Aptamer closed type cell screening method, nucleic acid aptamer prepared by method and application of nucleic acid aptamer

Abstract

The invention provides an aptamer closed type cell screening method, a nucleic acid aptamer prepared through the method and application, and the method comprises the following steps: screening by using a Cell-SELEX method, co-incubating a closed aptamer and positively screened cells, and pre-closing the positively screened cells; during forward screening, a library and excessive closed aptamers are added, so that enrichment of aptamers combined with closed targets in the library is inhibited, and enrichment of aptamers combined with unclosed targets in the library is promoted. According to the method disclosed by the invention, the nucleic acid aptamer specifically bound with a protein target with relatively low cell membrane surface abundance can be found, so that a low-abundance potential tumor marker is found, and more potential molecular targets and characteristic molecular tools are provided for precise diagnosis and targeted therapy of tumors. The gastric cancer specific nucleic acid aptamer is screened out through the method, and a molecular tool which is high in specificity, strong in affinity, high in thermal stability and easy to store and modify is provided for specific recognition of gastric cancer cells and discovery of potential gastric cancer biomarkers.

Description

Technical Field

[0001] The present invention relates to the technical field of aptamers, and in particular to a method for screening aptamer closed cells and a nucleic acid aptamer prepared by the method and its application. Background Art

[0002] Gastric cancer originates from the gastric mucosal epithelial tissue. It is a malignant tumor with high morbidity and mortality. It ranks among the top five in the world in terms of new cancer cases and deaths, of which more than 40% are new cases in China. Gastric cancer patients in my country have the characteristics of high morbidity, low early diagnosis rate and short survival. The existing early diagnosis methods and treatment methods for gastric cancer are difficult to meet the clinical needs of patients. At present, there is a lack of molecular targets for targeted treatment of gastric cancer, and there are few targeted drugs approved for gastric cancer indications, mainly including anti-HER2 antibodies, anti-angiogenesis targeted drugs and PD-1 / PD-L1 inhibitors. Therefore, the discovery of new tumor markers and their molecular probes is of great significance for promoting the precise diagnosis and treatment of gastric cancer.

[0003] Aptamers are ssDNA or RNA obtained in vitro through systematic evolution of ligands by exponential enrichment (SELEX), usually 25-60 nt in length, and can bind to targets with high specificity and affinity. The traditional in vitro screening method for aptamers is to obtain aptamers that specifically bind to target molecules by cyclic enrichment of the initial library in vitro. When used in vivo, the aptamers obtained by this method may face the problem of decreased specificity and affinity due to changes in the binding environment and different conformations of the target molecule in vitro and in vivo.

[0004] Cell screening (Cell-SELEX technology) is a method for screening nucleic acid aptamers based on membrane proteins on the surface of living cells. Unlike traditional SELEX, which has clear target molecules and known properties, cell screening can simultaneously screen multiple membrane proteins with unknown information and in their natural conformations, providing a new way to discover biomarkers and even drug targets. Nucleic acid aptamer cell screening technology has solved the problems faced by aptamers obtained by in vitro screening in in vivo applications to a certain extent, but it also has many limitations. Since its discovery, cell screening technology has been widely used in the fields of tumor cell identification and capture, discovery of potential tumor markers, etc., but long-term research results have accumulated and found that the targets bound by nucleic acid aptamers obtained by this method have certain selectivity. Nucleic acid aptamers for certain protein targets are easier to be screened out in different laboratories, from different initial libraries, and in different screening experiments, for example, aptamers that specifically bind to proteins such as PTK7 and CD71.

[0005] Therefore, developing a new method for screening potential protein targets is a technical problem that needs to be solved urgently. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a closed cell screening method for nucleic acid aptamers that is different from conventional screening, and nucleic acid aptamers obtained by screening using the method and their applications. The present invention solves the problem that the prior art cannot effectively screen out nucleic acid aptamers of potential protein targets.

[0007] On the one hand, the present invention provides an aptamer-blocked cell screening method, comprising screening using the Cell-SELEX technology, wherein before forward screening, a blocking aptamer is co-incubated with positive screening cells to pre-block the positive screening cells; during forward screening, a library and an excess of blocking aptamers are added to inhibit the enrichment of nucleic acid aptamers that bind to blocked targets in the library and promote the enrichment of nucleic acid aptamers that bind to unblocked targets in the library.

[0008] Further, the following steps are included:

[0009] S1: initial library synthesis;

[0010] S2: adding blocking aptamers to pre-block the positive sieve cells to obtain pre-blocked positive sieve cells;

[0011] S3: Add the initial library to the reverse screening cells for reverse screening, and collect the supernatant; then add the supernatant and excess blocked aptamers to the pre-blocked positive screening cells for forward screening, separate the nucleic acid aptamers bound to the positive screening cells, and obtain the primary library by PCR amplification;

[0012] Alternatively, the initial library and excess blocked aptamers are added to pre-blocked positive screening cells for forward screening, and the nucleic acid aptamers bound to the positive screening cells are separated; then, the initial library and the excess blocked aptamers are added to the reverse screening cells for reverse screening, and the supernatant is collected and amplified by PCR to obtain the primary library;

[0013] S4: repeat step S3 to obtain a ssDNA enriched library;

[0014] S5: The ssDNA enriched library is sequenced and affinity tested to screen out nucleic acid aptamers that specifically bind to potential targets of positive screening cells.

[0015] Preferably, in step S1, the sequences of the initial library and the primers used are as follows:

[0016] 5'-CGTACGGTCGACGCTAGC(N44)CACGTGGAGCTCGGATCC-3'

[0017] 5'-FAM-CGTACGGTCGACGCTAGC-3'

[0018] 5'-biotin-GGATCCGAGCTCCACGTG-3'; or

[0019] 5'-AGACCGAGCTGAGTCAGTAT(N47)AGAGTAGTGAGTGCGAGAAC-3'

[0020] 5'-FAM-AGACCGAGCTGAGTCAGTAT-3'

[0021] 5'-biotin-GTTCTCGCACTCACTACTCT-3'

[0022] Preferably, in step S2, the positive screening cells include tumor cells, and more preferably, the tumor cells are gastric cancer cells;

[0023] Preferably, in step S2, the blocking aptamer comprises a nucleic acid aptamer targeting a known protein target of a positive screening cell;

[0024] More preferably, the known protein targets include PTK7, CD71, PTPRF, and ITGA3;

[0025] More preferably, the blocking aptamer comprises one or more of CY1-1f, XQ2d, ZAJ4a, and ZAJ2c, wherein the sequence of CY1-1f is shown in SEQ ID NO.2, the sequence of XQ2d is shown in SEQ ID NO.24, the sequence of ZAJ4a is shown in SEQ ID NO.25, and the sequence of ZAJ2c is shown in SEQ ID NO.26;

[0026] Preferably, in step S3, the mass ratio of the blocking aptamer to the initial library is 10-20:1;

[0027] Preferably, step S11 of primary screening is also included between steps S1 and S2: the initial library and positive screening cells are co-incubated, the nucleic acid aptamers bound to the positive screening cells are separated, and PCR amplification is performed to obtain a primary library.

[0028] In a second aspect of the present invention, a nucleic acid aptamer is provided, which is prepared by the above screening method;

[0029] Preferably, the nucleotide sequence of the nucleic acid aptamer includes any one of the following 1)-5):

[0030] 1) The nucleotide sequence is any one of SEQ ID NO.1-23;

[0031] 2) A nucleotide sequence having the same or similar function obtained by modifying the nucleotide sequence of sequence 1);

[0032] 3) A nucleotide sequence having the same or similar function obtained by deleting or adding nucleotides from the nucleotide sequence of sequence 1);

[0033] 4) One or more positions of the nucleotide sequence of sequence 1) are connected to a signal molecule, an active molecule and / or a functional group to obtain a nucleotide sequence with the same or similar function;

[0034] 5) The nucleotide sequence of sequence 1) is replaced by nucleotides to obtain a nucleotide sequence that has a homology of more than 80% with sequence 1) and has the same or similar function.

[0035] In a specific embodiment of the present invention, the modification includes inverted dT, phosphorylation, methylation, amination, sulfhydrylation, and isotopization.

[0036] In a specific embodiment of the present invention, the signal molecule includes a fluorescein molecule or a biotin molecule.

[0037] In a specific embodiment of the present invention, the functional groups include radioactive substances, therapeutic substances, nanomaterials, biological enzymes, cells and / or vesicles.

[0038] The third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned nucleic acid aptamer.

[0039] The fourth aspect of the present invention provides a detection reagent, comprising the above-mentioned nucleic acid aptamer.

[0040] The fifth aspect of the present invention provides a kit comprising the above-mentioned nucleic acid aptamer.

[0041] In a fifth aspect, the present invention provides the use of the above-mentioned nucleic acid aptamer, drug composition, detection reagent, and kit in any one of the following (1)-(5):

[0042] (1) Preparing products for identifying or assisting in identifying tumor cells;

[0043] (2) Preparation of products for molecular typing of tumor cells;

[0044] (3) Preparation of products for tumor diagnosis or tumor in vivo imaging;

[0045] (4) Preparation of products for tumor-related tumor markers or molecular probes;

[0046] (5) Preparation of targeted drugs for treating tumors;

[0047] Preferably, the tumor cells are gastric cancer cells and the tumor is gastric cancer.

[0048] The technical principle of the present invention is: in the prior art, nucleic acid aptamers are generally obtained through forward screening and reverse screening, but the nucleic acid aptamers obtained by this method are selective for certain targets. Under different initial libraries and experimental conditions, nucleic acid aptamers for certain targets are easier to be screened out, which may be due to the greater expression abundance of these targets or the stronger ability to bind to nucleic acids. Therefore, the types of specific nucleic acid aptamers screened by the existing methods are greatly affected by the abundance of their target expression, and many sequence information binding to proteins with relatively low abundance cannot be effectively screened out. After reaction research and experiments, the inventor found that by using an aptamer-enclosed cell screening method for cells, that is, adding an aptamer with a known target to competitively block its target, the sequence targeting the same target in the screening library cannot be combined with it and cannot be enriched, while the sequence binding to other targets can be smoothly enriched, thereby obtaining nucleic acid aptamers that specifically bind to protein targets with relatively low abundance on the cell membrane surface, discovering low-abundance potential tumor markers, and providing more potential molecular targets and characteristic molecular tools for accurate diagnosis and targeted treatment of diseases.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) The present invention provides an aptamer-enclosed cell screening method, which can discover nucleic acid aptamers that specifically bind to protein targets with relatively low abundance on the cell membrane surface, thereby discovering low-abundance potential tumor markers, providing more potential molecular targets and characteristic molecular tools for accurate diagnosis and targeted treatment of tumors.

[0051] (2) The present invention screened out a series of gastric cancer-specific nucleic acid aptamers that bind to human gastric cancer cells with high affinity and high specificity and can bind to different targets, providing a molecular tool with high specificity, strong affinity, high thermal stability, and easy storage and modification for the specific identification of gastric cancer cells and the discovery of potential biomarkers for gastric cancer.

[0052] (3) The nucleic acid aptamer of the present invention can be widely used in tumor cell molecular typing, gastric cancer cell specific capture and tumor in vivo imaging, as well as the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a diagram of the flow cytometry results of the screening process in Example 1;

[0054] Figure 2 There is a highly homologous fragment of about 10 nt in length in the enriched library 1 in Example 1;

[0055] Figure 3 Flow cytometry characterization diagram of the candidate aptamer and truncated optimized sequence in Example 2;

[0056] Figure 4 Cell affinity characterization diagram of the candidate aptamer and truncated optimized sequence in Example 2;

[0057] Figure 5 This is a flow cytometry test result diagram of the nucleic acid aptamer competition binding experiment in Example 2;

[0058] Figure 6 This is a graph showing the serum stability test of CY1-1f and its modified chains in Example 2;

[0059] Figure 7 This is a cell affinity characterization diagram of the CY1-1f modified chain in Example 2;

[0060] Figure 8 is a graph showing the binding ability of the nucleic acid aptamer in Example 2 to different tumor cell lines;

[0061] Fig. 9 The specific capture of free tumor cells by nucleic acid aptamer CY1-1f in Example 3;

[0062] Fig.10 This is a diagram of the application of nucleic acid aptamer CY3-6a in in vivo imaging in Example 4;

[0063] Fig.11 Flow chart of the screening method in Example 1. DETAILED DESCRIPTION

[0064] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0065] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods. The cells used in the present invention were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0066] Technical terms

[0067] The term "aptamer", also known as nucleic acid aptamer, refers to an oligonucleotide fragment that can bind to a specific target with high affinity and specificity. The target can be metal ions, small molecules, macromolecules, cells, tissues, and mixed targets. Aptamers are folded into a specific three-dimensional structure, and bind to the target with high affinity and specificity through spatial configuration complementarity, mainly short single-stranded nucleotide sequences (single-stranded DNA or RNA).

[0068] The term "screening" refers to the process of separating the target-oligonucleotide complex from the free nucleotides by reacting the target with a large-capacity random oligonucleotide library, and then amplifying the oligonucleotides forming the complex using PCR in vitro amplification technology. The above screening steps are repeated many times to finally obtain a nucleotide aptamer that has a specific binding effect with the target.

[0069] The term "positive screening", also known as "positive screening", refers to the target. When cells are positively screened, a target cell line can be selected.

[0070] The term "reverse screening" is also known as "reverse screening", which means that the target is a potential interferent, or a blank matrix (solid phase or background matrix). When performing cell reverse screening, similar cell lines can be selected.

[0071] The term "SELEX technology" refers to the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This technology can be used to screen out nucleic acid aptamers (Aptamers) that are specific and highly affinity with the target substance from a random single-stranded nucleic acid sequence library. The basic process of SELEX is to synthesize a single-stranded oligonucleotide library by chemical methods, mix it with the target substance, and the oligonucleotide chain binds to the target substance to form a complex. After removing the unbound nucleotide chain, the bound nucleotide chain is separated from the target substance. PCR amplification is performed using this oligonucleotide molecule as a template, and its product is used for the next cycle of screening. After several cycles, nucleic acid aptamers that can bind to the target substance with high affinity and high specificity can be obtained. If living cells are used in the SELEX process, it is called Cell-SELEX technology. Please refer to the literature "Cell-SELEX, an Effective Way to the Discovery of Biomarkers and Unexpected Molecular Events" or "Development of DNA aptamers using Cell-SELEX".

[0072] The term "blocking aptamer" refers to a nucleic acid aptamer that can bind to a known target of a cell, competitively block the cell target, inhibit the binding of nucleic acid aptamers in the library that have the same target as the blocking aptamer, and then screen out nucleic acid aptamers that bind to potential targets.

[0073] Example 1 Nucleic Acid Aptamer Screening

[0074] 1. Cell culture

[0075] The specific culture conditions of different cells are shown in Table 1. The complete culture medium used was added with 10% FBS and 100U / mL penicillin and streptomycin, and the cells were cultured in a carbon dioxide cell culture incubator with a CO2 concentration of 5%. The digestion solution used for cell passage was 0.25% Trypsin-EDTA, and the cryopreservation solution used for cell freezing was a commercial serum-free cell cryopreservation solution.

[0076] Table 1 Cell culture conditions

[0077]

[0078] 2. Library synthesis and solution preparation

[0079] 1) Library synthesis: To prevent DNA contamination in different screening experiments, the present invention used two initial DNA libraries Lib80YY and LibHZ3 with different primer sequences (the former was used for screening of AGS cells, and the latter was used for screening of MKN45 cells). The library sequences and related primer sequence information are shown in Table 2. All sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0080] Table 2 DNA library and primer sequence information

[0081]

[0082] Wherein, N represents a random base, and the numerical subscript of N represents the number of random bases.

[0083] 2) Solution preparation

[0084] a) Washing buffer (WB): Add 2.25 g D-(+)-glucose and 2.5 mL 1 M MgCl2 to 500 mL DPBS and store in a 4°C refrigerator.

[0085] b) Binding buffer (BB): Add 100 μL of bovine serum albumin (BSA, 10 mg / mL) and 20 μL of herring sperm DNA (hsDNA, 5 mg / mL) to 880 μL of WB to form 1 mL of BB and store in a 4°C refrigerator.

[0086] 3. Conventional cell screening

[0087] 1) Cell preparation: Newly revived cells were passaged twice or more before use. Cells with good growth status were selected for plating 24-36 hours in advance. The number of cells plated was such that the cell confluence reached 90-95% or more after 24-36 hours and the cells were in the logarithmic growth phase. This routine cell screening used AGS as positive screening cells and GES-1 as negative screening cells.

[0088] 2) Library preparation: The first round of library Lib80YY input was 2OD, and the library input was reduced in rounds according to the selection of screening pressure. The initial library (pool 0) powder was centrifuged at high speed and then dissolved in about 100 μL DPBS. After vortex mixing, it was denatured and renatured (denatured at 95℃ for 5 minutes, cooled on ice for 5 minutes, and renatured at room temperature for 15 minutes), and then an appropriate amount of pre-cooled BB was added for use.

[0089] 3) Forward screening: In order to fully enrich the ssDNA bound to the target cells, only forward screening was performed in the first two rounds of screening without reverse screening. Take out the positive screening cell AGS prepared in advance, discard the culture medium, wash twice with 2mL WB (pre-cooled at 4℃), add the pre-treated library in step 2) and incubate on a shaker at 4℃ for 1h, and the incubation time decreases with each round. After the incubation, discard the supernatant and wash thoroughly with 2mL WB. The washing intensity increases with each round to fully remove the ssDNA that is not bound to the positive screening cells or is not firmly bound. After washing, discard the supernatant, add an appropriate amount of ultrapure water, collect the positive screening cells into a clean EP tube with a cell scraper, denature at 95℃ for 10min, and fully centrifuge to remove the precipitate after denaturation. Take the scraped supernatant for later use.

[0090] 4) Reverse screening: Reverse screening starts from the third round and is performed before the third round of forward screening. Take out the GES-1 cells prepared in advance, discard the culture medium, wash as in the forward screening, add the pre-treated library in step 2) and incubate on a shaker at 4°C for 1 hour. The incubation time increases with each round. After the incubation, transfer the supernatant to the treated positive screening cell dish for the third round of forward screening.

[0091] 4. Screening of aptamer-enclosed cells

[0092] 1) Cell preparation: Same as conventional cell screening.

[0093] 2) Preparation of library and blocking aptamer: The input amount of the first round library Lib80YY is 2OD, and the input amount of the library is reduced round by round according to the selection of screening pressure. Except for the first round of screening (also called primary screening), blocking aptamers (aptamers with known targets that can competitively block their targets) are added to the forward screening incubation library, and the input amount is 10-20 times that of the library. No blocking aptamer is added for reverse screening. The initial library and blocking aptamer are dissolved in DPBS, vortexed and mixed, and then renatured, and then an appropriate amount of pre-cooled BB is added for standby use.

[0094] 3) Forward screening: In the first round of forward screening, in order to retain as many single copy sequences in the initial library that can bind to target cells as possible, no reverse screening was performed, and no blocking aptamers were added during the screening process.

[0095] Before the second round of forward screening, reverse screening was performed without blocking aptamers. After the reverse screening was completed, the supernatant was taken, and the blocking aptamer was added to make the final concentration 20 times that of the input library. The supernatant was transferred to the positive screening cell culture dish after blocking incubation and incubated on a shaker at 4°C for 30 minutes. The blocking incubation method of the positive screening cells was as follows: the positive screening cells were incubated in BB containing 200nM blocking aptamers for 20 minutes, and the supernatant was removed after the incubation without washing.

[0096] The operation after the positive screening incubation is completed is the same as the conventional cell screening in step 3.

[0097] 5. Preparation of ssDNA library after screening

[0098] 1) PCR1: 1 / 4 volume of 5×PCR mix was added to the scraped supernatant obtained after screening to prepare a PCR1 amplification system. The amplification program was as shown in Table 3, and the number of cycles was 8.

[0099] 2) PCR2: Take 1 μL of PCR1 product, add 10 μL of 5×PCR mix and 39 μL of DEPC water to prepare a PCR system with a total volume of 50 μL, and divide it equally into 5 PCR tubes. Prepare another group without adding PCR1 product as a negative control to monitor whether there is DNA aerosol contamination during the operation. The amplification program is shown in Table 3. The number of cycles in the 5 groups is 8, 12, 14, and 16, respectively, and the negative control group is 16 cycles. The PCR2 product was characterized by 8% nucleic acid denaturing PAGE gel electrophoresis, and the optimal amplification cycle number with the highest yield and the least by-products was obtained based on the analysis of the characterization results.

[0100] Table 3 PCR amplification program

[0101]

[0102] 3) PCR3: Take 10 μL of PCR1 product and add 100 μL 5×PCR mix and 390 μL DEPC water to prepare a PCR system with a total volume of 500 μL. The amplification program is shown in Table 3. The number of cycles is the optimal number of cycles obtained by PCR2 characterization (usually 12 to 14 cycles are optimal according to experience).

[0103] 4) Single-strand preparation: Add 70 μL of streptavidin agarose bead suspension to a clean microcolumn tube to be filtered, filter the liquid under pressure, and keep the beads on the upper part of the filter. Wash the column with beads twice with 100 μL DPBS. After washing, filter the PCR3 product solution through the beads under pressure twice. At this time, you can observe the green fluorescence of the beads under ultraviolet light. Wash the column twice with 100 μL DPBS, rinse the microcolumn with 100 μL 0.2M NaOH solution, and collect the effluent, which contains ssDNA.

[0104] 5) Desalting: Pre-wash the NAP-5 desalting column with no less than 15 mL of ultrapure water, add 100 μL of the effluent containing ssDNA to the desalting column, then add 400 μL of ultrapure water, and add 600 μL of ultrapure water to elute the ssDNA after the liquid stops flowing out, and collect the effluent, the volume is about 600 μL. Use UV260 to calibrate the ssDNA content. After the effluent is vacuum-dried, store it at -40°C, which is the ssDNA library used for the next round of screening after one enrichment. Fig.11 A flow chart of the screening process is shown.

[0105] 6. Flow cytometry and high-throughput sequencing

[0106] 1) Flow cytometry

[0107] a) Cell preparation: Select target cells with good growth status and plate them according to the required cell quantity one day in advance. When ready to be used, the cell confluence should reach about 85%. First, discard the culture medium in the dish, rinse the bottom of the dish with DPBS twice, add an appropriate amount of trypsin-free digestion solution and digest at 37°C for 5-10 minutes. After digestion, collect the cell suspension in a clean EP tube, centrifuge at 1000rpm for 3 minutes, discard the supernatant, wash the cell pellet once with WB, and finally add a slightly excess of BB to resuspend the cells according to the volume of 100μL of each flow cytometry sample for later use.

[0108] b) Library preparation: Add appropriate amount of DPBS to the enriched library 1, enriched library 2, and enriched library 3) in the freeze-dried library after each round of preparation (including step 2) to a final concentration of 1 μM, denature in a 95°C metal bath for 5 min, and immediately cool on ice for 5 min, and finally renature at room temperature for 15 min for use.

[0109] c) Incubation and washing: Add the prepared cells and library to a total volume of 100 μL per sample and a library concentration of 200 nM, incubate on ice for 30 minutes, vortex the sample every few minutes to prevent the cells from sinking to the bottom. After the incubation, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, wash twice with 200 μL WB, and finally add 100-200 μL WB to resuspend the cell pellet and prepare for the machine.

[0110] d) Detection and data processing: When performing flow cytometry, first use blank cells to select parameters so that the background fluorescence intensity is between 102-103. After adjusting the voltage, keep it unchanged and detect the sample to be tested. The flow cytometry results are analyzed using FlowJo 10. The test results are shown in Figure 1 , Figure 1 A is the flow cytometry result of the screening process of enrichment library 1. Figure 1 B is the result of flow cytometry detection of enrichment library 2 screening process, Figure 1C is the flow cytometry detection result of enrichment library 3 screening process.

[0111] 2) High-throughput sequencing

[0112] a) Enriched library 1: AGS was used as the positive screening cell and GES-1 was used as the negative screening cell for conventional screening. After 9 rounds, enriched library pools 1-9 were obtained. After PCR amplification reaction, PCR bands with no non-specific bands, clear target bands, and bright lanes were selected for amplification. After amplification, they were sequenced by Sangon Biotech (Shanghai) Co., Ltd. A total of 6821 single sequences were measured through high-throughput sequencing. All measured sequences were multi-sequence aligned using clustal omega and then seqlog was performed using weblogo3 to analyze the results (see Figure 2 ) showed that there was a highly homologous fragment of about 10 nt in the obtained enriched library, which indicated that most of the sequences in the library belonged to the same family and might bind to the same type of target. After analysis, it was found that it might bind to the PTK7 target.

[0113] b) Enrichment library 2: AGS was used as positive screening cell, GES-1 as negative screening cell, and CY1-1f as blocking aptamer for blocking screening. After 5 rounds, enrichment library pool 2-5 was obtained. After PCR amplification reaction, PCR bands with no non-specific bands, clear target bands, and bright lanes were selected for amplification. After amplification, sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. A total of 87,457 single sequences were measured through high-throughput sequencing. After analysis, it was found that there were almost no sequences in the same family as the blocking aptamer CY1-1f.

[0114] c) Enrichment library 3: MKN45 was used as the positive screening cell and AGS was used as the negative screening cell. The specific aptamers CY1-1f, XQ2d (referenced to Journal of the American Chemical Society, 2019, 141(27): 10760-10769, whose nucleotide sequence is shown in SEQ ID NO: 24), ZAJ4a (referenced to Analytical Chemistry, 2023, whose nucleotide sequence is shown in SEQ ID NO: 25), and ZAJ2c (referenced to ACS Applied Bio Materials, 2022, 5(7): 3461-3468, whose nucleotide sequence is shown in SEQ ID NO: 26) of the target that are known to be easily screened and identified by conventional cell screening methods based on previous experimental experience were selected as blocking aptamers for blocking screening. After 5 rounds, enrichment library pool 3-5 was obtained. After PCR amplification reaction, PCR bands without non-specific bands, clear target bands, and bright lanes were selected for amplification. After amplification, the DNA was sequenced by Sangon Biotech (Shanghai) Co., Ltd. A total of 809,867 unique sequences were obtained through high-throughput sequencing.

[0115] Example 2 Optimization and characterization of candidate nucleic acid aptamers and their application in preliminary molecular typing of tumor cells

[0116] 1. Optimization of candidate nucleic acid aptamers and characterization of their binding ability to target cells

[0117] 1) Enrichment library 1: Based on the enrichment of high-throughput sequencing and sequence homology analysis, three candidate nucleic acid aptamers (AGS-CY1-1, AGS-CY1-4, AGS-CY1-7) were selected and truncated and optimized (sequence information is shown in Table 4). The aptamers were labeled with FAM fluorescein and the candidate aptamers were determined by flow cytometry ( Figure 3 A), truncated optimized sequence ( Figure 3 B) Binding to target cells.

[0118] 2) Enrichment Library 2: Based on the enrichment of high-throughput sequencing and sequence homology analysis, 5 candidate nucleic acid aptamers (AGS-CY2-1, AGS-CY2-2, AGS-CY2-3, AGS-CY2-11, AGS-CY2-24) were selected and truncated and optimized (sequence information is shown in Table 4). The aptamers were labeled with FAM fluorescein and the candidate aptamers were determined by flow cytometry ( Figure 3 C), truncated optimized sequence ( Figure 3 D) Binding to target cells.

[0119] 3) Enrichment Library 3: Based on the enrichment of high-throughput sequencing and sequence homology analysis, 9 candidate nucleic acid aptamers were selected and truncated and optimized (sequence information is shown in Table 4). The aptamers were labeled with FAM fluorescein, and their binding to target cells was determined by flow cytometry ( Figure 3 E).

[0120] Table 4. Nucleic acid aptamer and its truncated chain sequence information

[0121]

[0122]

[0123]

[0124] 2. Affinity characterization of candidate aptamers and their truncated optimized chains

[0125] Flow cytometry was used to detect the dissociation constant of the binding of nucleic acid aptamers to target cells. The specific experimental method was the same as the flow cytometry detection in step 6 of Example 1. The final concentrations of the aptamers during incubation were set to 0 nM, 1.56 nM, 3.12 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM. The nucleic acid aptamers at different concentration gradients were respectively incubated with 2×10 5 The target cells were incubated on ice for 30 min, washed, and tested on the flow cytometer. The flow cytometer data were processed with FlowJo software and plotted with GraphPad Prism 8 software. The horizontal axis is the concentration of the nucleic acid aptamer (unit: nM), and the vertical axis is the average fluorescence intensity after deducting the background fluorescence value. The test results are shown in Figure 4 As shown. Figure 4 It can be seen that the dissociation constants (K D values) were 2.69±0.27nM, 3.69±0.63nM, 1.27±0.19nM, and 4.96±0.86nM, respectively, indicating that they all have strong affinities with target cells.

[0126] 3. Competition Binding Experiment

[0127] Flow cytometry was used to perform nucleic acid aptamer competition binding experiments. The specific experimental steps were the same as the flow cytometry detection in step 6 of Example 1, except that in addition to the aptamer to be tested labeled with FAM, a 20-fold excess concentration of a non-fluorescent labeled competitive aptamer was added to the competitive analysis sample. Whether there was a competitive relationship between the two aptamers was determined by analyzing the relationship between whether the competitive aptamer was added and whether the ability of the aptamer to be tested to bind to the target cell changed.

[0128] The experimental results showed that there was no competitive binding relationship between the aptamer CY1-1f obtained in the first conventional screening and CY2-1d, CY2-2c, CY3-2a and CY3-6a ( Figure 5 -A); There is no competition between the aptamers CY2-1d and CY2-2c obtained in the first blocking screening, and their binding ability is not inhibited by CY1-1f, CY3-2a and CY3-6a ( Figure 5 -B); the aptamers CY3-2a and CY3-6a obtained in the second closed screening have a competitive relationship with the binding of cells. Excess CY3-6a can completely inhibit the binding of CY3-2a to target cells, but excess CY3-2a has no inhibitory effect on the binding of CY3-6a to target cells, which indicates that the two may bind to the same target, and the binding ability of CY3-6a to the target is stronger than that of CY3-2a. However, there is no competitive relationship between these two aptamers and CY1-1f, CY2-1d and CY2-2c ( Figure 5 -C). In summary, there is no competitive binding relationship among aptamers CY1-1f, CY2-1d, CY2-2c, and CY3-6a, but there is competitive binding between CY3-2a and CY3-6a, and the latter has stronger binding ability.

[0129] 4. Serum stability testing and optimization of aptamer CY1-1f

[0130] Since natural nucleic acid aptamers are usually easily degraded by nucleases in the complex environment of the body, their stability is poor, which makes their half-life in the body short and their application is greatly restricted. Therefore, it is necessary to characterize and optimize the serum stability of the aptamer. Here, CY1-1f is used as an example to detect and optimize the serum stability of the aptamer.

[0131] 1) Preparation of test samples: The nucleic acid aptamers to be tested were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the nucleic acid aptamers to be tested were prepared with a concentration of 2 μM using freshly prepared DMEM medium containing 10% FBS, and then divided into 8 200 μL PCR tubes for standby use, with 17 μL in each aliquot.

[0132] 2) Incubation: Each sample was incubated at 37°C for 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h. After the incubation, the sample was quickly removed and stored at -80°C.

[0133] 3) Detection: After all samples are incubated, they are taken out together, denatured in a 95°C metal bath for 10 min, and 8% nucleic acid urea denatured PAGE gel is prepared for electrophoresis detection. After the electrophoresis, imaging detection is performed using a gel imager.

[0134] Imaging results (see Figure 6) showed that CY1-1f was continuously degraded in complete medium, the fragment length gradually shortened, and was almost completely degraded in about 48 hours. However, the aptamer modified with 3'inverted dT still maintained good fragment integrity after incubation in complete medium for 48 hours, and the serum stability was significantly improved. At the same time, affinity characterization (see Figure 7 ) showed that the modified aptamer still maintained good affinity with the target cells, and the dissociation constant (K D value) was 2.33±0.43nM.

[0135] 5. Application of preliminary molecular typing

[0136] Based on the above results, five sequences were selected: CY1-1f, CY2-1d, CY2-2c, CY3-2a, and CY3-6a for further flow characterization applications, such as Figure 8 As shown, the five truncated and optimized sequences have different binding abilities to different tumor cell lines, which can preliminarily realize the molecular typing of different tumor cells.

[0137] Example 3 Specific capture of free tumor cells by nucleic acid aptamer CY1-1f

[0138] The nucleic acid aptamer was used to specifically capture the positive cells in the mixed cells. The positive screening cells AGS were selected as positive cells in the mixed cell sample, and the human B lymphoma cells Ramos were selected as negative cells. First, the binding of the two cells to CY1-1f was investigated by flow cytometry. Ramos and CY1-1f almost did not bind ( Fig. 9 A), which can be used as negative cells. Flow cytometry was then used to detect the ability of CY1-1f to distinguish positive cells from negative cells in mixed samples ( Fig. 9 B), it can selectively bind to positive cells but not negative cells in a mixed cell population, thereby achieving differentiation between the two.

[0139] 1) Preparation of aptamer CY1-1f and magnetic bead conjugates

[0140] First, synthesize the aptamer CY1-1f and the control con chain with two T bases added to the 5' end and modified with biotin, and then renature (heat at 95℃ for 5min, on ice for 5min, and at room temperature for 15min) for use. Take an appropriate amount of streptavidin magnetic beads with a concentration of 10mg / mL according to the volume of 2μL of each sample, rinse once with DPBS, and perform coupling incubation in a BB system with an aptamer concentration of 500nM and a total volume of 100μL. Incubate at room temperature for 30min, and mix from time to time to prevent the magnetic beads from sinking to the bottom. After the incubation, remove the supernatant, rinse the magnetic beads with WB containing 0.01% Tween 3 times, and obtain CY1-1f-beads and con-beads for use.

[0141] 2) Specific capture of gastric cancer cell AGS by magnetic beads coupled with nucleic acid aptamers

[0142] a) Cell preparation: Select AGS cells with good growth status and plate them at least one day in advance. When the cell confluence reaches about 80%, take out the cells and rinse them twice with DPBS, add trypsin-free cell digestion solution and digest them at 37℃ for 5min. After digestion, centrifuge to remove the digestion solution, wash the cells once with WB, and then add 100-fold diluted Hoechst 33342 (100X) dye solution to stain at 37℃ for 10min to distinguish the nucleus staining of positive AGS cells from negative cells. After staining, centrifuge to remove the dye solution, wash the cells twice with WB, add an appropriate amount of BB to resuspend, and count the cells. Take another negative cell Romas, remove the culture medium, wash the cells twice with WB, resuspend them with BB, and count the cells. Prepare mixed cell samples with positive cell proportions of 0%, 5%, and 50%, respectively, and the total number of cells in each sample is 2105.

[0143] b) Cell capture: Add equal amounts of CY1-1f-beads to mixed cell samples with 0%, 5%, and 50% positive cells, and add con-beads to mixed cell samples with 50% positive cells as a control group, incubate at 4°C for 30 minutes, and mix occasionally. After incubation, wash three times with WB containing 0.01% Tween, and finally add 20 μL WB to resuspend for later use.

[0144] c) Observation under a fluorescence microscope: 10 μL of the captured sample obtained in the previous step was placed on a glass slide, covered with a cover glass, and observed and recorded under a fluorescence microscope.

[0145] The ability of the aptamer coupled to magnetic beads to capture positive cells was tested in mixed cell samples with different positive cell ratios. The positive cells were distinguished from the unstained negative cells by nuclear fluorescence staining. A random sequence was used as the control group (con group). The capture results are shown in Figure 2. Fig. 9 As shown in C, the conjugate of CY1-1f and magnetic beads has a specific capture effect only on positive cells, but not negative cells. No cells were captured in the control group. As the proportion of positive cells in the mixed cell sample increased, the number of cells captured by the CY1-1f group gradually increased, but the capture rate of positive cells was still low.

[0146] Example 4 Application of nucleic acid aptamer CY3-6a in in vivo imaging of mouse tumor tissue

[0147] 1. Construction of nude mouse subcutaneous xenograft tumor model:

[0148] A cage of 5 4-week-old male nude mice was used. MNK45 cells with good growth status were plated at least one day in advance. When the cell confluence reached about 90%, the cells were taken out. The culture medium was first aspirated and discarded. The bottom of the dish was rinsed with DPBS twice, and trypsin was added for digestion for 2-3 minutes. After the digestion was completed, the culture medium was added to terminate the digestion, the supernatant was removed by centrifugation, and the cells were resuspended in the culture medium and then counted. Each nude mouse was subcutaneously injected with 200 μL 5×10 6 cells, and the tumor diameter reaches 0.5-1.0 cm in about 2-3 weeks and can be used for in vivo imaging.

[0149] 2. In vivo imaging

[0150] Synthesize CY3-6a aptamer labeled with cy5.5 at the 5' end and control con chain labeled with cy5.5 at the 5' end, renature in advance, and inject 100 μL of 2 nmol fluorescent labeled CY3-6a or con chain into each nude mouse through the tail vein. Use IVIS Lumina III imaging system to collect fluorescence signals of nude mice at 0min, 10min, 30min, 60min, 120min, 360min, and 600min after tail vein injection, respectively. The excitation wavelength is 660nm, and the emission light signal around 710nm is collected. Use the imaging system's own software to process the imaging data.

[0151] Imaging results ( Fig.10 ) showed that 10 minutes after injection, the fluorescently labeled ssDNA reached all parts of the mouse body through the blood circulation, and compared with the control group, the aptamer CY3-6a had a specific targeting effect on the tumor tissue (circled area in the figure), and the fluorescent signal could still be observed in the tumor tissue 10 hours after injection.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for screening aptamer-enclosed cells, comprising screening using the Cell-SELEX method, characterized in that: Before forward screening, the blocking aptamer is co-incubated with the positive screening cells to pre-block the positive screening cells; during forward screening, the library and excess blocking aptamer are added to inhibit the enrichment of nucleic acid aptamers in the library that bind to the blocked target and promote the enrichment of nucleic acid aptamers in the library that bind to the unblocked target.

2. The method for screening aptamer-enclosed cells according to claim 1, characterized in that: The following steps are involved: S1: initial library synthesis; S2: adding blocking aptamers to pre-block the positive sieve cells to obtain pre-blocked positive sieve cells; S3: Add the initial library to the reverse screening cells for reverse screening, and collect the supernatant; then add the supernatant and excess blocked aptamers to the pre-blocked positive screening cells for forward screening, separate the nucleic acid aptamers bound to the positive screening cells, and obtain the primary library by PCR amplification; Alternatively, the initial library and excess blocked aptamers are added to pre-blocked positive screening cells for forward screening, and the nucleic acid aptamers bound to the positive screening cells are separated; then, the initial library and the excess blocked aptamers are added to the reverse screening cells for reverse screening, and the supernatant is collected and amplified by PCR to obtain the primary library; S4: repeat step S3 to obtain a ssDNA enriched library; S5: Sequencing and affinity testing of the ssDNA enriched library to screen out nucleic acid aptamers that specifically bind to potential targets of positive screening cells; Preferably, in step S1, the sequences of the initial library and the primers used are as follows: 5'-CGTACGGTCGACGCTAGC(N44)CACGTGGAGCTCGGATCC-3' 5'-FAM-CGTACGGTCGACGCTAGC-3' 5'-biotin-GGATCCGAGCTCCACGTG-3'; or 5'-AGACCGAGCTGAGTCAGTAT(N47)AGAGTAGTGAGTGCGAGAAC-3' 5'-FAM-AGACCGAGCTGAGTCAGTAT-3' 5'-biotin-GTTCTCGCACTCACTACTCT-3' Preferably, in step S2, the positive screening cells include tumor cells; more preferably, the tumor cells are gastric cancer cells; Preferably, in step S2, the blocking aptamer comprises a nucleic acid aptamer targeting a known protein target of a positive screening cell; More preferably, the known protein targets include PTK7, CD71, PTPRF, and ITGA3; More preferably, the blocking aptamer comprises one or more of CY1-1f, XQ2d, ZAJ4a, and ZAJ2c, wherein the sequence of CY1-1f is shown in SEQ ID NO.2, the sequence of XQ2d is shown in SEQ ID NO.24, the sequence of ZAJ4a is shown in SEQ ID NO.25, and the sequence of ZAJ2c is shown in SEQ ID NO.26; Preferably, in step S3, the mass ratio of the blocking aptamer to the initial library is 10-20:1; Preferably, step S11 of primary screening is also included between steps S1 and S2: the initial library and positive screening cells are co-incubated, the nucleic acid aptamers bound to the positive screening cells are separated, and PCR amplification is performed to obtain a primary library.

3. A nucleic acid aptamer, characterized in that: Prepared by the screening method according to claim 1 or 2; Preferably, the nucleotide sequence of the nucleic acid aptamer includes any one of the following 1)-5): 1) The nucleotide sequence is any one of SEQ ID NO. 1-23; 2) A nucleotide sequence having the same or similar function obtained by modifying the nucleotide sequence of sequence 1); 3) A nucleotide sequence having the same or similar function obtained by deleting or adding nucleotides from the nucleotide sequence of sequence 1); 4) One or more positions of the nucleotide sequence of sequence 1) are connected to a signal molecule, an active molecule and / or a functional group to obtain a nucleotide sequence with the same or similar function; 5) The nucleotide sequence of sequence 1) is replaced by nucleotides to obtain a nucleotide sequence that has a homology of more than 80% with sequence 1) and has the same or similar function.

4. The nucleic acid aptamer according to claim 3, characterized in that: The modifications include inverted dT, phosphorylation, methylation, amination, sulfhydrylation, and isotopization.

5. The nucleic acid aptamer according to claim 3, characterized in that: The signal molecule includes a fluorescein molecule or a biotin molecule.

6. The nucleic acid aptamer according to claim 3, characterized in that: The functional groups include radioactive substances, therapeutic substances, nanomaterials, biological enzymes, cells and / or vesicles.

7. A pharmaceutical composition comprising the nucleic acid aptamer according to any one of claims 3 to 6.

8. A detection reagent comprising the nucleic acid aptamer according to any one of claims 3 to 6.

9. A kit comprising the nucleic acid aptamer according to any one of claims 3 to 6.

10. Use of the nucleic acid aptamer according to any one of claims 3 to 6, the pharmaceutical composition according to claim 7, the detection reagent according to claim 8, or the kit according to claim 9 in any of the following (1) to (5): (1) Preparing products for identifying or assisting in identifying tumor cells; (2) Preparation of products for molecular typing of tumor cells; (3) Preparation of products for tumor diagnosis or tumor in vivo imaging; (4) Preparation of products for tumor-related tumor markers or molecular probes; (5) Preparation of targeted drugs for treating tumors; Preferably, the tumor cells are gastric cancer cells; and the tumor is gastric cancer.

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