Covalent nucleic acid aptamer and application thereof

Abstract

The invention relates to a covalent nucleic acid aptamer. The covalent nucleic acid aptamer comprises any one of S2 nucleic acid aptamer DNA sequence as shown in SEQ ID NO: 1, S2 nucleic acid aptamer DNA sequence as shown in SEQ ID NO: 2; the DNA sequence of the S2v1 aptamer is as shown in SEQ ID NO: 2; and the DNA sequence of the S2v2 aptamer is as shown in SEQ ID NO: 3. The aptamer also comprises phosphorothioate modification at the 3'end of the S2v2 and addition of an electrophilic group. In the aptamer, six T basic groups are added at the 3'end of S2v2, and phosphorothioate modification is carried out. Further, the aptamer reacts with 4-(bromomethyl) benzenesulfonyl fluoride to modify an electrophilic group, and the aptamer can form covalent binding with cells. The nucleic acid aptamer has high specificity, high affinity and no immunogenicity, can be chemically synthesized in vitro, can target bladder cancer cells, and is relatively strong in binding capacity. In addition, the aptamer can kill or inhibit bladder cancer cells, the effect is obvious, and targeted therapy of bladder cancer is facilitated.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to covalent nucleic acid aptamers and their applications. Background Art

[0002] Aptamers are single-stranded DNA or RNA molecules that interact specifically with their target, identified from a library of oligonucleotides using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. Aptamers possess high affinity and form thermodynamically stable three-dimensional structures, such as stem-loops, hairpins, and G-quadruplexes, enabling them to specifically recognize and tightly bind to target molecules through hydrogen bonding, electrostatic interactions, van der Waals forces, and ionic bonds. Aptamers can be rapidly obtained through in vitro screening methods and are chemically stable, making them suitable for structural or functional enhancement through chemical modification.

[0003] Bladder cancer is one of the most common malignant tumors of the urinary system, with a high mortality rate in late-stage patients. Therefore, developing new methods to screen for bladder cancer and early detection and treatment is crucial to reducing the mortality rate of bladder cancer. Currently, the main methods for detecting bladder cancer include: 1) imaging examinations: ultrasound, CT, KUB+IVU, MRI; 2) (fluorescence) cystoscopy, diagnostic transurethral resection (TUR); 3) urine cytology;

[0004] 4) Bladder cancer tumor marker detection. However, existing methods still have many limitations. For example, imaging and cystoscopy are expensive, complex, and painful for patients, making them unsuitable for early screening. Exfoliative cytology also has low sensitivity. Furthermore, existing tumor markers used in clinical testing suffer from insufficient sensitivity and specificity, necessitating the simultaneous detection of multiple markers to improve the reliability of the results. Therefore, there is a need to discover new bladder cancer tumor markers to develop novel bladder cancer detection strategies.

[0005] Aptamers offer significant advantages in cancer detection: high thermal stability, high biocompatibility, low cost, non-invasive and painless detection, and high sensitivity and specificity. Aptamers have the potential to be developed as molecular tools for early screening of bladder cancer. While aptamers offer numerous advantages over antibodies, their current application remains limited, primarily due to the lack of stability in their binding to the target and their susceptibility to environmental influences. Therefore, further research on aptamers is needed. Summary of the Invention

[0006] The purpose of this application is to provide a covalent nucleic acid aptamer, including any of the following:

[0007] A nucleic acid aptamer, the DNA sequence of which is shown in SEQ ID NO: 1;

[0008] A nucleic acid aptamer, the DNA sequence of which is shown in SEQ ID NO: 2;

[0009] The nucleic acid aptamer has a DNA sequence as shown in SEQ ID NO: 3.

[0010] The nucleic acid aptamer further comprises a 3' end of the nucleic acid aptamer shown in SEQ ID NO: 3 modified with phosphorothioate to add an electrophilic group.

[0011] In the above-mentioned nucleic acid aptamer, six T bases are added to the 3' end of the nucleic acid aptamer shown in SEQ ID NO: 3, and phosphorothioate modification is performed.

[0012] In the above-mentioned nucleic acid aptamer, the electrophilic group includes one or more of a benzenesulfonyl fluoride group, an N-hydroxysuccinimide group, and an acrylamide group.

[0013] Among the above-mentioned nucleic acid aptamers, the nucleic acid aptamer shown in SEQ ID NO: 3 is modified with phosphorothioate and a benzenesulfonyl fluoride group is added to obtain a nucleic acid aptamer whose DNA sequence is shown in SEQ ID NO: 4.

[0014] The above-mentioned nucleic acid aptamer also includes a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer obtained by connecting a signal molecule, an active molecule, a functional group or a radioactive nuclide to one end or the middle of any of the nucleic acid aptamers.

[0015] The application of the above-mentioned nucleic acid aptamers in identifying and binding to bladder cancer cells or renal cancer cells is also within the scope of protection of this application.

[0016] As certain embodiments of the present application, the application is the application of the nucleic acid aptamer in recognizing and binding to bladder cancer cells.

[0017] As certain embodiments of the present application, the application is the application of the nucleic acid aptamer in recognizing and binding to renal cancer cells.

[0018] As certain embodiments of the present application, the bladder cancer cells are T24 cells.

[0019] In certain embodiments of the present application, the renal cancer cells are 786O cells.

[0020] As certain embodiments of the present application, the application is the use of a nucleic acid aptamer with a DNA sequence such as that shown in SEQ ID NO:1 in recognizing and binding to bladder cancer cells. As certain embodiments of the present application, the application is the use of a nucleic acid aptamer with a DNA sequence such as that shown in SEQ ID NO:2 in recognizing and binding to bladder cancer cells. As certain embodiments of the present application, the application is the use of a nucleic acid aptamer with a DNA sequence such as that shown in SEQ ID NO:3 in recognizing and binding to bladder cancer cells. As certain embodiments of the present application, the application is the use of a nucleic acid aptamer with a DNA sequence such as that shown in SEQ ID NO:4 in recognizing and binding to bladder cancer cells.

[0021] The application of the above-mentioned nucleic acid aptamers in killing or inhibiting bladder cancer cells is also within the scope of protection of this application.

[0022] As certain embodiments of the present application, the bladder cancer cells are T24 cells.

[0023] As certain embodiments of the present application, the application is the use of a nucleic acid aptamer with a DNA sequence as shown in SEQ ID NO: 1 in killing or inhibiting bladder cancer cells. As certain embodiments of the present application, the application is the use of a nucleic acid aptamer with a DNA sequence as shown in SEQ ID NO: 2 in killing or inhibiting bladder cancer cells. As certain embodiments of the present application, the application is the use of a nucleic acid aptamer with a DNA sequence as shown in SEQ ID NO: 3 in killing or inhibiting bladder cancer cells. As certain embodiments of the present application, the application is the use of a nucleic acid aptamer with a DNA sequence as shown in SEQ ID NO: 4 in killing or inhibiting bladder cancer cells.

[0024] The second object of the present application is to provide a molecular tool for detecting bladder cancer cells and renal cancer cells to mark bladder cancer cells and renal cancer cells, comprising the above-mentioned nucleic acid aptamer.

[0025] The third object of the present application is to provide a drug comprising the above-mentioned nucleic acid aptamer.

[0026] The fourth object of the present application is to provide the application of the above-mentioned drug, which is the application in the preparation of drugs for treating bladder cancer or drugs for killing and inhibiting bladder cancer cells.

[0027] As certain embodiments of the present application, the bladder cancer cells are T24 cells.

[0028] The beneficial effects of this application are as follows: 1. The nucleic acid aptamer of this application has high affinity, is non-immunogenic, can be chemically synthesized in vitro, can target bladder cancer cells, and has strong binding ability. The nucleic acid aptamer of this application can also kill or inhibit bladder cancer cells with significant effects, which is conducive to the targeted treatment of bladder cancer.

[0029] 2. The nucleic acid aptamer of the present application introduces electrophilic groups through chemical modification, establishing a covalent bond between the sequence and the target protein, which can further improve the affinity and targeted binding stability of the nucleic acid aptamer to the target, and improve the detection sensitivity of the nucleic acid aptamer and its ability to kill and inhibit tumor cells.

[0030] 3. The marker of this application can specifically identify bladder cancer cells.

[0031] 4. The drug of the present application can kill or inhibit bladder cancer cells and can be used alone or in combination with other drugs to treat bladder cancer. It has stable properties, obvious effects and no side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the secondary structure diagram of the S2 nucleic acid aptamer of this application;

[0033] Figure 2 The secondary structure diagram of the S2v1 and S2v2 nucleic acid aptamers of this application;

[0034] Figure 3 This is a flow chart of the cell-based in vitro screening in Example 1;

[0035] Figure 4 S2 specifically binds to bladder cancer cells. Figure 4 A is fluorescence microscope imaging, Figure 4 B is the determination of the dissociation constants of S2 with T24 and SV-huc-1;

[0036] Figure 5 Flow cytometry analysis of S2 binding to different cells;

[0037] Figure 6 This is an analysis diagram of S2 specific binding to bladder cancer tissue;

[0038] Figure 7 The CCK8 test results of Example 5 are shown below:

[0039] Figure 8 This is a diagram showing the binding effects of S2, S2v1, and S2v2 on T24 cells;

[0040] Figure 9 Figure 2 is the PAGE analysis result of benzenesulfonyl fluoride modification of S2v3;

[0041] Figure 10 This is a flow cytometry analysis of the binding of several nucleic acid aptamers to T24 cells in this application;

[0042] Figure 11 This is the CCK8 detection result of Example 8. DETAILED DESCRIPTION

[0043] Here, exemplary embodiments are described in detail. The embodiments described in the embodiments do not represent all embodiments consistent with the present application. Unless otherwise specified, the experimental methods used in the examples of the present application are conventional methods.

[0044] Based on in vitro screening and high-throughput sequencing technology, the S2 sequence was isolated and identified with a length of 80 nt, and the secondary structure of S2 was predicted using mFold, such as Figure 1 The S2 sequence was truncated to obtain 67nt S2v1 and 46nt S2v2, respectively. The secondary structures of S2v1 and S2v2 are shown in Figure 2 shown.

[0045] The nucleic acid aptamer of the present application further comprises six T bases added to the 3' end of S2v2, and is modified with phosphorothioate to add an electrophilic group. The electrophilic group includes a benzenesulfonyl fluoride group, an N-hydroxysuccinimide group, and an acrylamide group, preferably a benzenesulfonyl fluoride group.

[0046] S2v3 is obtained by adding six T bases to the 3' end of S2v2, performing phosphorothioate modification, and adding a benzenesulfonyl fluoride group. This can further improve the targeted binding stability of the nucleic acid aptamer to cells.

[0047] The specific sequence of the covalent nucleic acid aptamer is as follows:

[0048] The S2 aptamer DNA sequence is (SEQ ID NO: 1):

[0049] 5'-AGCGTCGAATACCACTACAGGCACTGGCTGCGGAATTTCCTCATT

[0050] TATGAATATATCTAACTAATGGAGCTCGTGGTCAG-3';

[0051] The S2v1 aptamer DNA sequence is (SEQ ID NO: 2):

[0052] 5'-ACCACTACAGGCACTGGCTGCGGAATTTCCTCATTTATGAATATAT

[0053] CTAACTAATGGAGCTCGTGGT-3';

[0054] The S2v2 aptamer DNA sequence is (SEQ ID NO: 3):

[0055] 5'-GGCTGCGGAATTTCCTCATTTATGAATATATCTAACTAATGGAGCT-3';

[0056] The S2v3 aptamer DNA sequence is (SEQ ID NO: 4):

[0057] 5'-GGCTGCGGAATTTCCTCATTTATGAATATATCTAACTAATGGAGC

[0058] T SF T SF T SF T SF T SF TT-3'.

[0059] The cell sources in the examples of this application are as follows:

[0060] SV-huc-1 human ureteral epithelial immortalized cells were purchased from Wuhan Punosai Life Science Technology Co., Ltd.;

[0061] T24 human bladder transitional cell carcinoma cells were purchased from the Cell Bank of the Chinese Academy of Sciences;

[0062] 5637 human bladder cancer cells were purchased from Wuhan Punosai Life Science Technology Co., Ltd.;

[0063] BIU87 human bladder cancer cells were obtained from Jiangsu Institute of Tumor Biotherapy;

[0064] 786O human renal clear cell adenocarcinoma cells were purchased from Wuhan Punosai Life Science Technology Co., Ltd.;

[0065] HCT8 human ileocecal cancer cells were purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0066] Malignant bladder cancer cells were provided by the Department of Urology, Affiliated Hospital of Xuzhou Medical University.

[0067] Example 1: Screening and preparation of nucleic acid aptamers

[0068] In the screening, bladder cancer cell T24 was used as the target cell, and normal bladder epithelial cell SV-HUC-1 was used as the control cell. The full length of the random library is 80nt, with a fixed sequence of 20nt on each side of the sequence for PCR amplification, and the middle 40nt sequence is composed of four random bases. The library was synthesized by Suzhou Beixin Biomedical Technology Co., Ltd. The screening process is briefly described as follows: T24 cells and SV-HUC-1 cells were cultured in a 37°C, 5% CO2 incubator. At the beginning of the screening, the cells were scraped off with a cell scraper and placed in a 1.5mL centrifuge tube and washed once with PBS. 1nmol of library sequence was diluted with 50μL 2×binding buffer (DPBS, 100μg / mL yeast tRNA, 0.1% BSA 4mM MgCl2), annealed at 95°C for 5 minutes, and placed on ice for 10 minutes. The library sequence was then combined with 1×107 T24 cells were incubated at 4°C for 1 hour. After the reaction, they were washed three times with PBS. Finally, 100 μL of DNase-free water was added to resuspend the cells, heat-treated at 95°C for 10 minutes, centrifuged at 13,000 rpm for 5 minutes, and the supernatant containing the eluted DNA was collected. The recovered product was used as a template for PCR amplification to enrich the active sequence. In the third round of screening, SV-HUC-1 cells were introduced for negative screening. In order to obtain sequences with high affinity, the amount of T24 cells was gradually reduced during the screening, from 1×10 7 (First round) reduced to 6×10 5 (Round 11); the incubation time was also reduced from 1 hour in the first round to 40 minutes. Finally, the screening products of round 11 were sequenced to identify the active sequence. Figure 3 shown.

[0069] Example 2: Optimization of nucleic acid aptamers and detection of affinity

[0070] (A) Fluorescence analysis of the binding of S2 sequence to T24 and SV-HUC-1 cells:

[0071] (1) Nucleic acid preparation: Take 100 μL 2X binding buffer (containing yeast tRNA) and add 5 μL (concentration of 10 μM) Cy5-labeled S2 nucleic acid aptamer, denature at 95°C for 5 minutes, add 95 μL serum-free culture medium after 10 minutes on ice, and store at room temperature. (2) Prepare T24 and SV-HUC-1 cells in advance and plant them on 12-well plate slides. Wash twice with PBS, add 200 μL nucleic acid aptamer mixture prepared in step (1) respectively, incubate at 37°C in the dark for 30 minutes, and wash twice with PBS. Then use 100×Hoechst33342 to stain the nucleus for 15 minutes, clamp the slide and cover it on a slide with anti-fluorescence quencher, fix it, and use a focusing microscope (Zeiss, LSM880) for fluorescence imaging. The results are as follows Figure 4 As shown in A.

[0072] pass Figure 4 A shows that the S2 nucleic acid aptamer specifically binds to bladder cancer cells T24 and hardly binds to normal cells SV-HUC-1.

[0073] (B) Flow cytometric analysis of the binding of different concentrations of S2 aptamers to T24 and SV-HUC-1 cells: Cy5-labeled S2 aptamers were incubated with T24 and SV-HUC-1 cells at 37°C for 30 min, respectively. The binding strength of the two cells to different concentrations of S2 aptamers was statistically analyzed based on the flow cytometric analysis results. The results are shown in Figure 2. Figure 4 As shown in B.

[0074] pass Figure 4 As shown in Figure B, the S2 aptamer binds strongly to T24 cells, with the equilibrium dissociation constant calculated using GraphPad being 83.21±35.85 nM. Furthermore, S2 slightly binds to SV-HUC-1 with increasing concentrations, but this is significantly different from T24.

[0075] Example 3: Analysis of S2 specific recognition and binding ability

[0076] Flow cytometry analysis of the binding ability of Cy5-labeled S2 nucleic acid aptamers to different cells. 250nM Cy5-labeled S2 sequence was incubated with SV-HUC-1 cells, T24 cells, 5637 cells (bladder cancer), BIU87 cells (bladder cancer), 786-O cells (kidney cancer) and HCT8 cells (colon cancer) at 37°C for 40 minutes, and then analyzed by flow cytometry. The results showed that S2 nucleic acid aptamers can bind to a variety of cells with different strengths, such as Figure 5 As shown, 786-O and T24 cells had stronger binding abilities to S2 nucleic acid aptamer.

[0077] Example 4: Experiment on the specific binding of S2 to human bladder cancer tissue

[0078] Bladder cancer tissue and adjacent adjacent tissue from two bladder cancer patients (patient 1, pathology number: 3512954; patient 2, pathology number: 3512605, provided by the Department of Urology, Affiliated Hospital of Xuzhou Medical University) were collected. The tissues were first fixed with 4% paraformaldehyde for one week, with the paraformaldehyde solution replaced every two days. The tissues were then paraffin-embedded and sectioned. Before aptamer binding to the tissues, dewaxing, hydration, and antigen retrieval were performed. The following steps were performed: 200 mL of xylene, 100%, 95%, 90%, 85%, 80%, and 75% ethanol, and PBS were added to a histochemical cassette. Tissue sections were immersed in xylene and various concentrations of ethanol for 5 minutes each. Afterwards, the sections were washed three times with PBS for 3 minutes each. Antigen retrieval was performed using citrate buffer. The tissues were then labeled with 250 nM Cy5-labeled S2 aptamer and mounted with mounting medium containing DAPI. Finally, fluorescence microscopy (Zeiss, Axio Obserer7) was used to image the cells. Figure 6 As shown in the figure, it was found that the binding of S2 to the two bladder cancer tissues was significantly higher than that to the adjacent adjacent tissues.

[0079] Example 5: Experiment on the killing effect of S2

[0080] 3×10 3T24 cells were seeded into 96-well plates and cultured overnight. T24 cells were incubated with 2μM Scrambled and 2μM S2, respectively. After 48h and 96h of culture, the effects of the two sequences on T24 cell proliferation were analyzed using the CCK8 assay. The test results are shown in Figure 2. Figure 7 As shown, the statistical results showed that compared with the scrambled treatment group, the 2 μM S2 treatment group significantly inhibited the proliferation of T24 cells, S2 had a killing effect on T24 cells, and within a reasonable time, the longer the time, the more obvious the inhibitory effect of S2 on T24 cells.

[0081] Example 6: Analysis of the binding ability of S2, S2v1 and S2v2

[0082] 250nM Cy5-modified Scrambled, S2, S2v1, and S2v2 sequences were diluted in 100μL of binding buffer and annealed (95°C for 5 minutes, then on ice for 10 minutes). 100μL of 1640 medium was added to the system, followed by incubation with T24 cells for 30 minutes and flow cytometry analysis. The results are shown in Figure 2. Figure 8 As shown, it was found that the binding levels of S2v1 and original S2 to T24 cells were basically the same, and the affinity level of S2v2 was improved.

[0083] Example 7: Preparation and Binding Ability Analysis of S2v3 Aptamer

[0084] Six Ts were added to the 3' end of the S2v2 sequence, and phosphorothioate modification was performed between the six Ts to obtain PS-S2v2. 4-(Bromomethyl)benzenesulfonyl fluoride was dissolved in acetonitrile and the electrophilic group of benzenesulfonyl fluoride (SF) was modified in the nucleic acid aptamer sequence by reacting with phosphorothioate to obtain S2v3 (SF-S2v2) nucleic acid aptamer. After SF modification, the results were analyzed using 10% PAGE. Figure 9 As shown, it was found that the molecular weight of the modified sequence increased, indicating that the modification of the electrophilic group was successful.

[0085] Flow cytometry was used to analyze the binding ability of Cy5-labeled S2v2, PS-S2v2, and S2v3 aptamers to T24 cells. The aptamers were diluted in binding buffer to a final concentration of 250 nM and incubated with T24 cells for 30 minutes. The binding levels of different aptamers to T24 cells were then analyzed using flow cytometry. The results are shown in Figure 2. Figure 10 As shown in the flow cytometry analysis, the S2v3 aptamer has better binding ability to T24 cells than S2v2, and SF modification improves the targeting binding ability of the aptamer.

[0086] Example 8: Experiment on the killing effect of S2v3

[0087] 3×103 T24 cells were seeded into 96-well plates and cultured overnight. T24 cells were incubated with 0.5 μM and 1 μM Scrambled and S2v3, respectively, and the effects of the two sequences on T24 cell proliferation were analyzed using CCK8 assay at 72 hours. The test results are shown in Figure 2. Figure 11 As shown, the statistical results showed that compared with the scrambled treatment group, the 1 μM S2v3 treatment group significantly inhibited cell proliferation.

[0088] The above are only preferred embodiments of the present application and do not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A covalent nucleic acid aptamer, characterized in that Include any of the following: A nucleic acid aptamer, the DNA sequence of which is shown in SEQ ID NO: 1; A nucleic acid aptamer, the DNA sequence of which is shown in SEQ ID NO: 2; The nucleic acid aptamer has a DNA sequence as shown in SEQ ID NO:

3.

2. The covalent nucleic acid aptamer according to claim 1, wherein The method further comprises performing phosphorothioate modification on the 3' end of the nucleic acid aptamer shown in SEQ ID NO: 3 to add an electrophilic group; Preferably, 6 T bases are added to the 3' end of the nucleic acid aptamer shown in SEQ ID NO: 3, and phosphorothioate modification is performed.

3. A covalent nucleic acid aptamer according to claim 2, characterized in that: The electrophilic group includes one or more of a benzenesulfonyl fluoride group, an N-hydroxysuccinimide group, and an acrylamide group.

4. The covalent nucleic acid aptamer according to claim 3, characterized in that The nucleic acid aptamer shown in SEQ ID NO: 3 is modified with phosphorothioate and a benzenesulfonyl fluoride group is added to obtain a nucleic acid aptamer whose DNA sequence is shown in SEQ ID NO:

4.

5. The covalent nucleic acid aptamer according to any one of claims 1 to 4, characterized in that It also includes connecting a signal molecule, an active molecule, a functional group or a radioactive nuclide to one end or the middle of any of the nucleic acid aptamers to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer.

6. Use of the covalent nucleic acid aptamer according to claim 1 or 4 in recognizing and binding bladder cancer cells or renal cancer cells; Preferably, the bladder cancer cells are T24 cells; Preferably, the renal cancer cells are 786O cells.

7. Use of the covalent nucleic acid aptamer according to claim 1 or 4 in killing or inhibiting bladder cancer cells; Preferably, the bladder cancer cells are T24 cells.

8. A marker for detecting bladder cancer cells or renal cancer cells, comprising the nucleic acid aptamer according to claims 1 to 5.

9. A medicine comprising the nucleic acid aptamer according to claims 1 to 5.

10. The use of a medicine according to claim 9, characterized in that: The application is in the preparation of drugs for treating bladder cancer or drugs for killing or inhibiting bladder cancer cells; Preferably, the bladder cancer cells are T24 cells.

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