Nucleic acid aptamers targeting p53-R175H and their protein-targeted degradation chimeras and their applications

Abstract

The present invention belongs to the field of biomedicine technology, and relates to a nucleic acid aptamer targeting p53-R175H and its protein targeted degradation chimera and application. A protein targeted degradation chimera DNA nucleic acid aptamer targeting p53-R175H, wherein the DNA nucleic acid aptamer is connected to a ligand of an E3 ubiquitin ligase through a connecting molecule, and the DNA nucleic acid aptamer is a nucleic acid aptamer targeting p53-R175H. The protein targeted degradation chimera targeting p53-R175H provided by the present invention has higher affinity and high serum stability, thereby realizing precision medicine for p53-R175H mutant tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and relates to a nucleic acid aptamer targeting p53-R175H and a protein-targeted degradation chimera thereof and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] TP53 is the most commonly mutated gene, and most mutations are missense mutations, which are usually clustered in several hotspots within the DNA binding domain of the p53 protein, including R175, G245, R248, R249, R273, and R282. These hotspot mutations not only eliminate the tumor suppressor function of wild-type p53, but also acquire oncogenic functions to promote lesion transformation, cancer growth, metastasis, and drug resistance. Wild-type p53 is rapidly degraded under physiological conditions, but p53 mutants are highly expressed and abnormally stable in cancer cells. Therefore, specific degradation of p53 mutants is a promising cancer treatment strategy. Although targeting p53 mutants is hailed as the holy grail of precision medicine, there are still no related drugs on the market.

[0004] The p53-R175H mutation has the highest incidence in various types of cancer. Compared with other mutants, p53-R175H is more likely to promote tumor initiation, metastasis and drug resistance, leading to the worst prognosis of cancer. Proteolysis targeting chimeras (PROTACs) are heterogeneous bifunctional molecules that recruit target proteins with one molecule and E3 ligases with another molecule, and then induce ubiquitination and degradation of target proteins through the ubiquitin proteasome system. However, according to the inventors' research, PROTACs directly targeting p53 mutants have not been reported yet, and the main reason is that it is difficult to find suitable ligands for p53 mutants. Nucleic acid aptamers are short single-stranded oligonucleotides generated by artificial system evolution method (SELEX) of ligand index enrichment. Although there are RNA nucleic acid aptamers that specifically target p53-R175H, the aptamers are extremely unstable in serum, which seriously limits their scope of application. Summary of the invention

[0005] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a nucleic acid aptamer targeting p53-R175H and its protein targeted degradation chimera and application. The protein targeted degradation chimera targeting p53-R175H provided by the present invention has higher affinity and high serum stability, thereby achieving precision medicine for p53-R175H mutant tumors.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] On the one hand, a nucleic acid aptamer targeting p53-R175H is shown in SEQ ID NO.1.

[0008] On the other hand, a method for modifying the above-mentioned nucleic acid aptamer targeting p53-R175H comprises the following steps:

[0009] Obtaining the sequence of the RNA aptamer targeting the p53-R175H mutation and constructing the three-dimensional conformation of the RNA aptamer targeting the p53-R175H mutation;

[0010] The molecular dynamic simulation method was used to analyze the binding sites of RNA aptamers and p53-R175H mutants, and the key amino acid residues and nucleotides for the interaction between RNA aptamers and p53-R175H mutants were obtained;

[0011] On the basis of maintaining the three-dimensional structure of the RNA aptamer, the nucleotides are replaced one by one, and the nucleic acid aptamer targeting p53-R175H is obtained according to the binding of the replaced aptamer to the p53-R175H mutant.

[0012] In a third aspect, a protein targeted degradation chimera targeting p53-R175H includes a DNA nucleic acid aptamer, which is connected to a ligand of an E3 ubiquitin ligase via a connecting molecule, and the DNA nucleic acid aptamer is the above-mentioned nucleic acid aptamer targeting p53-R175H.

[0013] In a fourth aspect, a method for preparing the above-mentioned protein targeted degradation chimera targeting p53-R175H comprises the following steps:

[0014] synthesizing a single-stranded DNA containing an azide modification, wherein the single-stranded DNA comprises a nucleotide linker molecule and a DNA nucleic acid aptamer, and the azide group is located on the linker molecule;

[0015] The azide-modified single-stranded DNA is coupled with the ligand of the alkyne-modified E3 ubiquitin ligase through a click reaction to obtain the product.

[0016] In a fifth aspect, a chimera of the protein targeted degradation targeting p53-R175H is used in the preparation of drugs for degrading diseases related to p53-R175H mutation.

[0017] In a sixth aspect, an anti-tumor drug comprises an active ingredient and at least one inactive drug ingredient, wherein the active ingredient is the above-mentioned protein targeted degradation chimera targeting p53-R175H.

[0018] The beneficial effects of the present invention are:

[0019] The present invention uses the method of molecular dynamic simulation to guide the optimization of nucleic acid aptamers, and the obtained nucleic acid aptamers have higher affinity and significantly improved serum stability. Using the optimized aptamer as a ligand, the first chimeric degrader that selectively degrades the p53-R175H mutant was developed, thereby achieving precise treatment of patients with p53-R175H mutations.

[0020] The protein targeted degradation chimera targeting p53-R175H provided by the present invention has good degradation efficiency and anti-tumor cell effect, wherein 500nM chimera molecule can effectively and significantly inhibit the proliferation of p53-R175H mutant tumor cells, but has no inhibitory effect on p53 wild-type and other mutation site tumor cells. Moreover, simple nucleic acid aptamers can hardly inhibit the proliferation of p53-R175H mutant tumor cells at the same concentration; at the same time, the chimera molecule can significantly increase the sensitivity of p53-R175H mutant tumor cells to the chemotherapy drug cisplatin, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 The DNA aptamer p53m-DA and its azide-modified form (N 3 -p53m-DA) mass spectrum, A is p53m-DA, B is N 3 -p53m-DA;

[0023] Figure 2 The alkynyl-modified CRBN ligand in Example 1 of the present invention 13 C NMR spectrum;

[0024] Figure 3 The molecular simulation diagram of the binding of RNA nucleic acid aptamer p53m-RA and p53-R175H in Example 1 of the present invention, A is an interaction site analysis diagram, and B is a helical conformation diagram;

[0025] Figure 4 The molecular simulation diagram of the binding of p53m-DA and p53-R175H obtained by the nucleic acid aptamer modification strategy guided by molecular dynamic simulation in Example 1 of the present invention, A is an interaction site analysis diagram, and B is a helical conformation diagram;

[0026] Figure 5Figure 1 is a graph showing the affinity test results of the nucleic acid aptamers p53m-RA, p53m-DA and p53-R175H (A) and p53m-DA and p53-R175H, p53-WT (B) in Example 1 of the present invention;

[0027] Figure 6 Figure 1 is a graph showing the serum stability test result of the p53m-DA nucleic acid aptamer in Example 1 of the present invention, A is an electrophoresis graph, and B is a curve graph;

[0028] Figure 7 The structure diagram (A) of the p53-R175H degradation chimera (dp53m) constructed with p53m-DA as a ligand in Example 1 of the present invention and the electrophoresis result diagram (B) of the non-denaturing SDS-PAGE showing the electrophoresis results before and after the connection of the aptamer and the CRBN ligand;

[0029] Figure 8 This is a chemical reaction diagram for synthesizing the degradation chimera dp53m by click reaction in Example 1 of the present invention;

[0030] Fig. 9 The results of affinity test between the degradation chimera dp53m and p53-R175H and p53-WT in Example 1 of the present invention;

[0031] Fig.10 Figure 1 is a graph showing the degradation effect of the degradation chimera dp53m in Example 1 of the present invention on p53-R175H or p53-WT in H1299 cells, A is an electrophoresis graph, and B is a curve graph;

[0032] Fig.11 This is a diagram showing the degradation effect of the degradation chimera dp53m in Example 1 of the present invention on p53 degradation of SKBR3, A549 and H1975 cells with different mutations;

[0033] Fig.12 This is a diagram showing the degradation effect of the degradation chimera dp53m in Example 1 of the present invention on common p53 hotspot mutations;

[0034] Fig.13 The results of the cell proliferation experiment of the degradation chimera dp53m in Example 1 of the present invention on p53 wild-type or R175H mutant cells are shown in Figure A, H1299-p53-R175H, B H1299-p53-WT, C SKBR3, and D A549;

[0035] Fig.14Figures 1 and 2 are the results of the cell clone formation experiment of the degradation chimera dp53m in Example 1 of the present invention on p53 wild-type or R175H mutant cells, A is a microscope picture of H1299-p53-WT and H1299-p53-R175H, B is a bar graph of the cell numbers of H1299-p53-WT and H1299-p53-R175H, C is a microscope picture of A549 and SKBR3, and D is a bar graph of the cell numbers of A549 and SKBR3;

[0036] Fig.15 The graphs show the results of the killing experiment of the degradation chimera dp53m combined with cisplatin on p53 wild-type or R175H mutant cells in Example 1 of the present invention, A is H1299-p53-R175H, B is H1299-p53-WT, C is SKBR3, and D is A549. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0038] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0039] Since there are no reports of PROTACs targeting p53 mutants, although there are nucleic acid aptamers that specifically target p53-R175H, the aptamers are RNA aptamers and are extremely unstable in serum. The present invention proposes nucleic acid aptamers targeting p53-R175H and their protein-targeted degradation chimeras and applications.

[0040] A typical embodiment of the present invention provides a nucleic acid aptamer targeting p53-R175H, as shown in SEQ ID NO.1.

[0041] Another embodiment of the present invention provides a method for modifying the above-mentioned nucleic acid aptamer targeting p53-R175H, comprising the following steps:

[0042] Obtaining the sequence of the RNA aptamer targeting the p53-R175H mutation and constructing the three-dimensional conformation of the RNA aptamer targeting the p53-R175H mutation;

[0043] The molecular dynamic simulation method was used to analyze the binding sites of RNA aptamers and p53-R175H mutants, and the key amino acid residues and nucleotides for the interaction between RNA aptamers and p53-R175H mutants were obtained;

[0044] On the basis of maintaining the three-dimensional structure of the RNA aptamer, the nucleotides are replaced one by one, and the nucleic acid aptamer targeting p53-R175H is obtained according to the binding of the replaced aptamer to the p53-R175H mutant.

[0045] The present invention uses the method of molecular dynamics simulation to reconstruct the binding site between RNA aptamers and p53-R175H, and based on this, develops DNA aptamers with higher affinity and greatly improved serum stability. And through the optimized nucleic acid aptamer, a chimera that selectively degrades p53-R175H is developed, thereby realizing precision medicine for patients with p53-R175H mutant tumors.

[0046] Specifically, the three-dimensional conformation of the RNA aptamer targeting the p53-R175H mutation was first constructed using a computer modeling method, and the binding site between the RNA aptamer and the p53-R175H mutant was analyzed using a molecular dynamic simulation method to find out the key amino acid residues and nucleotides for the interaction between the two. After the prediction of the interaction and molecular dynamic simulation, we replaced the nucleotides one by one on the basis of maintaining the three-dimensional structure of the aptamer, and then analyzed the binding between the replaced aptamer and the p53-R175H mutant, and finally obtained the DNA nucleic acid aptamer targeting the p53-R175H protein, whose nucleotide sequence is 5′-attagcgcaccctcacagagggtgc-3′ (SEQ ID NO.1).

[0047] A third embodiment of the present invention provides a protein targeted degradation chimera targeting p53-R175H, including a DNA nucleic acid aptamer, which is connected to a ligand of an E3 ubiquitin ligase via a connecting molecule, and the DNA nucleic acid aptamer is the above-mentioned nucleic acid aptamer targeting p53-R175H.

[0048] In some embodiments, the linker molecule is at least one -CH 2 CH 2 O-unit or at least one deoxythymidine nucleotide (T). Preferably, the number of deoxythymidine nucleotides in the linker molecule is 1 to 5.

[0049] In order to improve the serum stability and affinity of nucleic acid aptamers, the present invention also performs a series of modifications on nucleic acid aptamers. In some embodiments, 2'-OMe or PS modification is added to the nucleotides.

[0050] In some embodiments, the 5' end of the DNA aptamer is linked to the ligand of the E3 ubiquitin ligase via a linker molecule.

[0051] In some embodiments, the ligand of the E3 ubiquitin ligase is a CRBN ligand or a VHL ligand.

[0052] A fourth embodiment of the present invention provides a method for preparing the above-mentioned protein targeted degradation chimera targeting p53-R175H, comprising the following steps:

[0053] synthesizing a single-stranded DNA containing an azide modification, wherein the single-stranded DNA comprises a nucleotide linker molecule and a DNA nucleic acid aptamer, and the azide group is located on the linker molecule;

[0054] The azide-modified single-stranded DNA is coupled with the ligand of the alkyne-modified E3 ubiquitin ligase through a click reaction to obtain the product.

[0055] In some embodiments, the conditions for the click reaction are: incubation at 35-40° C. for 4-6 hours.

[0056] A fifth embodiment of the present invention provides a use of the above-mentioned protein targeted degradation chimera targeting p53-R175H in the preparation of drugs for degrading diseases related to p53-R175H mutation.

[0057] Specifically, the p53-R175H mutation-related diseases include, but are not limited to, inflammation-related diseases, viral infection-related diseases, and tumors. The drug includes a single preparation or a combination with other chemotherapy drugs.

[0058] A sixth embodiment of the present invention provides an anti-tumor drug, comprising an active ingredient and at least one inactive drug ingredient, wherein the active ingredient is the above-mentioned protein targeted degradation chimera targeting p53-R175H.

[0059] In some embodiments, a pharmaceutical carrier is included. The pharmaceutical carrier is a liposome and / or an exosome.

[0060] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0061] Example 1

[0062] Optimization method of p53m-RA aptamer

[0063] The R175 of the crystal structure of p53 (1TSR, chain B) was mutated to H using the Swiss PDB Viewer, and then a 1μs molecular dynamics simulation was performed to obtain the equilibrium state of p53-R175mH. The 3D structure of p53m-RA was constructed using the online servers RNAComposer and 3DNA. All molecular dynamics simulations were performed by Amber20 using the Amber14 force field. After four rounds of energy minimization steps, the temperature of the system was gradually heated to 300K within 100ps, followed by 5ns NVT equilibrium and 5ns NPT equilibrium. Finally, the molecular dynamics simulation calculations of the product were performed at 300K and 1atm using the LINCS algorithm to restrict the hydrogen position to its equilibrium distance, which allowed the use of an integration time step of 2fs. The energy and coordinates were saved every 10ps for later analysis of the molecular dynamics simulation. All molecular dynamics simulations were performed on a high-performance computer cluster running the Linux operating system.

[0064] The binding sites of p53m-RA and p53-R175H mutant were analyzed by molecular dynamic simulation method, and the key amino acid residues and nucleotides for the interaction between the two were found, among which G5, C6, U23 and C25 were key nucleotides. After interaction prediction and molecular dynamic simulation, non-key nucleotides were replaced one by one on the basis of maintaining the three-dimensional structure of the aptamer, and then the binding of the replaced aptamer with the p53-R175H mutant was analyzed, and finally the DNA nucleic acid aptamer p53m-DA targeting p53-R175H protein was obtained, and its nucleotide sequence is shown in Table 1.

[0065] Synthesis of chimeric molecules:

[0066] (1) Design and synthesize a series of DNA aptamers with azido modification at the 5' end (a series of N 3 -p53m-DA, which is N 3 -p53m-DA-1, N 3 -p53m-DA-2, N 3 -p53m-DA-3、N 3 -p53m-DA-4,N 3 -p53m-DA-5), the nucleotide sequence of which is shown in Table 1.

[0067] Table 1 DNA aptamers with azido modification at the 5' end

[0068]

[0069] The above nucleotide sequences are provided by Sangon Biotech (Shanghai) Co., Ltd.; the underlined molecules are linkers, N 3The azide modification is linked to the linker molecule via a phosphoramidite monomer at the C6 amino group. Figure 1 Shown is a single-stranded DNA molecule containing an azide modification (N 3 -p53m-DA-1, the mass spectrometry results of this azide-modified single-stranded DNA molecule were used in the following examples.

[0070] (2) Chimera ligation reaction, such as Figure 8 Specifically, the azide-modified aptamer of step (1) and the alkyne-containing CRBN ligand were mixed at a molar ratio of 1:5 and in a reaction buffer (10 mM Tris-HCl, 10 mM CuSO 4 , 10 mM sodium isothiazine), incubated at 37°C for 5 hours, and then removed the excess CRBN ligand through an ultrafiltration tube with a molecular weight cutoff of 3K to obtain a protein targeted degradation chimera targeting p53-R175H, namely dp53m, such as Figure 7 As shown in A. The efficiency of the ligation reaction was detected by 20% non-denaturing SDS-PAGE gel. Figure 7 As shown in B, the migration speed of dp53m is slower than that of the aptamer p53m-DA, indicating that the ligation is successful.

[0071] Synthesize alkyne-modified CRBN ligand molecules, specifically:

[0072] a.

[0073] Triethylamine (TEA, 1.2mmol) is added to 3-aminopropyne (1mmol) in dichloromethane (DCM, 10mL), and stirred at 0°C. Subsequently, a DCM solution of bromoacetyl bromide is added dropwise to the reaction solution. The mixture is stirred at room temperature for at least 2 hours. Finally, the residue is washed twice with petroleum ether, and the product is purified by flash chromatography.

[0074] b.

[0075] 4-Hydroxyisobenzofuran (3 mmol) was mixed with 3-aminopiperidine (3 mmol) in anhydrous toluene (20 mL), and TEA (3.36 mmol) was added to the mixture and stirred at 110°C for 12 hours. The mixture was cooled to room temperature and filtered. The obtained filter cake was washed with flash chromatography (3:10) solution for 10 minutes, and then the crude product was purified by flash chromatography.

[0076] c.

[0077] To K dissolved in DMF (5 mL) 2 CO 32-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindole-1,3-dione was added to a suspension of 2-bromo-N-(prop-2-yn-1-yl)acetamide (6mmol) and 2-bromo-N-(prop-2-yn-1-yl)acetamide (2mmol) and reacted at room temperature. After two hours, the mixture was diluted with EtOAc, washed once with water, and then washed twice with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. Purification by column chromatography (EtOH / DCM) produced a beige solid product, i.e., an alkynyl-modified CRBN ligand, a CRBN ligand of 13 C NMR spectrum Figure 2 shown.

[0078] Figure 3 A is the structure of p53m-RA and analysis of its interaction sites with p53-R175H protein, and areas a and b are magnified images. Figure 3 B is the helical conformation of the p53m-RA model structure in the 500ns molecular dynamics simulation, which relaxes along the time trajectory. Figure 4 A is the structure of p53m-DA and analysis of its interaction sites with p53-R175H protein, and areas a and b are magnified images. Figure 4 B is the helical conformation of the p53m-DA model structure in the 500ns molecular dynamic simulation. It has always maintained a stable helical conformation, suggesting that p53m-DA may have good affinity and stability.

[0079] Example 2

[0080] p53 affinity test:

[0081] The affinity of p53m-RA, p53m-DA and dp53m for wild type (p53-WT) or mutant (p53-R175H) was determined by microscale thermophoresis (MST). Purified p53-WT and p53-R175H proteins (aa: 94-293) were serially diluted and mixed with 200nM Cy3-labeled aptamer and dp53m. After incubation for 30 minutes, the mixture was added to the hydrophobic capillary and then measured by Monolith NT 115 (NanoTemper Technologies), and the Kd value was calculated by MO affinity analysis software (NanoTemper Technologies). The results are shown in Figure 5 As shown in the figure, the affinity of the modified aptamer p53m-DA was significantly improved, and its affinity for p53-R175H far exceeded that of p53-WT. Fig. 9 It also showed that the chimera dp53m constructed with the modified aptamer had a good affinity for p53-R175H.

[0082] Example 3

[0083] Serum stability test of p53m-DA:

[0084] 100 ng of p53m-DA prepared in Example 1 was dissolved in DMEM medium containing 15% serum, incubated at 37°C, and 5 μL of sample was taken out at different time points, and 5 μL of loading solution was added, firstly it was quickly frozen with liquid nitrogen, and then stored at -80°C. Then 20% non-denaturing polyacrylamide gel electrophoresis was performed, and 10 μL of the previously frozen sample was dissolved and loaded into each well. The voltage was 150 V, and the electrophoresis was performed for about 1 hour, and then it was stained with SYBR gold nucleic acid gel dye, and finally placed in the Bio-RAD instrument for imaging, and the results are as follows Figure 6 As shown, p53m-DA has good serum stability.

[0085] Example 4

[0086] Degradation performance test of dp53m on p53-R175H protein:

[0087] A concentration gradient of 62.5nM, 125nM, 250nM, 500nM and 1000nM dp53m solution was prepared and transfected into H1299 cells (H1299-p53-WT) overexpressing wild type (p53-WT) or mutant (p53-R175H) H1299 cells (H1299-p53-R175H) or A549, SKBR3, H1975 cells by liposome transfection, and the cells were lysed 24 hours later, and the changes of p53 protein were detected by Western blot. Alternatively, H1299 cells were transfected with several hotspot mutations of p53 for 24 hours, and then 1000nM dp53m was transfected, and the cells were lysed 24 hours later, and the changes of p53 protein were detected by Western blot. Fig.10 and Fig.11 The results of dp53m degradation of p53 in different cells indicate that dp53m has a better degradation effect on p53-R175H in different cells. Fig.12 The results of dp53m degradation of different hotspot mutants indicate that dp53m characteristically degrades p53-R175H but not other hotspot mutations.

[0088] Example 5

[0089] dp53m test on cell proliferation:

[0090] The cell proliferation was detected using the CCK-8 kit. The specific process was as follows: 1×10 4Each well was incubated with H1299-p53-WT, H1299-p53-R175H, A549 or SKBR3 (100 μL / well) and incubated at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator for 12 hours; the dp53m prepared in Example 1 was transfected into the wells respectively, and then the cells were placed at 37°C, 5% CO 2 The cells were cultured in a cell culture incubator for a certain period of time; CCK-8 solution was added to each well, and after incubation at 37°C for 4 hours, the optical density of each well was measured at a wavelength of 450 nm using an ELISA reader. Fig.13 As shown, dp53m can significantly inhibit the growth of tumor cells containing p53-R175H mutation, but has no effect on the growth of p53-WT tumor cells.

[0091] In addition, the effect of dp53m on tumor cell proliferation was detected by clone formation assay. The specific process was as follows: 1×10 3 Each of H1299-p53-WT, H1299-p53-R175H, A549 or SKBR3 was placed at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator; the dp53m prepared in Example 1 was transfected into the wells every 2 days, and then the cells were placed at 37°C, 5% CO 2 After culturing in a cell culture incubator for 2 to 3 weeks, the cells were fixed with 4% paraformaldehyde, stained with 0.4% crystal violet, and photographed under an inverted microscope to count the number of clones formed. Fig.14 As shown, dp53m can significantly inhibit the colony formation of tumor cells containing p53-R175H mutation, but has no effect on the colony formation of p53-WT tumor cells.

[0092] Example 6

[0093] Combination effect test of dp53m and cisplatin:

[0094] Add 1 × 10 4 Each well was incubated with H1299-p53-WT, H1299-p53-R175H, A549 or SKBR3 (100 μL / well) and incubated at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator for 12 hours; the cells were treated with dp53m prepared in Example 1 and cisplatin at a fixed molar ratio (1:10), and then the cells were placed at 37°C, 5% CO 2After 48 hours of culture in a cell culture incubator, cell proliferation was detected using a CCK-8 kit. The percentage of live cells was converted to a decimal number to calculate Fa. The combination index (CI) was calculated using CompuSyn software, where drug interactions were divided into synergistic effects (CI < 1), additive effects (CI = 1), or antagonistic effects (CI > 1). The results are shown in Fig.15 As shown, dp53m can increase the sensitivity of p53-R175H mutant tumor cells to cisplatin, but has no effect on the sensitivity of p53-WT cells to cisplatin.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nucleic acid aptamer targeting p53-R175H, characterized in that: As shown in SEQ ID NO.

1.

2. A method for modifying a nucleic acid aptamer targeting p53-R175H according to claim 1, characterized in that: The steps include: Obtaining the sequence of the RNA aptamer targeting the p53-R175H mutation and constructing the three-dimensional conformation of the RNA aptamer targeting the p53-R175H mutation; The molecular dynamic simulation method was used to analyze the binding sites of RNA aptamers and p53-R175H mutants, and the key amino acid residues and nucleotides for the interaction between RNA aptamers and p53-R175H mutants were obtained; On the basis of maintaining the three-dimensional structure of the RNA aptamer, the nucleotides are replaced one by one, and the nucleic acid aptamer targeting p53-R175H is obtained according to the binding of the replaced aptamer to the p53-R175H mutant.

3. A protein-targeted degradation chimera targeting p53-R175H, characterized in that: The invention comprises a DNA nucleic acid aptamer, which is connected to a ligand of an E3 ubiquitin ligase via a connecting molecule. The DNA nucleic acid aptamer is the nucleic acid aptamer targeting p53-R175H according to claim 1.

4. The protein targeted degradation chimera targeting p53-R175H as claimed in claim 3, characterized in that: The linking molecule is at least one -CH2CH2O- unit or at least one deoxythymidine nucleotide; or, adding 2'-OMe or PS modifications to the nucleotides of the linker molecule; Alternatively, the 5' end of the DNA aptamer is linked to the ligand of the E3 ubiquitin ligase via a linker molecule; Alternatively, the ligand of the E3 ubiquitin ligase is a CRBN ligand or a VHL ligand.

5. A method for preparing the protein targeted degradation chimera targeting p53-R175H according to claim 3, characterized in that: The steps include: synthesizing a single-stranded DNA containing an azide modification, wherein the single-stranded DNA comprises a nucleotide linker molecule and a DNA nucleic acid aptamer, and the azide group is located on the linker molecule; The azide-modified single-stranded DNA is coupled with the ligand of the alkyne-modified E3 ubiquitin ligase through a click reaction to obtain the product.

6. The method for preparing the protein targeted degradation chimera targeting p53-R175H as claimed in claim 5, characterized in that: The conditions for the click reaction are: incubation at 35-40 °C for 4-6 hours.

7. Use of the protein targeted degradation chimera targeting p53-R175H according to any one of claims 3 to 4 in the preparation of H1299 cells and SKBR3 cells drugs for degrading p53-R175H mutation.

8. An anti-tumor drug comprising an active ingredient and at least one inactive ingredient, characterized in that: The active ingredient is the protein targeted degradation chimera targeting p53-R175H as described in any one of claims 3-4.

9. The antitumor drug according to claim 8, characterized in that: Includes a pharmaceutically acceptable carrier.

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