherein the RA16-A sequence is RA16 with 20 poly A added to the 3' end, the nucleotide sequences of the four single-stranded DNAs of the DNA tetrahedron carrier are respectively as shown in SEQ ID NO: 1-4, and the molar ratio of RA16-A to the DNA tetrahedron carrier is 1:1. The present invention solves the problem that the bispecific RNA aptamer RA16 has a short circulation time and is easy to degrade in vivo. The RA16 loaded with a DNA nanostructure can significantly increase the circulation time of RA16 in cells and target tissues in vivo, increase the enrichment and survival time of RA16 in cells or tumor tissues, successfully achieve long-term enrichment of RA16 in tumor areas, and can be used as a tumor diagnostic reagent for in vivo imaging.

Description

Technical Field

[0001] The present invention belongs to the field of bionanomedicine, and specifically relates to a targeted delivery system. More specifically, the present invention relates to a copolymer of RA16-A and a DNA tetrahedron carrier, and a preparation method and application thereof. Background Art

[0002] Lung cancer is the most common cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) accounts for 85%–90% of lung cancer cases. Traditional cancer therapies, such as chemotherapy and radiotherapy, can cause severe side effects, including gastrointestinal discomfort, organ damage, and a significant impact on quality of life.

[0003] Currently, new therapies for non-small cell lung cancer have been most commonly used in the past few decades for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) for lung cancer treatment. These inhibitors have greatly reduced systemic toxicity and significantly improved patient survival compared to chemotherapy for patients with advanced non-small cell lung cancer containing EGFR mutations, so targeted therapy is very necessary.

[0004] Aptamers are a type of single-stranded oligonucleotide fragments (DNA or RNA) that can fold into a specific spatial structure in a biological environment. They can bind to specific targets with high affinity through spatial complementarity, electrostatic interaction, van der Waals forces, hydrogen bonds, and other interactions. Because aptamers have the specificity to bind to target substances, compared with antibodies, they have a smaller molecular weight, are easier to synthesize, and are non-immunogenic; compared with small interfering RNA (siRNA), which is also a nucleic acid, their action forms and scope of action are more diverse. Therefore, aptamers are very valuable for developing targeted diagnostic or targeted therapeutic preparations that specifically bind to target tissues. At the same time, aptamers have a small molecular weight, are easy to synthesize chemically, and are easy to store and transport. They have certain advantages over antibodies and small nucleic acids, which are also nucleic acids. Therefore, aptamers are widely used in in vitro diagnosis, in vivo imaging, and the development of targeted preparations. So far, RNA aptamer drugs have achieved good results in the diagnosis and treatment of some diseases. The FDA has approved pegaptanib (trade name Macugen), a nucleic acid aptamer against VEGF developed by Eyech / Pfizer, for the treatment of age-related macular degeneration.

[0005] In previous studies, our R&D team reported the first in vivo SELEX technology using a polyethylene glycol (PEG)-modified RNA library directly in nude mice bearing human non-small cell lung cancer, enriching the RNA aptamer RA16 (CN201610168486.9) with dual specific functions of targeting and tumor inhibition for human non-small cell lung cancer. This technology not only effectively reduces the nonspecific binding of the screening library to high-abundance non-target proteins in the previous in vitro screening process, but also improves the screening probability and pertinence of the target sequence specifically binding to the in situ tissue. Moreover, due to the introduction of PEG modification in the screening process, the screened RNA aptamers are easier to carry out various modifications and couplings, and are not easy to lose their biological properties during the modification process, and have great potential for later development. However, although we have partially modified RA16 and enhanced its tissue stability and half-life, due to the easy degradation and easy clearance of RNA itself, we still need to continue to explore and further improve its stability, drug loading capacity and biocompatibility, and further develop a nanoscale targeted drug delivery system that is more suitable for clinical use.

[0006] Although many aptamers have been screened out through SELEX technology, only a handful of aptamers have been used in clinical diagnosis and targeted therapy. Many aptamers that have been screened out in large quantities and at great cost cannot be truly applied to clinical diagnosis and treatment due to factors such as poor stability. Therefore, using drug delivery systems to increase the half-life of in vivo circulation and drug stability has become a feasible method. Compared with synthetic materials, the superior biocompatibility of carriers derived from endogenous materials is very beneficial in the development of nanomedicine. Therefore, using DNA nanomaterials with nanoscale size, high drug loading capacity, biocompatibility, high affinity for target recognition after adhering to aptamer molecules, and good cell internalization efficiency as carriers has great development prospects. Summary of the invention

[0007] Purpose of the invention: In view of the above-mentioned problems existing in the prior art, the main purpose of the present invention is to solve the defects of the prior art. The present invention provides a copolymer of RA16-A and DNA tetrahedron carrier, which is a targeted tumor-suppressing bispecific aptamer nanomedicine. The system uses the DNA tetrahedron structure as a DNA carrier, which is the smallest DNA nanocarrier unit with a fixed spatial structure. It is successfully loaded with the bispecific aptamer RA16 obtained by in vivo screening for targeting and inhibiting human non-small cell lung cancer.

[0008] To achieve the above object, the present invention provides a copolymer of RA16-A and a DNA tetrahedral carrier, wherein the RA16-A is prepared by adding 20 poly As at the 3' end of RA16, and the nucleotide sequences of the four single-stranded DNAs of the DNA tetrahedral carrier are respectively shown in SEQ ID NOs: 1-4, wherein any one of the four single-stranded DNAs has 20 poly Ts added at the 3' end, and the molar ratio of RA16-A to the DNA tetrahedral carrier is 1:1.

[0009] In a specific embodiment of the present invention, complementarity with RA16 is achieved by adding 20 poly Ts to the 3' end of the nucleotide sequence of SEQ ID NO: 4.

[0010] Preferably, any one of the four single-stranded DNAs and RA16-A is fluorescently labeled before copolymerization.

[0011] The fluorescent label is any one of FITC, Alexa 488, Cy3, Cy5 and Cy7. In the specific implementation, the labeled nucleotide chain is directly used to participate in annealing, and the fluorescent labeled copolymer is obtained by self-assembly.

[0012] The present invention further proposes a method for preparing the copolymer of RA16-A and DNA tetrahedron carrier, which is characterized by comprising the following steps:

[0013] (1) Synthesis of RA16-A: The corresponding template strand DA16-T was synthesized by PCR and then transcribed in vitro;

[0014] (2) Loading of RA16-A by DNA nanocarrier: Four complementary single-stranded DNAs were loaded with RA16-A through base complementarity by heating and then annealing in a PCR program to obtain a copolymer of RA16-A and DNA tetrahedral carrier, wherein the nucleotide sequences of the four single-stranded DNAs are shown in SEQ ID NOs: 1-4, respectively.

[0015] The annealing procedure is: 80-95°C for 10-20 min, 4°C for 30-40 min.

[0016] The present invention further proposes the use of the RA16-A and DNA tetrahedral carrier copolymer in the preparation of a diagnostic reagent targeting non-small cell lung cancer.

[0017] The present invention also proposes the use of the copolymer of RA16-A and DNA tetrahedron carrier in the preparation of a drug for inhibiting non-small cell lung cancer.

[0018] On the other hand, the present invention proposes the use of the above-mentioned RA16-A and DNA tetrahedral carrier copolymer in biological detection, in vivo imaging, gene carrier, and drug delivery.

[0019] Furthermore, the present invention provides a drug targeting non-small cell lung cancer, which is a complex of the above copolymer and any one of chemotherapeutic drugs, siRNA or monoclonal antibodies for non-small cell lung cancer.

[0020] Beneficial effects: The present invention mainly solves the problem of short in vivo circulation time and easy degradation of the bispecific RNA aptamer RA16. The use of RA16 loaded with DNA nanostructures can significantly increase the circulation time of RA16 in cells and target tissues in the body, increase the enrichment and survival time of RA16 in cells or tumor tissues, and successfully achieve long-term enrichment of RA16 in tumor areas, which can be used as a tumor diagnostic reagent for in vivo imaging. In addition, the nucleic acid aptamer itself can inhibit tumor cell growth. By coupling with DNA tetrahedrons, it can be further loaded with lung cancer chemotherapy drugs, siRNA, monoclonal antibodies, etc., to achieve long-term targeted drug delivery. The nanoparticles have been successfully synthesized and verified by establishing an animal model of non-small cell tumors to have the following advantages:

[0021] 1) The nanosystem obtained by the present invention is easy to prepare, the raw materials are easily available, the method is cleverly designed, the conditions are mild, the operation is simple, the yield is high, and the mass production can be expanded, and it can be effectively applied to industrial production;

[0022] 2) The nanoparticles obtained by the present invention are easily taken up by cells and have good biocompatibility, and are easy to achieve clinical transformation;

[0023] 3) Under the condition of intravenous administration, the targeting of RA16 can be ensured while improving the ability of RA16 to enrich in the tumor area and the plasma half-life;

[0024] 4) It can be further coupled and loaded with lung cancer chemotherapy drugs, siRNA, monoclonal antibodies, etc. to achieve long-term tumor-targeted drug delivery and increase the drug concentration and action time at the tumor site. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Agarose gel electrophoresis of DA16-T and RA16-A;

[0026] Figure 2 Schematic diagram of the preparation of DTA-16 nanodrug;

[0027] Figure 3 Native-page representation diagram of the DTA-16 synthesis process;

[0028] Figure 4 AFM stereogram;

[0029] Figure 5 Dynamic light scattering and Zeta potential characterization of DTA-16 nanoparticles;

[0030] Figure 6 Confocal microscopy of DTA-16 nanoparticle cellular uptake;

[0031] Figure 7 DTA-16 nanoparticle cell viability bar graph, where C: control; R: RA16; D: DTN; A: DTA-16;

[0032] Figure 8 Distribution of DTA-16 nanoparticles in vivo. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments. The embodiments will help to understand the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0034] On the one hand, the present invention provides a copolymer of RA16-A and DNA tetrahedral carrier, which specifically comprises the following steps:

[0035] 1) Synthesis and characterization of RA16-A

[0036] The RA16-A sequence was obtained by adding 20 poly A residues to the 3' end of RA16. The corresponding template chain DA16-T was synthesized by PCR and then transcribed in vitro.

[0037] 2) Loading of RA16-A by DNA nanocarrier

[0038] The PCR program was used to heat up and then anneal the four complementary oligonucleotide chains (called P1, P2, P3, P4-T, where P1-P4 is 64bp long and P4-T is 63+20bp) and RA16-A to complete the loading through base complementarity. This system is called DTA-16.

[0039] Among them, P1, P2, P3, and P4 each have a pairing region, and they are paired complementary to form a regular tetrahedron. The poly-T of P4 extends outward from the tetrahedron and is complementary to the poly A of RA16-A. They are paired together to form DTA-16 (such as Figure 2 shown).

[0040] 2) Characterization of DTA-16 nanoparticles

[0041] The successful synthesis of nanoparticles was confirmed by Native Page gel electrophoresis, the size distribution was measured by dynamic light scattering (DLS, Zetasizer Nano Instruments), the surface electrical properties were determined by zeta potential, and the size and morphology of nanoparticles were observed by atomic force microscopy (AFM).

[0042] The second aspect of the present invention includes determining the effect of a single carrier on cell viability at the cellular level, and the cell's ability to take up the nanoparticles. Specifically:

[0043] 1) The cytotoxicity of the nanoparticles was detected using CCK8 detection reagent.

[0044] 2) The nanoparticles are loaded with fluorescent groups and co-incubated with cells, and the uptake of the nanoparticles by cells is observed using a laser confocal microscope.

[0045] The third aspect of the present invention includes a method for diagnosing a nude mouse non-small cell lung cancer model using the nano drug shown in the invention as a fluorescent diagnostic probe, wherein a humanized tumor model is prepared by subcutaneous injection of human large cell lung cancer cells (H460 cells), and the tumor is imaged in vivo by intravenous injection of the nano drug. Specifically:

[0046] 1) Establishment of animal models of non-small cell tumors

[0047] The humanized non-small cell lung cancer model was established by subcutaneously injecting human large cell lung cancer cells (H460 cells) into 5-week-old Balb / c nude mice.

[0048] 2) In vivo fluorescence imaging

[0049] The nanoparticles were fluorescently labeled and the fluorescence distribution of the mice at different time points was observed using a small animal fluorescence imaging system after administration via the tail vein in model mice. Tissues were obtained 72 hours after administration to observe the fluorescence distribution of each tissue.

[0050] The nucleotide sequences involved in the following examples are shown in Table 1:

[0051] Table 1 Sequence table of nucleic acid chains

[0052]

[0053]

[0054] The present invention is described in detail below through specific embodiments.

[0055] Example 1 Synthesis and characterization of RA16-A.

[0056] (1) Preparation and characterization of DA16-T

[0057] DA16-T is the template chain of RA16-A, and the RA16-A sequence is derived from RA16 with 20 poly A residues added to the 3' end.

[0058] Prepare 1000 μL reaction system according to the conditions in Table 2:

[0059] Table 2

[0060]

[0061]

[0062] After the reaction system was prepared, one group was used as a blank control without adding DA16 template.

[0063] The reaction conditions are shown in Table 3:

[0064] Table 3

[0065]

[0066] After the PCR reaction was completed, agarose electrophoresis (120V, 40min) was performed to observe the product amplification effect. The target band was 123bp.

[0067] The PCR product was purified using the SanPrep column-based PCR product purification kit according to the manufacturer's instructions and the gel was run to verify the band position.

[0068] (2) In vitro transcription of RA16

[0069] RA16 with amino-modified side chains was obtained, and the transcription system is shown in Table 4:

[0070] Table 4

[0071]

[0072]

[0073] Place the reaction system in a 37°C water bath. After the overnight reaction, place at 95°C for 5 minutes to terminate in vitro transcription. Take 1 μL of in vitro transcription product and judge whether the transcription is successful by the brightness and migration position after the agarose electrophoresis well, and calculate the amount of RNA product. Use the phenol / chloroform extraction standard procedure to purify RA16, and finally add three times the volume of anhydrous ethanol, 1 / 10 volume of 3M NaAc (pH5.2), and 10 μL of nucleic acid precipitation agent. Place at -20°C or -80°C for precipitation for 2 hours before use, or store at -80°C for at least three months.

[0074] (3) RNA resuspension

[0075] The RNA after the above ethanol precipitation was first centrifuged at 4°C, 12000rpm for 10 minutes, and the supernatant was discarded. It was then washed once with 70% cold ethanol and centrifuged at 4°C, 12000rpm for 10 minutes. The supernatant was discarded and the RNA precipitate was placed at room temperature to dry. After the precipitate was basically dry, it was suspended in RNA refolding buffer.

[0076] The results are attached Figure 1 As shown, the correct position assignment of the DA16-T chain indicates successful amplification, and the correct position assignment of the RA16-A chain indicates successful transcription.

[0077] Example 2 Synthesis, characterization and drug loading capacity of DNA tetrahedrons.

[0078] (1) Synthesis and Native PAGE Characterization:

[0079] The concentration of each chain and RA16-A was tested by spectrophotometer, where the concentration of each chain ranged from 12nM to 25nM and the buffer was 1xTM buffer + Mg 2+ The ratio of each chain was 1:1:1:1 in equimolar amount, and P1, P2 double-chain structures, P1, P2, P3 triple-chain systems, P1+P2+P3+P4 (DTN) framework structure and P1+P2+P3+P4-T+RA16-A (DTA-16) were synthesized in 50 μL system respectively.

[0080] Among them, 1xTM buffer + Mg 2+ By 10xTM buffer + Mg 2+ Dilution was obtained. Among them, 10X TMbuffer was prepared by the following method: 968.8 mg Tris and 4.066 g MgCl2·6H2O were accurately weighed, poured into a 50 mL centrifuge tube, 40 mL deionized water was added, and the mixture was shaken and mixed, and the pH value was adjusted to 8 with glacial acetic acid.

[0081] The annealing schedule is shown in Table 5 below.

[0082] Table 5

[0083]

[0084] Prepare a 10% Native-page gel. Load each prepared sample onto the gel according to the standard gel running procedure: Use 4S red (Shanghai Biotechnology) staining solution, stain the gel in the dark for 20 minutes, and observe it in a gel imaging instrument.

[0085] The schematic diagram of the synthesis steps is shown in the attached Figure 2 The synthesis results are shown in the attached Figure 3As shown, the largest nucleic acid band in the marker is 500bp. We found that from P1, P1+P2 to DTN, DTA-16, the band gradually increased and eventually exceeded the position of the 500bp marker. In addition, a bright single band appeared in each lane, indicating that the structure was well synthesized. This result shows that the tetrahedron was successfully synthesized, and the connection with RA16 will not affect the integrity of the overall structure of the DNA tetrahedron. The half-life and in vivo stability of RA16 after complexing with DTN were significantly improved. In fact, each DNA chain from P1 to P4 can be modified with ploy-T.

[0086] (2) Atomic force microscopy characterization structure:

[0087] According to the above sample preparation steps, DTN and DTA-16 were synthesized in a 50 μL system. The two samples to be tested were diluted to a final concentration of 20 nM. After passing through a 0.22 μm filter membrane, 5 μL of the diluted sample was taken with a pipette and tilted onto the surface of the mica sheet to maximize the surface area. Oven drying, when the liquid on the surface of the mica sheet evaporates, the sample preparation is complete and can be tested immediately or stored for later use. Using an atomic force microscope, the surface flatness of the mica sheet is scanned to detect the appearance of the nanoparticles. It is worth noting that only the Z-axis of the AFM data is meaningful and represents the actual size.

[0088] As attached Figure 4 As shown, atomic force microscopy imaging shows that the DNA vector is a tetrahedral structure. From the brightness of the ordinate, it can be seen that the size of the DNA tetrahedron is about 5nm, and the height after connecting RA16 is about 8nm.

[0089] (3) DLS detection of particle size distribution and zeta potential

[0090] Use about 1 ml of 1×TM buffer + Mg 2+ Dilute the above sample 10-20 times, pipette and add it to a clean sample pool; use Malvern Nano ZS90 particle size and potential meter to measure particle size distribution and Zeta potential. Figure 5 As shown in Figure 2, DLS results show that after RA16 is connected to DTN, the hydrodynamic radius becomes larger and the distribution becomes narrower, which further proves that the DTN tetrahedron is successfully synthesized and RA16 is successfully connected to the DTN tetrahedron. Figure 5 As shown, the Zeta potential shows that the surface of the nanoparticles is negatively charged, which also proves that the synthesis is successful and the structure is relatively stable.

[0091] Example 3 Cytotoxicity and cellular uptake of DTA-16

[0092] (1) The specific experimental steps for cell uptake are as follows:

[0093] 1) The folding and pairing process of DTA-16 was completed according to the synthesis steps in Example 2.

[0094] 2) Labeling NHS-FITC fluorescence, add fluorescent dye (molar ratio 1: 10) to 1M NaHCO3 buffer system, incubate at 4℃ for 2h, and then ultrafilter with YM-3KDa~10KDa ultrafiltration tube to remove excess fluorescent dye. RA16 is labeled with water-soluble FITC-NHS using a similar method.

[0095] 3) Cell culture and plating

[0096] The cells were cultured in an incubator at 37°C and 5% CO2. When the confluence reached 90%, H460 cells (purchased from ATCC) were cultured at 5×10 4 ~1ⅹ10 5 The cells were subcultured at a density of 10 cells / well into confocal dishes and cultured for 24 h. The cells were observed under an inverted microscope. If the cells were in good condition and the cell number was appropriate, subsequent experiments could be performed.

[0097] 4) Drug administration

[0098] The culture medium in the culture dish was aspirated and discarded, and 200 μL PBS was added to wash two to three times. The 200 nM sample mixture (Control group RA16 group, DTN group, DTA-16 group) prepared with serum-free culture medium was mixed with a vortex mixer and added to the center of the confocal culture dish. After culturing for 4 hours, it was washed 1-3 times, and at least 200 μl of cell nuclear staining working solution (hoechst 33242) was added to each dish. After staining at 37°C for 15 minutes, it was washed three times with 1ⅹ PBS, and 200 μl~300 μl of phenol red-free culture medium was added to each dish. Observe and take pictures under a confocal microscope.

[0099] As attached Figure 6 As shown, the fluorescence of RA16 is FITC, the fluorescence of DTN is Cy3 (by labeling one of the nucleotide chains more than once before polymerization), and the blue is the fluorescence of the cell nucleus. The results show that DTA-16 does not affect the uptake of RA16 by cells, and can be used for intracellular targeted drug delivery therapy later.

[0100] (2) Vector safety testing

[0101] The cell density was 6.5 × 10 4 / mL BEAS-2B, H460; the density is 6.5×10 4 / mL of cell mixture was added to a 96-well plate, 100μL was added to each well, and 100μL PBS was added to each well around the 96-well plate. The 96-well plate with cells was placed in a CO2 incubator for 24h. Nanoparticles were synthesized according to the above method, and each sample prepared with serum-free medium was added according to the following groups: 1) Control+PBS, 2) DNA nanocage (i.e. DTN): the concentration of DTN in 100 μL system was 100nM, 3) the concentration of DTA-16 in 100μL system was 100nM, after incubation in the incubator for 6h, washed twice with serum-free medium, and 100μl serum-free medium containing 10μl CCK8 solution was added to each well to detect cell viability.

[0102] The results are as follows Figure 7 As shown, the ingested nanoparticles still maintain high cell activity, indicating that the particles have good safety and high biosafety.

[0103] Example 4 Distribution and tumor targeting of DTA-16 in vivo

[0104] (1) Establishment of animal model

[0105] H460 cells in the logarithmic growth phase were digested, centrifuged, resuspended in PBS, washed twice with PBS, and resuspended in serum-free 1640 medium to a cell concentration of 2×10 6 pcs / 100μl;

[0106] Five-week-old Balb / c nude mice were selected and 100 μl of cell suspension was injected subcutaneously under the axilla of the right forelimb of the mice. The formation of a skin papule was observed at the injection site and gently pressed with a sterile cotton swab to avoid tumor cell exudation.

[0107] (2) Sample preparation

[0108] Prepare each group of samples according to the above method, and label the water-soluble Cy5.5 NHS on RA16, prepare a 1mg / ml Cy5.5NHS DMSO solution, combine the system in the PCR tube in a 1.5ml EP tube, and label at room temperature for 1.5 to 2h in a 1MNaHCO3, pH=8.3-9.0 buffer environment at a molar ratio of RA16:Cy5.5 of 1:10. Purify the unlabeled fluorescent molecules according to the ultrafiltration method in Example 3.

[0109] (3) Drug administration and imaging

[0110] Physiological saline was added to the two groups of samples to prepare the drug solution, and 5 μl was taken out of the tube and diluted 10 times, and the exposure value of in vivo imaging was adjusted. The control group was injected with 100 μl of physiological saline through the tail vein; RA16 and DTA-16 groups were injected with 0.5 nmol, and the whole mouse was imaged at fixed exposure values ​​at time intervals of 0, 12, 24, 36, 48h, and 72h. After the fluorescence weakened, the mice were killed and the main tissues (brain, heart, liver, spleen, lung, kidney, muscle) and tumors were taken for material distribution analysis by in vitro fluorescence imaging.

[0111] As attached Figure 8 The results showed that both the RA16 group and the DTA group had a trend of tumor enrichment, but the DTA-16 group had a faster tumor enrichment rate than the RA16 group, and the in vivo imaging still had a sustained tumor enrichment ability after 48 hours. When the organs were taken after 72 hours, it was obvious that the fluorescence intensity of the enrichment in the tumor site of the DTA-16 group was brighter, proving that this system can help nucleic acid aptamers prolong the in vivo metabolism time and increase the half-life, thereby increasing the possibility of using RA16 aptamers as diagnostic probes.

[0112] In summary, the present invention provides a method for preparing and applying DNA nanomaterials coupled to RNA aptamers, and further applied as targeted diagnostic reagents, providing new means for the diagnosis and treatment of human non-small cell lung cancer, the method is simple in process, easy to operate, high in safety, low in cost, and short in cycle. In addition, the system can be further applied to lung cancer chemotherapy drugs, siRNA, monoclonal antibodies, etc., to form targeted drugs to achieve targeted treatment of lung cancer. The nano drug has a high loading rate, the DNA tetrahedron-aptamer copolymer is successfully prepared, and the preparation method is simple and reproducible. The system can be taken up by tumor cells, significantly improve the stability and half-life of the aptamer RA16, and has high biocompatibility through in vitro and in vivo experiments. At the same time, the system can couple a variety of fluorescent molecules to obtain fluorescent tetrahedron-aptamer copolymers for in vivo imaging of tumors. The in vivo imaging results show that the half-life of the fluorescent tetrahedron-aptamer using the system is about three times higher than that of the simple fluorescent aptamer.

[0113] The present invention provides a preparation idea and method for a targeted tumor-suppressing bispecific aptamer nanomedicine. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies. Sequence Listing <110> Suzhou Ark Biotechnology Co., Ltd. <120> A copolymer of RA16-A and DNA tetrahedral carrier and its preparation method and application <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 63 <212> DNA <213> P1 Nucleotide Sequence (Artificial Sequence) <400> 1 tgggtagtca gcgctaccac acacagaact ccctacgaca ttggcatgag atactcggag 60 acc 63 <210> 2 <211> 63 <212> DNA <213> P2 Nucleotide Sequence (Artificial Sequence) <400> 2 gtctccgagt atctcatgcc catttgtgcc tagggtatca cgctttatct gtagctcgcc 60 ccc 63 <210> 3 <211> 63 <212> DNA <213> P3 Nucleotide Sequence (Artificial Sequence) <400> 3 ggagttctgt gcttggatca cgcgtcggtt gttgtgatac cctaggcaca aatgatgtcg 60 tag 63 <210> 4 <211> 63 <212> DNA <213> P4 Nucleotide Sequence (Artificial Sequence) <400> 4 ggggcgagct acagataaag aacaaccgac gcgtgatcca atgtggtagc gctgactacc 60 cac 63 <210> 5 <211> 83 <212> DNA <213> P4-T Nucleotide Sequence (Artificial Sequence) <400> 5 ggggcgagct acagataaag aacaaccgac gcgtgatcca atgtggtagc gctgactacc 60 cacttttttt tttttttttt ttt 83 <210> 6 <211> 44 <212> DNA <213> 16-TRP Nucleotide Sequence (Artificial Sequence) <400> 6 ctactgacgt gatgcaatgc actctttttt tttttttttt tttt 44 <210> 7 <211> 34 <212> DNA <213> FP nucleotide sequence (Artificial Sequence) <400> 7 cactaatacg actcactata gggagagaac aatg 34 <210> 8 <211> 103 <212> DNA <213> DA16 Nucleotide Sequence (Artificial Sequence) <400> 8 cactaatacg actcactata gggagagaac aatgacctgc ggtgccaagc cgtcgggtta 60 tgttgatctc ctcaaggacg agtgcattgc atcacgtcag tag 103

Claims

1. A copolymer of RA16-A and DNA tetrahedral carrier, characterized in that: The RA16-A is a DNA tetrahedron vector with 20 poly As added to the 3' end of RA16. The nucleotide sequences of the four single-stranded DNAs of the DNA tetrahedron vector are shown in SEQ ID NOs: 1-4, respectively, wherein any one of the four single-stranded DNAs has 20 poly Ts added to the 3' end, and the four single-stranded DNAs each have a pairing region, which are complementary and paired to form a regular tetrahedron. The added 20 poly-Ts extend outward from the tetrahedron and are complementary to the 20 poly As of RA16-A, and are paired and combined together. The molar ratio of RA16-A to the DNA tetrahedron vector is 1:1, and any one of the four single-stranded DNAs and RA16-A is fluorescently labeled before copolymerization.

2. The RA16-A and DNA tetrahedral carrier copolymer according to claim 1, characterized in that: The fluorescent label is any one of FITC, Alexa 488, Cy3, Cy5 and Cy7.

3. The method for preparing the copolymer of RA16-A and DNA tetrahedral carrier according to claim 1, characterized in that: The steps include: (1) Synthesis of RA16-A: The corresponding template strand DA16-T was synthesized by PCR and then transcribed in vitro; (2) Loading of RA16-A by DNA nanocarrier: Four complementary single-stranded DNAs were loaded with RA16-A through base complementarity by heating and annealing in a PCR program to obtain a copolymer of RA16-A and DNA tetrahedral carrier, wherein the nucleotide sequences of the four single-stranded DNAs are shown in SEQ ID NOs: 1-4, respectively.

4. The method according to claim 3, characterized in that The annealing procedure is: 80~95℃ 10~20 min, 4℃ 30~40 min.

5. Use of the copolymer of RA16-A and DNA tetrahedron carrier according to claim 1 in the preparation of a diagnostic reagent targeting non-small cell lung cancer.

6. Use of the RA16-A and DNA tetrahedral carrier copolymer according to claim 1 in the preparation of biological detection, in vivo imaging, gene carrier, and drug delivery products.

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