Screening and application of esophageal squamous carcinoma cell aptamer

Abstract

The invention discloses screening and application of an esophageal squamous carcinoma cell aptamer, and belongs to the technical field of nucleic acid. The technical problem to be solved in the invention is how to obtain the aptamer specifically targeting esophageal squamous carcinoma cells and to prepare a reagent for identifying and diagnosing esophageal squamous carcinoma or esophageal squamous carcinoma cells. In order to solve the technical problem, the invention provides a nucleic acid aptamer ESCwl-7a, ESCwl-68a or ESCwl-75a and a composition composed of the nucleic acid aptamer ESCwl-75a, ESCwl-68a or ESCwl-75a. The nucleic acid aptamer obtained through screening has the advantages of being low in immunogenicity, easy to modify and mark, stable in property and good in reproducibility; compared with an antibody, the stability is good, and the synthesis cost is low. And further characterization and target identification can be carried out on the properties of the nucleic acid aptamer subsequently, so that the exploration and discovery of tumor markers are facilitated. Meanwhile, the aptamer can be used as a high-efficiency molecular probe tool with strong penetrating power for early diagnosis of tumors, and also can be combined with drugs for tumor targeted therapy.

Description

Technical Field

[0001] The present application belongs to the field of nucleic acid technology, and specifically relates to the screening and application of nucleic acid aptamers for esophageal squamous cell carcinoma cells. Background Art

[0002] Esophageal cancer is the ninth most common digestive tract tumor in the world. According to the latest global cancer data statistics in 2022, the number of deaths from esophageal cancer in 2022 was 445,100, ranking seventh among all cancers. In 2022, the number of cases of esophageal cancer in my country was 224,000, and the number of deaths was 187,500, accounting for 42.13% of the global number of deaths from esophageal cancer. At the same time, esophageal cancer also showed a trend of "high incidence and high mortality". Esophageal cancer can be divided into squamous cell carcinoma, adenocarcinoma and other types according to histological characteristics. Esophageal squamous cell carcinoma (ESCC) accounts for about 90% of esophageal cancer in China. It has a high degree of malignancy and a poor prognosis. The five-year survival rate is 10%, and the early symptoms are not obvious. When most patients are discovered, they are already in the middle and late stages. More than 75% of ESCC are diagnosed in stage III or IV. Currently, the clinical treatments for ESCC mainly include surgery, radiotherapy, chemotherapy and neoadjuvant therapy, but there are few reports on targeted treatments for esophageal squamous cell carcinoma, and there is no effective targeted drug yet.

[0003] Aptamers are a segment of RNA or single-stranded DNA that has the ability to specifically bind to a specific target molecule. The number of bases is usually between dozens and a hundred, and they form different secondary and tertiary structures by folding. Aptamers can bind to a wide range of targets, including small molecules, proteins, cells, and tissues. The high specificity and high affinity recognition and binding between aptamers and these target molecules make them promising biosensors and functional regulators for these targets. Compared with antibodies, aptamers have the advantages of small molecular weight, easy synthesis, easy modification, low cost, and low immunogenicity. Therefore, they have good development prospects in the fields of medicine such as new drug development, drug targeting carriers, and biomedical diagnosis (including disease diagnosis and molecular imaging).

[0004] Cell-SELEX of nucleic acid aptamers uses the whole living cell as the target, and uses the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology to screen out one or more single-stranded DNAs that can specifically recognize the target cell surface markers. Cell screening does not require the knowledge of the target, and can screen a variety of receptors present on the cell surface, so a group of nucleic acid aptamer probes can be obtained, which can specifically recognize the biomarkers on cancer cells and distinguish them from normal cells based on differences at the molecular level. Summary of the invention

[0005] The technical problem to be solved by this application is: how to obtain a nucleic acid aptamer that specifically targets esophageal squamous cell carcinoma cells and use it to prepare a reagent for identifying and diagnosing esophageal squamous cell carcinoma or esophageal squamous cell carcinoma cells. To solve this technical problem, this application provides the following technical solutions: The present application provides a composition, which comprises at least two of the nucleic acid aptamers ESCwl-7a, ESCwl-68a and ESCwl-75a. The nucleic acid aptamer ESCwl-7a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.1; The nucleic acid aptamer ESCwl-68a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.2; The nucleic acid aptamer ESCwl-75a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO. 3.

[0006] The present application also provides a nucleic acid aptamer, wherein the nucleic acid aptamer includes any one of the nucleic acid aptamers ESCwl-7a, ESCwl-68a or ESCwl-75a. The nucleic acid aptamer ESCwl-7a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.1; The nucleic acid aptamer ESCwl-68a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.2; The nucleic acid aptamer ESCwl-75a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.3.

[0007] The present application also provides nucleic acid aptamer derivatives, which are obtained by performing the following operations on each single nucleic acid aptamer constituting the nucleic acid aptamers ESCwl-7a, ESCwl-68a and ESCwl-75a: connecting fluorescein, anti-tumor drugs, radioactive elements, biological enzymes, biotin and / or nanomaterials to one end or the middle of the single nucleic acid aptamer to obtain a nucleic acid aptamer derivative having the same function as the single nucleic acid aptamer.

[0008] Furthermore, the composition, nucleic acid aptamer, and nucleic acid aptamer derivative are used to distinguish tumor cells from healthy cells.

[0009] Furthermore, the tumor cells include, but are not limited to, human esophageal squamous cell carcinoma cells, human cervical cancer cells, human breast cancer cells, human colorectal cancer cells, human bladder cancer cells, and human acute lymphoblastic leukemia cells.

[0010] Furthermore, the human esophageal squamous cell carcinoma cells include human esophageal squamous cell carcinoma cells KYSE510, human esophageal squamous cell carcinoma cells KYSE30, and human esophageal squamous cell carcinoma cells KYSE150.

[0011] Furthermore, the human cervical cancer cells include human cervical cancer cells Hela.

[0012] Furthermore, the human breast cancer cells include human breast cancer cells SUM159.

[0013] Furthermore, the human colorectal cancer cells include human colorectal cancer cells HCT-8.

[0014] Furthermore, the human bladder cancer cells include human bladder cancer cells YTS-1.

[0015] Furthermore, the human acute lymphoblastic leukemia cells include human acute lymphoblastic leukemia cells CEM.

[0016] In some embodiments of the present application, fluorescein isothiocyanate (FITC) is linked to the single nucleic acid aptamer.

[0017] The present application also provides an application, wherein the application is an application of the composition, the nucleic acid aptamer and / or the nucleic acid aptamer derivative in any of the following: (B1) Use in the preparation of products for identifying or assisting in the identification of tumor cells; (B2) Application in identifying or assisting in identifying tumor cells; (B3) Use in the preparation of products for identifying or assisting in the identification of tumor patient samples; (B4) Application in identifying or assisting in identifying samples from tumor patients; (B5) Application in the preparation of products for imaging tumor cells (B6) Application in imaging of tumor cells; (B7) Use in the preparation of a product for imaging tumor patient samples (B8) Application in imaging of tumor patient samples; (B9) Use in the preparation of products for molecular typing or auxiliary molecular typing of tumor cells and / or tumor patients; (B10) Application in molecular typing or auxiliary molecular typing of tumor cells and / or tumor patients; (B11) Use in the preparation of products for treating tumors; (B12) Use as a drug in the treatment of tumors; (B13) Application in tumor-related physiological and pathological research; (B14) Application in the preparation of products for tumor-related physiological and pathological research.

[0018] Furthermore, the products include but are not limited to: detection reagents, imaging reagents, drugs, and research models.

[0019] Specifically, in the present application, the products described in (B1) and (B3) can be biosensors. The working principle of the applications (B1) to (B4) is that when the nucleic acid aptamer specifically binds to the target substance, its configuration changes accordingly. This characteristic is used to develop an electrochemical sensor based on the change of nucleic acid aptamer configuration, namely an E-AB (Electrochemical aptamer-based) sensor. It has broad application prospects in fields such as medical diagnosis.

[0020] Specifically, in the present application, the products described in (B5) and (B7) can be imaging agents. The working principle of the applications (B5) to (B8) is that after the nucleic acid aptamer binds to the target substance, the real-time monitoring and imaging of the target molecule is achieved through signal transduction mechanisms such as fluorescence and chemiluminescence.

[0021] Specifically, in the present application, the product described in (B12) can be a drug delivery carrier. The applications described in (B12) and (B13) can be achieved through drug delivery. The working principle of drug delivery utilizes nucleic acid aptamers to bind to specific target cells or tissues for drug delivery. By combining drugs with nucleic acid aptamers, drugs can be accurately delivered to diseased cells or tissues, thereby improving the efficacy of drugs and reducing side effects.

[0022] The present application also provides a reagent, the active ingredients of which include the composition, the nucleic acid aptamer and / or the nucleic acid aptamer derivative.

[0023] The present application also provides the use of the reagent in any of the following: (C1) Application in identifying or assisting in identifying tumor cells; (C2) Application in identification or assisting identification of tumor patient samples; (C3) Application in imaging of tumor cells; (C4) Application in imaging of tumor patient samples; (C5) Application in molecular typing or auxiliary molecular typing of tumor cells and / or tumor patients; (C6) Application in the preparation of drugs for treating tumors; (C7) Application in tumor-related physiological and pathological research.

[0024] Furthermore, the reagents include but are not limited to molecular typing reagents, imaging reagents, biosensors, drug delivery vectors, gene therapy vectors, and the like.

[0025] Furthermore, in the application, the tumor cells include but are not limited to human esophageal squamous cell carcinoma cells, human cervical cancer cells, human breast cancer cells, human colorectal cancer cells, human bladder cancer cells, and human acute lymphoblastic leukemia cells.

[0026] Furthermore, the human esophageal squamous cell carcinoma cells include human esophageal squamous cell carcinoma cells KYSE510, human esophageal squamous cell carcinoma cells KYSE30, and human esophageal squamous cell carcinoma cells KYSE150.

[0027] Furthermore, the human cervical cancer cells include human cervical cancer cells Hela.

[0028] Furthermore, the human breast cancer cells include human breast cancer cells SUM159.

[0029] Furthermore, the human colorectal cancer cells include human colorectal cancer cells HCT-8.

[0030] Furthermore, the human bladder cancer cells include human bladder cancer cells YTS-1.

[0031] Furthermore, the human acute lymphoblastic leukemia cells include human acute lymphoblastic leukemia cells CEM.

[0032] Furthermore, the tumor patients include but are not limited to patients with esophageal squamous cell carcinoma, cervical cancer, breast cancer, colorectal cancer, bladder cancer, and acute lymphoblastic leukemia.

[0033] The sample includes, but is not limited to, lesion sampling from a tumor patient, such as tissue.

[0034] Furthermore, the esophageal squamous cell carcinoma patient samples include but are not limited to: patient lesion samples and / or patient esophageal samples.

[0035] The composition, nucleic acid aptamer, nucleic acid aptamer derivative and / or reagent of the application can be used for the capture, enrichment and / or purification of esophageal squamous cell carcinoma cells and samples, and can also be used for esophageal squamous cell carcinoma imaging or esophageal squamous cell carcinoma detection and identification, and can also be used for molecular typing of esophageal squamous cell carcinoma cells or patients, and can also be used for the diagnosis or treatment of esophageal squamous cell carcinoma.

[0036] The present application also provides a method for identifying or assisting in identifying tumor cells, the method comprising contacting the tumor cells with the composition, the nucleic acid aptamer, the nucleic acid aptamer derivative and / or the reagent to identify or assist in identifying whether the cells are tumor cells.

[0037] Furthermore, the tumor cells are in vitro cells.

[0038] Furthermore, the tumor cells include, but are not limited to, human esophageal squamous cell carcinoma cells, human cervical cancer cells, human breast cancer cells, human colorectal cancer cells, human bladder cancer cells, and human acute lymphoblastic leukemia cells.

[0039] The composition, nucleic acid aptamer, nucleic acid aptamer derivative and / or reagent binds to the tumor cell (with strong binding affinity). The nucleic acid aptamer is connected to a marker, and detecting the content of the marker on the surface of the cell to be tested can reflect whether the nucleic acid aptamer binds to the cell to be tested.

[0040] Furthermore, in the method described above, the tumor cells include but are not limited to: human esophageal squamous cell carcinoma cell KYSE30 and human esophageal squamous cell carcinoma cell KYSE150.

[0041] The beneficial technical effects achieved by this application are as follows: This application is based on the traditional Cell-SELEX technology, combined with UMI and high-throughput sequencing technology to select multiple nucleic acid aptamers that specifically identify esophageal squamous cell carcinoma cells. The nucleic acid aptamers screened in this application have the characteristics of low immunogenicity, easy modification and labeling, stable properties and good reproducibility; compared with antibodies, they have good stability and low synthesis cost. Further characterization of the properties of nucleic acid aptamers and identification of targets can be carried out in the future, which will help explore and discover tumor markers. At the same time, nucleic acid aptamers can be used as efficient and penetrating molecular probe tools for early diagnosis of tumors, and can also be combined with drugs for targeted tumor therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Flow chart for preparing probe packages and specific recognition molecules for esophageal squamous cell carcinoma cells.

[0043] Figure 2Summary of screening pressure for ZEC400.

[0044] Figure 3 Summary of screening pressures for ZEC398.

[0045] Figure 4 The screening pressure summary for KYSE30.

[0046] Figure 5 Summary of screening pressure for KYSE150.

[0047] Figure 6 The binding ability of each round of screening library to ZEC400, ZEC398, KYSE150 and KYSE30 cells was detected by flow cytometry.

[0048] Figure 7 The results of molecular typing of esophageal squamous cell carcinoma cells and normal esophageal epithelial cells using the probe package. A: Abundance map of the top 100 nucleic acid sequences of KYSE30. B: Abundance map of the top 100 nucleic acid sequences of HEEC. C: Differential analysis map of the top 100 nucleic acid sequences of KYSE30 and HEEC. D: Heat map of differential analysis of the top 100 nucleic acid sequences of KYSE30 and HEEC.

[0049] Figure 8 The results of flow cytometry detection of nucleic acid aptamers for distinguishing esophageal squamous cell carcinoma cells KYSE30 and normal esophageal epithelial cells. A: Flow cytometry detection of the binding ability of candidate aptamers to KYSE30 cells, KYSE150 cells and HEEC cells. B: Statistics of the ratio of KYSE30 cells, KYSE150 cells and HEEC cells to candidate aptamers. Ratio = F (aptamer) / F (random sequence), F is the fluorescence intensity. C: Flow cytometry detection of the dissociation constant (K) of ESCwl-1a, ESCwl-7a, ESCwl-68a and ESCwl-75a aptamers to KYSE30 cells d ). D: The specific sequences of aptamers ESCwl-1a, ESCwl-7a, ESCwl-68a and ESCwl-75a after truncation are listed. They were commissioned to be synthesized by a third-party biological company, and fluorescein isothiocyanate (FITC) was labeled at the 5' end of the aptamer.

[0050] Fig. 9 Flow cytometry was used to detect the binding of nucleic acid aptamers ESCwl-7a, ESCwl-68a, ESCwl-75a and random aptamers to different cells. DETAILED DESCRIPTION

[0051] In order to achieve accurate molecular typing and efficacy monitoring of esophageal squamous cell carcinoma, this paper develops specific recognition molecules and probe packages for esophageal squamous cell carcinoma cells, such as Figure 1 , which will provide available molecular tools for early diagnosis and target discovery of esophageal squamous cell carcinoma. In this study, the library obtained by nucleic acid aptamer screening of esophageal squamous cell carcinoma cell lines (KYSE30 and KYSE150) and esophageal squamous cell carcinoma clinical prognosis primary cells (ZEC400 and ZEC398) was used to prepare esophageal squamous cell carcinoma nucleic acid aptamer probe packages. Through molecular level differential expression analysis, the probe package can distinguish between esophageal squamous cell carcinoma cell line KYSE30 and normal esophageal epithelial cells HEEC.

[0052] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0053] The experimental methods in this application, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0054] The reagent preparation method in this application and the reagent purchase sources are as follows: The preparation methods or sources of the reagents used in this application are as follows: Wash Buffer: Add 2.5 mL 1M MgCl2 and 2.25 g D-glucose to 500 mL DPBS solution, mix well and store in a 4°C refrigerator.

[0055] Binding Buffer: 1 mL of Binding Buffer is composed of 880 µL of Washing Buffer, 100 µL of 10 mg / L herring sperm DNA (HsDNA) and 20 µL of 5 mg / L bovine serum albumin (BSA), and is prepared for immediate use.

[0056] 5× PCR Mix solution: Each 1000 µL 5× PCR Mix solution is composed of 120 µL upstream primer PZJS (final concentration: 6 µM), 120 µL downstream primer PTSAB (final concentration: 6 µM), 150 µL dNTP (final concentration: 375 µM), 25 µL Taq DNA polymerase, 500 µL 10× Buffer and 85 µL ultrapure water. Mix well and store in a -40°C refrigerator.

[0057] 5× Library Construction PCR Mix-1 Solution: Each 1000 µL 5× Amplification PCR Mix-1 solution is composed of 60 µL upstream primer PZJS (final concentration: 3 µM), 60 µL downstream primer HTSP2w (final concentration: 3 µM), 150 µL dNTP (final concentration: 375 µM), 25 µL Taq DNA polymerase, 500 µL 10× Buffer and 205 µL ultrapure water. Mix well and store in a -40°C refrigerator.

[0058] Library construction PCR Mix-2 solution: Each 165 µL of library construction PCR Mix-2 solution is composed of 60 µL 2.5mM dNTP, 100 µL 10×Buffer and 5 µL Taq DNA polymerase. Mix well and store in a -40℃ refrigerator.

[0059] Table 1 Reagents involved in this application

[0060] In this application, the human esophageal squamous cell carcinoma cells KYSE30, human esophageal squamous cell carcinoma cells KYSE150, human esophageal squamous cell carcinoma cells KYSE510, human cervical cancer cells Hela, human ovarian cancer cells 3AO, human gastric cancer cells MGC803, human breast cancer cells SUM159, human colorectal cancer cells HCT-8, human bladder cancer cells YTS-1, and human acute lymphoblastic leukemia cells CEM are owned by the Hangzhou Institute of Medicine, Chinese Academy of Sciences. ZEC400 and ZEC398 primary cells were provided by Zhejiang Cancer Hospital. HEEC cell line was purchased from Mingzhou Biotechnology, item number MZ-0720. The cell lines and primary cells used in this application are all derived from humans.

[0061] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0062] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. The t-test was used for the test, and P < 0.05 (*) indicated a significant difference, and P < 0.001 (***) indicated a very significant difference.

[0063] Example 1 Screening and enrichment of nucleic acid aptamer probes 1.1 Cell culture The cell lines used in the experiment were cultured in a constant temperature incubator at 37°C and a CO2 concentration of 5%. We used commercial serum-free cell freezing solution to freeze the cells, which can greatly improve the efficiency and reliability of cell freezing. The preparation method of the complete cell culture medium is to add 10% FBS and 5% 100 U / mL double antibody (streptomycin and penicillin) to 500 ml of the basic culture medium, mix well and store in a 4°C refrigerator. Specific cell information and culture medium types are shown in Table 1-1.

[0064] Table 1-1 Cell lines and culture conditions used in the experiment

[0065] 1.2 Cell screening process (taking the first round of screening of ZEC398 cells as an example) (1) Preparation of library and primers: The initial library is 2OD, and 25µL of DPBS is added to dilute the initial library. Denature at 95°C for 5 min, then quickly move to ice and cool for 5 min to allow ssDNA to form a natural conformation. Finally, place at room temperature for 15 min. Remove the primers and dilute to 50µM with ddH2O.

[0066] Table 1-2 Library and primer sequences

[0067] (2) Observe the ZEC398 cells and select cells with a cell density of 80%-90% or more and good cell growth. Discard the culture medium and add 2 mL of pre-cooled Wash Buffer to wash the remaining culture medium. Repeat the washing process 2-3 times.

[0068] (3) Add Binding Buffer to the DPBS-diluted library to 2 mL, then transfer to a ZEC398 cell culture dish. Incubate the dish at 4°C for 60 min, shaking every 3 to 5 min and keeping away from light.

[0069] (4) After the incubation, discard the supernatant and wash the remaining culture medium with 2 mL Wash Buffer. Repeat the washing for 3 times, and aspirate the remaining Wash Buffer for the last time. Then add 160 µL ddH2O (80 µL and 80 µL in two times) to the cell culture dish, scrape the cells in the dish with a cell scraper, and collect the cell mixture into a 1.5 mL EP tube.

[0070] (5) Heat the cell mixture in the 1.5 mL EP tube in a 95°C metal bath for 10 min, place on ice for 5 min, and place at room temperature for 10 min. Then centrifuge at 5500 rpm for 1 min to remove the cells and collect the supernatant. (160 µL of the first round supernatant and 160 µL of the lower layer of cells plus ddH2O are used for PCR separately) (6) 3 rounds of PCR (a) PCR1: Prepare PCR1 system 100 µL × 7 tubes: 140 µL 5× amplification PCR Mix solution, 560 µL denatured supernatant and ddH2O resuspended cell pellet solution.

[0071] PCR1 amplification conditions: denaturation temperature: 95°C, annealing temperature: 60°C, extension temperature: 72°C, and amplification round number 8. After PCR, centrifuge to remove the cell pellet, and then merge PCR1 into the same EP tube and mark it as PCR1.

[0072] (b) PCR2: Explore the number of PCR amplification rounds.

[0073] Using the PCR1 amplified fragment as template, prepare 50 µL of PCR2 system: 1 µL template, 10 µL 5× amplification PCR Mix solution, and 39 µL ddH2O.

[0074] Divide the prepared 50 µL PCR2 system into 5 tubes, with 10 µL in each tube.

[0075] PCR2 amplification conditions: denaturation temperature: 95°C, annealing temperature: 60°C, extension temperature: 72°C, and amplification rounds of 8, 10, 12, 14, and 16. Run the gel on 8% PAGE denaturing gel, observe the band results, and select the appropriate amplification rounds.

[0076] (c) PCR3: Using the PCR1 amplified fragment as template, prepare the PCR3 system 100 µL×10 tubes: 40 µL template, 200 µL 5× amplification PCR Mix solution, 760 µL ddH2O. PCR3 amplification conditions: denaturation temperature: 95°C, annealing temperature: 60°C, extension temperature: 72°C, and the appropriate number of amplification rounds (based on the PCR2 run results). After the PCR is completed, PCR3 is combined into the same EP tube and marked as PCR3.

[0077] (7) Single chain (a) Prepare the microcolumn first. Insert the filter in the 10 µL pipette tip into the 200 µL pipette tip. Take 60 µL of the sugar bead liquid and inject it into the bottom of the microcolumn. Repeat the washing twice with 100 µL DPBS.

[0078] (b) Pipette 100 µL of PCR3 product into the column and recover it. Repeat the previous step to ensure sufficient enrichment.

[0079] (c) Repeat the wash twice with 100 µL DPBS.

[0080] (d) Add 50 µL of 200 mM NaOH to the column, let it stand for 3 min, and then pump it into the NaOH collection tube. Repeat once. The PCR-amplified DNA is now concentrated in the eluate in the NaOH collection tube.

[0081] (8) Desalination: (a) Clean the desalting column first. After all the liquid in the column has dripped out, add ddH2O from the top until the column is just full. Wait for all the ddH2O to drip out from the bottom. Repeat this step 3 times for a total of 15 mL ddH2O.

[0082] (b) Adding single-stranded dissociation products: When the liquid level in the column is completely submerged in the desalting column and water stops dripping from the lower port, add 100 µL of single-stranded dissociation products from the upper port and wait for all the droplets to drip out from the lower port.

[0083] (c) Add ddH2O: When the liquid level in the column is completely submerged in the desalting column and water stops dripping from the lower port, add 400 µL ddH2O from the upper port and wait for all the droplets to drip out from the lower port.

[0084] (d) Elution: Wait until the liquid level in the column is completely submerged in the desalting column and the lower port stops dripping. Scrape off the water drops hanging from the lower port and prepare a 1.5 mL new EP tube at the lower port. Open the cap of the EP tube and add 600 µL ddH2O from the upper port to collect the eluate. Wait until 600 µL ddH2O is completely submerged in the column and the lower port stops dripping, then cover the cap of the EP tube.

[0085] (9) Use a DNA vacuum centrifugal concentrator to vacuum dry the obtained screening DNA library. Store the newly screened freeze-dried library at -40°C and wait for the next round of screening.

[0086] Polyacrylamide gel electrophoresis and imaging: First, the ratio of 8% PAGE denatured gel is: add 40µL 10% APS and 10µL TEMED to 5mL nucleic acid denatured polyacrylamide electrophoresis stock solution. After mixing evenly, pour it into the gel plate and wait for it to solidify. Pre-electrophoresis the sample for 10 minutes, then load the sample, electrophoresed at 300V, 40mA for 15 minutes, and finally observe the running gel under the gel imaging instrument.

[0087] 1.3 Enrichment of cell screening libraries In this experiment, a total of four cells were screened, of which KYSE30 and KYSE150 are esophageal squamous cell carcinoma cell lines; ZEC400 is a primary cell with a good prognosis; and ZEC398 is a primary cell with a poor prognosis. The conditions for the four cell pressure screening and enrichment are as follows Figures 2 to 5 As shown. Flow cytometry was used to detect the binding ability of each round of screening library to ZEC400, ZEC398, KYSE150 and KYSE30 cells. Figure 6 As shown, with the continuous increase of screening pressure, the four types of cells showed enrichment of the library in the third round, because the fluorescence intensity was significantly stronger than that in the first round, and reached the maximum enrichment in the fifth round.

[0088] 1.4 Cell flow sorting and library construction (1) Prepare the probe kit: Take 2.5 µL (concentration: 4 µM) from the first round of screening (R1) and the last round of screening (R5) of ZEC398, ZEC400, KYSE150, and KYSE30, respectively, and the final total volume is 20 µL. Then amplify through three rounds of PCR.

[0089] (a) PCR1: Prepare PCR1 system 100 µL × 5 tubes: 20 µL template, 100 µL 5× amplification PCR Mix solution, 380 µL ddH2O; PCR1 amplification conditions: denaturation temperature: 95°C, annealing temperature: 60°C, extension temperature: 72°C, and amplification round number: 8.

[0090] (b) PCR2: Using the PCR1 amplified fragment as template, prepare 50 µL of PCR2 system: 1 µL template, 10 µL 5× amplification PCR Mix solution, 39 µL ddH2O. Divide the prepared 50 µL PCR2 system into 5 tubes, 10 µL in each tube. The number of amplification rounds is 8, 10, 12, 14, and 16. PCR2 amplification conditions: denaturation temperature: 95°C, annealing temperature: 60°C, extension temperature: 72°C, amplification rounds: 8, 10, 12, 14, and 16. Run the gel on 8% PAGE denaturing gel, observe the band results, and select the appropriate number of amplification rounds.

[0091] (c) PCR3: Using the fragment amplified by PCR1 as the template, prepare the PCR3 system (100 µL × 10 tubes): 40 µL template, 200 µL 5× amplification PCR Mix solution, 760 µL ddH2O; the number of amplification rounds is 14.

[0092] (d) The amplified product of PCR3 is made into single strand, desalted and suspended for drying (the steps are the same as steps 7 to 9 of the first round of cell screening).

[0093] (e) The probe-coated single-stranded DNA was diluted with DPBS to a concentration of 4 µM, denatured at 95°C for 5 min, cooled on ice for 5 min, and left at room temperature for 15 min.

[0094] (2) Prepare a large dish for each of HEEC and KYSE30 cells, and select cells with a cell density of 80%-90% or more and good cell growth. Discard the culture medium, add 2 mL of pre-cooled DPBS to wash the remaining culture medium, and repeat the washing 2-3 times.

[0095] (3) Add 1 mL of enzyme-free digestion solution to each HEEC and KYSE30 culture dish to digest into a cell suspension, centrifuge and remove the supernatant. The cell concentration is controlled at 6 × 10 5 cells / mL. Then add 200uL Wash Buffer and repeat the washing for 3 times.

[0096] (4) Add 80ul Binding Buffer to the EP tubes of HEEC and KYSE30 respectively to resuspend the cells, then add 20uL of single-stranded probe package, incubate at 4℃ for 30min, and shake every 5 minutes.

[0097] (5) After incubation, wash twice with 200 µL Wash Buffer, then resuspend with 200 uL Wash Buffer. Finally, test the prepared samples on a flow cytometer. Prepare 8 Q-PCR tubes and add 10 µL ddH2O to each well for later use. Collect 6 PCR wells for HEEC and KYSE30, and sort 1000 positive cells from each PCR well.

[0098] (6) Heat the sorted cells at 95°C for 10 min, add TSlinker3, UMI and 1 / 10×T4 DNA ligase buffer at a final concentration of 80 nM, mix well, and incubate at 95°C for 3 min; 55°C for 3 min; and room temperature for 10 min. Then add 2.5uL 10×T4 DNA ligase buffer to each well, mix well, incubate at room temperature for 20 min, and shake every five minutes. Then add 1uL T4 DNA ligase to each well, mix well, and leave at room temperature for 30 min, shaking every five minutes. Finally, inactivate at 65°C for 10 min.

[0099] (7) Three rounds of PCR in each well (a) Prepare 30 µL of PCR1 system: 24 µL of ligation complex, 6 µL of 5× library construction PCR Mix-1 solution; PCR1 amplification conditions: denaturation temperature: 95°C, annealing temperature: 57°C, extension temperature: 72°C, and 10 amplification rounds.

[0100] (b) PCR2: Using the fragment amplified by PCR1 as the template, prepare 10 µL of PCR2 system: 2.5 µL template, 0.6 µL 5× library PCR Mix-1 solution, 5.25 µL ddH2O, and 1.65uL library PCR Mix-2 solution. PCR2 amplification conditions: denaturation temperature: 95°C, annealing temperature: 57°C, extension temperature: 72°C, and amplification rounds of 25. Run the gel on 8% PAGE denaturing gel and observe the band results to see if the UMI is successfully connected.

[0101] (c) PCR3: Using the fragment amplified by PCR1 as the template, prepare 50 µL of PCR3 system: 27.5 µL template, 3 µL 5× library construction PCR Mix-1 solution, 8.25uL library construction PCR Mix-2 solution, 11.25 µL ddH2O. The number of amplification rounds is 20.

[0102] (8) Perform high-throughput sequencing on the library construction products.

[0103] 1.5 Molecular typing of esophageal squamous cell carcinoma cells and normal esophageal epithelial cells using probe kits The first round screening (R1) and fifth round screening (R5) libraries of KYSE30, KYSE150, ZEC400 and ZEC398 were mixed together to form a new enrichment library. The aptamers obtained from the four esophageal squamous cell carcinoma cell screening were only partially identical. These different and identical aptamers were mixed to form an esophageal squamous cell carcinoma aptamer probe package. The probe package was then used for flow sorting and high-throughput sequencing of KYSE30 and HEEC.

[0104] The results are as follows Figure 7 As shown in Figures 2A and 2B, the abundance diagram of the top 100 nucleic acid aptamers in the high-throughput sequencing results of KYSE30 and HEEC was analyzed. The horizontal axis is the top 100 nucleic acid aptamers arranged from high to low, and the vertical axis is their copy number. Figure 7 As shown in C, the top 100 were analyzed for differences through bioinformatics. First, different colors represent different copy numbers. The redder the color of the dot, the higher the copy number; the bluer the color of the corresponding location, the lower the copy number. The upper left and upper right areas represent nucleic acid aptamers that can clearly distinguish KYSE30 and HEEC cells. That is, the upper right area represents nucleic acid aptamers with relatively high expression levels on KYSE30; the upper left area represents nucleic acid aptamers with relatively high expression levels on HEEC. Figure 7 As shown in D Figure 7The six repetitions of the aptamer copy number in the upper left and upper right of C are made into a heat map. Different colors represent different binding abilities. The redder the stronger the binding, the bluer the weaker the binding. It can be seen that these aptamers can clearly distinguish between esophageal squamous cell carcinoma cells KYSE30 and normal esophageal epithelial cells HEEC.

[0105] Example 2: Use of nucleic acid aptamers to differentiate esophageal squamous cell carcinoma cells KYSE30 from normal esophageal epithelial cells according to Figure 7 The experimental data finally selected nine specific aptamers with relatively high copy numbers, commissioned a third-party biological company to synthesize them, and labeled the 5' end of the aptamer with fluorescein isothiocyanate (FITC). The binding ability of the candidate aptamers to KYSE30 cells, KYSE150 cells and HEEC cells was detected by flow cytometry, and the blank cell ESCwl-cell group without adding nucleic acid aptamers was used as a control. The specific operation of the flow cytometry experiment is as follows: 1) Take a 10 cm × 2 cm dish of KYSE30 cells, KYSE150 cells or HEEC cells with a confluence of 80% to 90%, remove the medium, wash twice with DPBS buffer solution, add 1 mL of enzyme-free digestion solution, digest at 37°C for 2 min, remove the supernatant, blow off from the bottom of the dish with washing buffer, collect the cells, wash once with washing buffer, resuspend with binding buffer, and place on ice for later use. 2) Dilute the FITC-labeled aptamer to 4 μM with DPBS, mix well, denature at 95°C for 5 min, cool on ice for 5 min, and renature at room temperature for 15 min to obtain the aptamer solution.

[0106] 3) Take 5 μL of 4 μM nucleic acid aptamer solution and add it to 95 μL of cell suspension, shake and mix, incubate on ice for 30 min, and shake every 3 to 5 min to allow the aptamer to fully bind to the cells.

[0107] 4) After incubation, centrifuge to remove the supernatant, wash twice with 200 μL of washing buffer, and finally resuspend with 200 μL of washing buffer and detect on a flow cytometer.

[0108] The sequence of the control aptamer (ESCwl-ctrl) is: TCG CCT GAC GGC AGT CAT TTG GGA CCGCCC CTA CTG TCT CAT GG.

[0109] The results are as follows Figure 8As shown in A, the aptamer ESCwl-1a binds to all three cells, indicating that it has no specificity; the aptamers ESCwl-7a, ESCwl-68a and ESCwl-75a bind to KYSE30 and KYSE150 cells but not to HEEC cells, indicating that these three aptamers are specific to esophageal squamous cell carcinoma cells.

[0110] Will Figure 8 The flow fluorescence data of A is displayed by the signal-to-background ratio statistics, and the calculation formula is as follows, where F is the fluorescence intensity,

[0111] It can be seen that the ratios of aptamers ESCwl-7a, ESCwl-68a, and ESCwl-75a all reached 2 or above, further indicating the specific binding of these aptamers to KYSE30 and KYSE150 cells ( Figure 8 Middle B).

[0112] In order to detect the binding affinity of the aptamer, the concentration gradient dilution method was used to make the final concentrations in the incubation system 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, and 0.78nM, respectively. The aptamers were incubated with KYSE30 cells at 4°C for 30 minutes in a 100µL Binding Buffer incubation system. After the incubation, the cells were washed twice with WB, and then suspended with 200µL WB. Finally, the binding affinity with KYSE30 cells was detected by flow cytometry, with blank cells as the control. The equation Y=Bmax X / (K d +X) to fit the relationship between the cell fluorescence signal and the aptamer concentration, and further calculate the dissociation constant (K d ).

[0113] The results are as follows Figure 8 As shown in C, the K of aptamers ESCwl-1a, ESCwl-7a, ESCwl-68a and ESCwl-75a against KYSE30 cells d The values ​​were all in the nanomolar range, namely 5.0±0.9nM, 22.0±9.7nM, 33.1±19.4nM, and 63.3±40.6nM, respectively, indicating that these aptamers had a high affinity for KYSE30 cells.

[0114] Figure 8 Figure D lists the specific sequences of aptamers ESCwl-1a, ESCwl-7a, ESCwl-68a and ESCwl-75a after truncation, which were commissioned to a third-party biological company for synthesis, and fluorescein isothiocyanate (FITC) was labeled at the 5' end of the aptamer.

[0115] Example 3: Characterization of the binding of nucleic acid aptamers to different cells In order to verify the binding of the selected aptamers to different cells, three aptamers ESCwl-7a, ESCwl-68a and ESCwl-75a with good affinity and specificity were selected for further verification. Human esophageal squamous cell carcinoma cell KYSE510, human cervical cancer cell Hela, human ovarian cancer cell 3AO, human gastric cancer cell MGC803, human breast cancer cell SUM159, human colorectal cancer cell HCT-8, human bladder cancer cell YTS-1, and human acute lymphoblastic leukemia cell CEM were selected for flow cytometry detection. The method of flow cytometry detection refers to Example 2, and the only difference is that the cells to be tested are different. The results of flow cytometry detection show that the aptamers ESCwl-7a, ESCwl-68a and ESCwl-75a have greater binding to KYSE510, HCT-8, YTS-1, and CEM, and have relatively small binding to Hela, SUM159, 3AO and MGC803 ( Fig. 9 ), indicating that these aptamers are universal to different cancer types and their targets are expressed in different cancer types.

[0116] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims

1. A composition, characterized in that The composition comprises at least two of the nucleic acid aptamers ESCwl-7a, ESCwl-68a and ESCwl-75a, The nucleic acid aptamer ESCwl-7a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.1; The nucleic acid aptamer ESCwl-68a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.2; The nucleic acid aptamer ESCwl-75a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.

3.

2. A nucleic acid aptamer, characterized in that The nucleic acid aptamer includes any one of the nucleic acid aptamers ESCwl-7a, ESCwl-68a or ESCwl-75a, The nucleic acid aptamer ESCwl-7a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.1; The nucleic acid aptamer ESCwl-68a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.2; The nucleic acid aptamer ESCwl-75a is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO.

3.

3. A nucleic acid aptamer derivative, characterized in that: The nucleic acid aptamer derivative is obtained by performing the following operations on each single nucleic acid aptamer in the nucleic acid aptamers ESCwl-7a, ESCwl-68a and ESCwl-75a in claim 1 or 2: connecting fluorescein, anti-tumor drugs, radioactive elements, biological enzymes, biotin and / or nanomaterials to one end or the middle of the single nucleic acid aptamer to obtain a nucleic acid aptamer derivative having the same function as the single nucleic acid aptamer.

4. Application, characterized in that, The application is the application of the composition according to claim 1, the nucleic acid aptamer according to claim 2 and / or the nucleic acid aptamer derivative according to claim 3 in any of the following: (B1) Use in the preparation of products for identifying or assisting in the identification of tumor cells; (B2) Application in identifying or assisting in identifying tumor cells; (B3) Use in the preparation of products for identifying or assisting in the identification of tumor patient samples; (B4) Application in identifying or assisting in identifying samples from tumor patients; (B5) Application in the preparation of products for imaging tumor cells (B6) Application in imaging of tumor cells; (B7) Use in the preparation of a product for imaging tumor patient samples (B8) Application in imaging of tumor patient samples; (B9) Use in the preparation of products for molecular typing or auxiliary molecular typing of tumor cells and / or tumor patients; (B10) Application in molecular typing or auxiliary molecular typing of tumor cells and / or tumor patients; (B11) Use in the preparation of products for treating tumors; (B12) Use as a drug in the treatment of tumors; (B13) Application in tumor-related physiological and pathological research; (B14) Application in the preparation of products for tumor-related physiological and pathological research.

5. A reagent, the active ingredients of which include the composition according to claim 1, the nucleic acid aptamer according to claim 2 and / or the nucleic acid aptamer derivative according to claim 3.

6. Application, characterized in that, The application is the application of the reagent according to claim 5 in any of the following: (C1) Application in identifying or assisting in identifying tumor cells; (C2) Application in identification or assisting identification of tumor patient samples; (C3) Application in imaging of tumor cells; (C4) Application in imaging of tumor patient samples; (C5) Application in molecular typing or auxiliary molecular typing of tumor cells and / or tumor patients; (C6) Application in the preparation of drugs for treating tumors; (C7) Application in tumor-related physiological and pathological research.

7. The use according to claim 4 or 6, characterized in that: The tumor cells include, but are not limited to, human esophageal squamous cell carcinoma cells, human cervical cancer cells, human breast cancer cells, human colorectal cancer cells, human bladder cancer cells, and human acute lymphoblastic leukemia cells.

8. A method for identifying or assisting in identifying tumor cells, the method comprising contacting the tumor cells with the composition of claim 1, the nucleic acid aptamer of claim 2, the nucleic acid aptamer derivative of claim 3 and / or the reagent of claim 5 to identify or assist in identifying whether the cells are tumor cells.

9. The method according to claim 8, characterized in that The tumor cells include, but are not limited to, human esophageal squamous cell carcinoma cells, human cervical cancer cells, human breast cancer cells, human colorectal cancer cells, human bladder cancer cells, and human acute lymphoblastic leukemia cells.

10. The method according to claim 8 or 9, characterized in that: The tumor cells include but are not limited to: human esophageal squamous cell carcinoma cell KYSE30 and human esophageal squamous cell carcinoma cell KYSE150.

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