CN114127293B - A method for in vitro screening of DNA aptamers of PD-L1 and its application in cancer diagnosis - Google Patents

Abstract

A novel method for screening the high-affinity and specific DNA nucleic acid aptamer of the programmed cell death receptor 1-ligand 1 (PD-L1), abbreviated as "endo-SELEX" (Encoded-SELEX). The starting random DNA library of the present method comprises two random sequence regions and an immobilization region (i.e., an internal coding) containing a recognition site for a type II restriction enzyme located therebetween. The library was pre-enriched using MCP-SELEX, followed by incubation of the library with PD-L1, followed by addition of restriction enzymes. The sequence capable of binding to PD-L1 has its internal coding structure disrupted, so that the sequence is not cleaved by a restriction enzyme and PCR amplification is retained. The obtained aptamer has the characteristic of higher affinity as the length of the inner code is longer, and is convenient for rapidly selecting high-affinity aptamer. The fluorescent-labeled aptamer is successfully used for fluorescent imaging of PD-L1 expression levels of normal human tonsil tissues and various tumor tissue sections, has the performance equivalent to that of antibodies, and has a superior application value.

Description

A method for in vitro screening of DNA aptamers of PD-L1 and its application in cancer diagnosis application in

technical field

The invention relates to a method for in vitro screening of a broad-spectrum tumor marker programmed cell death receptor 1-ligand 1 (PD-L1) DNA nucleic acid aptamer with high affinity and specificity and its application in cancer diagnosis , belonging to the field of biotechnology.

Background technique

Programmed cell death receptor 1 (PD-1) is a cell surface receptor, type I transmembrane protein belonging to the immunoglobulin superfamily, expressed on T cells and pro-B cells. PD-1 is an immune checkpoint that prevents autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in the lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells). PD-1 binds two ligands, programmed cell death receptor 1-ligand 1 (PD-L1) and programmed cell death receptor 1-ligand 2 (PD-L2). PD-L1 is mainly expressed in antigen-presenting cells such as dendritic cells and various tumor cells. Normally, the immune system responds to foreign antigens accumulated in the lymph nodes or spleen, promoting the proliferation of antigen-specific T cells. The combination of PD-1 and PD-L1 can transmit inhibitory signals, reduce the activation and proliferation of T cells, and enable tumor cells to obtain immune escape. Both PD-1 and PD-L1 can be used as targets, and their antibodies have been proven to enhance anti-tumor, anti-infection, anti-autoimmune diseases and organ transplantation survival rates. Currently, antibody drugs for cancer treatment based on blocking PD-L1/PD-1 binding on the market include K drug Pembrolizumab (Clin. Cancer Res. 2017, 23, 5666–5670), O drug and T drug (Atezolizumab).

How to screen patients who may benefit from PD-1/PD-L1 inhibitor therapy is the most clinical concern. PD-L1 immunohistochemical detection is a simple and effective method to predict the efficacy of PD-1/PD-L1 inhibitors. Currently, indications for PD-1/PD-L1 inhibitors approved by FDA/NMPA include lung cancer, melanoma, and urothelial cancer. As a predictive marker for the efficacy of PD-1/PD-L1 immune checkpoint inhibitor drugs, PD-L1 detection has been approved by the FDA as a companion or supplementary diagnosis for immunotherapy. At present, there are five main types of PD-L1 immunohistochemical detection kits/antibodies: 22C3, 28-8, SP263, SP142 and 73-10, which are tested on two immunohistochemical platforms Dako and Ventana respectively. At present, PD-L1 immunohistochemical detection technology faces the following main detection problems. (1) Different antibodies require different detection platforms. For example, the 22C3 and 28-8 antibody detection uses the DAKO AutoStainer Link 48 platform, while the SP142 and SP263 antibody detection uses the VentanaBenchmark Ultra platform. (2) Different antibody test results have different interpretation standards, and the interpretation of PD-L1 requires a lot of training by professional pathologists to ensure the accuracy of interpretation. (3) There are differences in the detection results of different antibodies. For example, although studies have shown that the test results of 28-8, 22C3 and SP263 are highly consistent, they are only comparisons of specific tumor types. There are still considerable differences in the test results of different antibody types among individual tumor types. In addition, antibodies also have disadvantages such as high price, poor stability, easy inactivation, and large performance differences between batches.

Nucleic acid aptamer (aptamer) is a kind of short-chain nucleotide sequence (RNA or DNA) that can specifically bind various target molecules, usually obtained by in vitro screening technology called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) (Science, 1990, 249, 505-510). SELEX is an in vitro screening technique for screening nucleic acid aptamers with high affinity and specificity through multiple rounds of affinity enrichment of target molecules from chemically synthesized random nucleic acid libraries. Compared with antibodies, nucleic acid aptamers have the advantages of long shelf life, high stability, small batch-to-batch variation, low or no immunogenicity, low cost, and convenient chemical modification (Biotechnol. Adv. 2018, 37, 28- 50), has shown a good application prospect in the field of tumor diagnosis. However, there are no relevant literature reports on aptamer-based PD-L1 detection kits and their application to the detection of PD-L1 expression levels in human cancer tissue sections.

Traditional SELEX technology utilizes cellulose acetate membranes to isolate aptamers bound to protein targets. In order to improve the screening efficiency and performance of nucleic acid aptamers, a variety of improved SELEX technologies have been reported, such as Capture-SELEX, Capillary electrophoresis-SELEX, MB-SELEX, Microfluidic-SELEX, Cell-SELEX, Tissue-SELEX, etc. ( Int. J. Mol. Sci. 2017, 18, 2142-2160). However, each of these screening techniques has certain limitations: Capture-SELEX needs to fix the library, and the enrichment effect is not good due to the unavoidable self-dissociation phenomenon of the library, requiring multiple rounds of screening; Capillary electrophoresis-SELEX can When screening nucleic acid aptamers, it is often impossible to recognize the target protein in the natural state; MB-SELEX and Microfluidic-SELEX need to immobilize the target protein on magnetic beads, which partially changes the secondary structure of the protein, and there are interface steric hindrance and Interface adsorption, thus limiting the affinity and specificity of nucleic acid aptamers; Cell-SELEX and Tissue-SELEX respectively use cells or tissue slices to screen nucleic acid aptamers. Since cells or tissue slices are more complex, it is easy In the process, the enrichment of sequences with affinity to non-target targets occurs, resulting in disadvantages such as low enrichment efficiency.

In 2010, the US patent disclosed (patent publication number: US 2010/0152056A1) a nuclease-based SELEX technology. This method does not require solid-phase immobilization of libraries or targets, and does not require complex equipment, but requires the use of multiple nucleases. By using exonuclease (exonuclease I, III, T, or T7 exonuclease I) to degrade the sequence that cannot bind to the target, the sequence that can bind to the target is not degraded, thereby realizing the aptamer filter. The DNA library used in this method has a random sequence region between 5 and 1000 bases in length and fixed sequences (either 5' or 3' or 5' and 3'). It is necessary to add 5 to 30 bases of sequence to the 3' of the sequence using terminal transferase after each round of nuclease degradation reaction to allow PCR amplification of the sequence. Each nuclease has different requirements on the structure of nucleic acid. For example, exonuclease I can only degrade single-stranded DNA from the 3' end, while exonuclease III can only degrade double-stranded DNA along the direction from 3' to 5'. , and only blunt or 3' concave ends. Since the nucleic acid sequence not bound to the target can form a variety of secondary structures, such as single-stranded and double-stranded secondary structures with complementary ends, it is necessary to use multiple exonucleases at the same time to fully degrade the non-target bound nucleic acid sequence. sequence. The simultaneous use of multiple nucleases brings great difficulties to the optimization of nuclease reactions, because the structural selectivity of nucleases is closely related to the structure, composition and concentration of the substrate, and these factors cannot be predicted during the screening process. Moreover, this method must add a sequence for PCR amplification at the 3' end of the sequence after the nuclease reaction, and the operation is cumbersome.

The U.S. patent (U.S. Patent No.: US8680017B2) granted in 2014 overcomes the above-mentioned deficiency of adding a sequence for PCR amplification at the 3' end of the sequence after the nuclease reaction by using end-complementary library design. The DNA library used in this method has a central region of random sequences flanked by fixed sequences that are complementary in sequence. The complementary double-stranded region may or may not contain a recognition site for a restriction endonuclease, and the sequence that does not bind to the target is degraded by a restriction endonuclease or one or more endonucleases or exonucleases. However, recent studies have shown that the binding of the target to the sequence with the above structure will not destroy the double-stranded structure at the end of the sequence. In fact, the design of the terminal double-strand structure is often used to stabilize the structure of the aptamer and enhance the affinity of the target and the aptamer, such as the aptamer of cocaine and the aptamer of thrombin TBA29 and so on. This method therefore degrades a large number of high-affinity sequences. Moreover, even if the ends of the library are designed to be complementary sequences, due to the diversity of the secondary structure of the library and the mutual hybridization between the library sequences, it is impossible to ensure that most of the sequences in the library form a double-stranded DNA structure with complementary ends, and a large number of them do not bind to the target. The sequence cannot be degraded by double-stranded structure-specific nucleases. In order to solve this problem, it is necessary to use multiple nucleases at the same time, which not only makes it difficult to optimize the reaction conditions, but also causes the loss of the sequence that can bind to the target.

Therefore, there is a need for a new screening technology that can have the advantages of simple operation, high enrichment efficiency, strong specificity, and good compatibility with real samples. In addition, in all existing SELEX technologies, it is necessary to select the nucleic acid aptamer with the highest affinity through affinity testing of the candidate sequences, which is the most time-consuming and expensive technical step in the current SELEX technology due to heavy workload and high cost.

At present, there have been publicly reported DNA aptamers of human PD-L1, respectively using traditional cellulose acetate membrane-based SELEX (Molecular Therapy—Nucleic Acids, 2016,5,e397) or target-immobilized MB-SELEX Technology (Microchim Acta 2017, 184, 4029-4035) was screened, but none of them were applied to the detection of PD-L1 expression level in human cancer tissue sections. In 2018, American scholars reported the use of chemically modified libraries to screen PD-L1 DNA aptamers based on library-modified microbeads, which were applied to fluorescence imaging of human prostate cancer tissue sections, but there was no negative control and no nucleic acid was disclosed. Sequence information and chemical modification information of aptamers (Biochimie 2018, 145, 125-130).

Contents of the invention

In order to overcome the above-mentioned defects in the prior art, the present invention provides a high-affinity and specific DNA nucleic acid aptamer for in vitro screening of a broad-spectrum tumor marker programmed cell death receptor 1-ligand 1 (PD-L1) A new method, referred to as internal coding-SELEX (Encoded-SELEX). The starting random DNA library of this method comprises two random sequence regions and a fixed region (ie, internal coding) containing restriction endonuclease recognition sites located therebetween. The internal coding region hybridizes with a short-strand complementary sequence (cDNA) to form a double-stranded DNA that can be cut by type II restriction endonucleases. The library was pre-enriched by magnetic cross-linking precipitation-SELEX (MCP-SELEX, Anal.Chem.2019, 91, 13383-13389), and then the DNA library was incubated with PD-L1, and then the type II restriction enzyme Alu I was added . The sequence that can bind to PD-L1, its internal coding structure is destroyed, so the sequence is not cut by restriction endonuclease, and can be retained by PCR amplification. In each round of screening, MCP-SELEX was used to specifically enrich for sequences that produced changes in the internal coding structure induced by target binding, but not restriction endonucleases. The method of the invention avoids the immobilization of the library and the target on the solid phase, and only needs one restriction endonuclease to realize the separation of the sequence combined with the target, the operation is simple, and the experimental conditions are easy to optimize. Moreover, the candidate nucleic acid aptamers obtained in the present invention have the characteristics that the longer the internal coding length, the higher the affinity, which greatly accelerates the selection of high-affinity nucleic acid aptamers, which is not available in all current SELEX technologies. In addition, the method of the present invention uses MCP-SELEX to first enrich the library for 3 rounds, which greatly reduces the diversity of the library, helps to reduce the non-specific interaction between sequences, and promotes the recognition of restriction endonucleases in the sequence. Formed, thereby greatly screening efficiency. The method of the present invention also uses MCP-SELEX to efficiently capture the sequence that binds to the target after each round of restriction endonuclease reaction, and eliminates the sequence mutation induced by restriction endonuclease, thereby escaping from the restriction endonuclease reaction. non-specific sequence. However, in the prior art, nuclease-induced mutations and nuclease-resistant sequences are not removed from the enrichment library, so the screening efficiency is low. The preferred fluorescent-labeled nucleic acid aptamer has been successfully used for fluorescence imaging of PD-L1 expression levels in various tumor tissue sections. Its performance is comparable to that of antibodies, and the operation is extremely simple. It has great potential for PD-1/PD-L1 immunotherapy. Companion or supplementary diagnostics. The superior performance of the screened nucleic acid aptamer fully proves the feasibility of the method of the present invention. The prior art (US 2010/0152056A1; US8680017B2) only provides method descriptions, but has not verified the feasibility of the method through experiments.

Compared with the existing nuclease-based SELEX technology (US8680017B2), the inventive method has the following advantages:

1) The method of the present invention only needs a type II restriction endonuclease to conveniently and efficiently separate the target-bound sequence from the unbound sequence. However, the prior art requires a variety of nucleases, and it is difficult to optimize the conditions.

2) The library design of the method of the present invention is more conducive to the combination of the target and the sequence. The library design of the method of the present invention comprises two random sequence regions and a fixed region (that is, internal coding) containing type II restriction endonuclease recognition sites located therebetween. The internal coding region hybridizes with a short-strand complementary sequence (cDNA) to form a double-stranded DNA that can be cut by type II restriction endonucleases. The existing technology uses a library whose ends form complementary double strands, so the hybridization between the sequences is inevitable, resulting in the easy formation of cross-linked products of the sequences in the system, which not only hinders the combination between the target and the sequence, but also causes some sequences with affinity to be blocked. Degradation; In the method of the present invention, the recognition structure of type II restriction endonuclease is formed by using excessive short-chain cDNA to hybridize with the internal coding region of the library, not only the hybridization efficiency is high, but also the formation of the internal coding/cDNA double-stranded structure can be Reduce the interaction between sequences, which is conducive to the effective combination of the target and a single sequence.

3) The library design of the method of the present invention is more conducive to inducing a large secondary structure change in the recognition structure of type II restriction endonuclease when the target binds to the sequence, so that the sequence with affinity is screened out more efficiently. In the library design of the present invention, the random region is located on both sides of the internal coding, and the length is only 20 bases. The combination of the target and one or two random region sequences will cause a large change in the internal coding/cDNA double-stranded DNA structure, making This sequence is not degraded in restriction enzyme reactions. The random region in the prior art is located at the position of the "loop" in the "stem-loop structure" of the sequence. The combination of the target and the random region often does not cause changes in the structure of the double-stranded "stem", and even prolongs the length of the "stem", making the The sequence is still degraded in the restriction enzyme reaction, resulting in loss of the sequence with affinity.

4) The method of the present invention utilizes MCP-SELEX to efficiently capture the sequence bound to the target after each round of restriction endonuclease reaction, and eliminates the mutant sequence induced by the restriction endonuclease, thereby eliminating the The non-specific sequences escaped in the Dicer reaction, so the specificity of the screening is good. However, in the prior art, nuclease-induced mutations and nuclease-resistant sequences were not removed from the enrichment library.

5) The nucleic acid aptamers obtained according to the method of the present invention have the characteristics that the longer the internal coding length is, the higher the affinity is, and high-affinity nucleic acid aptamers can be easily selected by the length of the nucleic acid aptamers, without the need for a large number of candidate sequences one by one Perform an affinity test. All current SELEX technologies do not have this feature.

6) The best nucleic acid aptamer screened by the method of the present invention is used for the first time to specifically recognize PD-L1 in various tumor tissue sections, reaching a level comparable to that of PD-L1 antibodies, and the operation is extremely simple and fast, very effective It has the potential to be used as a companion or supplementary diagnosis for PD-1/PD-L1 immunotherapy.

Concrete experimental steps of the present invention: see Table 1 for the names, sequences and uses of all DNAs used. 1. Using PD-L1 as the target protein, three rounds of MCP-SELEX were used to prepare a pre-enriched library

The specific operation steps are as follows: the experimental parameters of each round of screening are shown in Table 3.

Step 1.DNA library heat treatment: DNA library diluted in 500 μL (microliter) of screening buffer solution (50mM (millimole per liter) 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 100mM NaCl, 1mM MgCl2, 5mM KCl , 1 mM CaCl2, pH=7.4), heated in a water bath at 95°C (Celsius) for 10 minutes, quenched on ice for 10 minutes, and stood at room temperature for 10 minutes.

Step 2. Activation of carboxyl-coated magnetic beads:

Step 2.1. Rotate and mix the carboxyl-coated magnetic beads at room temperature for 15 minutes.

Step 2.2. Pipette 10 μL of magnetic beads and 100 μL of 25 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH=5.0) to mix thoroughly for 10 minutes (minutes), discard the supernatant, and then wash with 100 μL of 25 mM MES solution twice.

Step 2.3. Use 25mM MES solution to freshly configure 50mg/mL (mg/ml) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution and 50mg/mL mL of N-hydroxysuccinimide (NHS) solution.

Step 2.4. Add 100 μL of the above EDC solution and 100 μL of the above NHS solution to the magnetic beads washed in 2.2, mix well, and shake at low speed for 30 minutes at room temperature.

Step 2.5. Place the centrifuge tube containing EDC/NHS/magnetic beads on a strong magnet for 1 minute, remove the supernatant and wash twice with 100 μL of 25 mM MES solution.

Step 2.6. Add 10 μL of screening buffer solution to the above-mentioned activated magnetic beads, so that the magnetic beads are evenly dispersed in the solution.

Step 3. Negative screening with activated magnetic beads: Add the heat-treated DNA library in step 1 to the activated magnetic beads, with a total volume of 500 μL, and incubate for 30 minutes at room temperature with rotation and mixing. After incubation, discard the magnetic beads and keep the supernatant.

Step 4. Incubate the library with the target protein PD-L1 after negative screening: add PD-L1 to the supernatant collected in step 3, and incubate for 30 minutes at room temperature with rotation and mixing. After incubation, discard the supernatant and keep the magnetic beads. Add 200 μL of screening buffer solution to the magnetic beads, incubate for 5 minutes at room temperature, place on a strong magnet for 1 minute, and repeat washing 4 times. Finally, 120 μL of screening buffer solution was added, heated with shaking at 95° C. for 20 minutes, magnetically separated for 1 minute, and the supernatant (eluate) was collected.

Step 5. Perform polymerase chain reaction (PCR) to amplify the DNA sequence in the eluate, using a biotin-labeled reverse primer.

Step 6. Preparation of single-stranded DNA:

Step 6.1. Prepare the column: place the streptavidin sepharose beads on a rotator at room temperature for 15 minutes, add 60 μl streptavidin sepharose beads to a 200 μl anti-aerosol tip, and use 200 μl The column was washed with a phosphate buffered saline solution (1×PBS/1M NaCl) containing 1M (moles per liter) NaCl.

Step 6.2. Passing the column: Add the PCR mixture to the column in batches with a pipette (100 μL each time), and use the pipette to apply pressure to fully mix the PCR mixture with the streptavidin sepharose beads, 3 After a few minutes, the mixture in the column was discharged out of the tip by applying pressure with a pipette gun, and then the column was washed with 200 μL of 1×PBS/1M NaCl. Repeat this operation until all the PCR solution has been added to the column.

Step 6.3. NaOH elution: After washing the column twice with 200 μL 1×PBS/1M NaCl, add 100 μL 30 mM NaOH to the column with a pipette gun, pressurize the NaOH solution out of the column with a pipette gun after 5 minutes, and collect In a clean centrifuge tube, the single-stranded DNA library.

Step 6.4. Library quantification: the above-mentioned single-stranded DNA library was neutralized with an equal amount of HCl, and the concentration was quantified by measuring the ultraviolet-visible absorption intensity at 260 nm (nanometer).

2. 4 rounds of Encoded-SELEX

The specific operation steps are as follows: The experimental parameters of each round are shown in Table 4.

Step 1. Restriction Enzyme-SELEX (RE-SELEX)

Step 1.1. DNA heat treatment and complementary hybridization: the DNA library and the complementary short-strand DNA (cDNA) of the intermediate immobilized sequence are prepared in the digestion buffer (10mM Tris-HCl, 10mM MgCl2, 1mM dithiothreitol, pH 7.5) The final concentration was 0.4 μM (micromoles per liter) library DNA solution. The above solution was heated in a water bath at 95°C for 10 minutes, and then slowly cooled to room temperature.

Step 1.2. Incubate the library with PD-L1: add PD-L1 to the above system at a final concentration of 0.2 μM, and incubate at room temperature for 1 hour. The specific experimental parameters are shown in Table 3.

Step 1.3. Enzyme digestion reaction: prepare an Alu I enzyme solution with a final concentration of 0.7 U/μL (activity unit/μL) in the enzyme digestion buffer solution. Add 50 μL of the newly prepared Alu I enzyme solution to the above 150 μL DNA solution, divide into two 100 μL PCR small tubes, set the heat treatment program of the PCR instrument as 37°C for 30 minutes; 80°C for 15 minutes; 4°C for 10 minutes, and the final obtained The digestion solution was stored in a refrigerator at 4°C for later use.

Step 1.4. Gel electrophoresis experiment: take 6 μL of enzyme digestion solution for gel electrophoresis experiment, see gel electrophoresis experiment for detailed steps.

Step 1.5. PCR amplification.

Step 1.6 Preparation of DNA single strands: For detailed steps, see Preparation of DNA single strands in MCP-SELEX.

Step 2.MCP-SELEX: See MCP-SELEX for detailed steps. The enriched library enters the next round of Encoded-SELEX. The first round of Encoded-SELEX did not perform this step.

3. High-throughput sequencing of enriched libraries.

4. Affinity and specificity tests for selecting candidate nucleic acid aptamer sequences.

5. The application of the obtained nucleic acid aptamer in the imaging of normal human tonsil tissue slices and cancer tissue slices.

Tissue section application experiments, using paraffin-embedded non-small cell lung cancer (NSCLC) tissue sections with high and no expression of PD-L1 expression, malignant melanoma tissue sections with high and no expression of PD-L1 expression, and normal human tonsil tissue sections, specifically The operation steps are as follows:

Step 1. Nucleic acid aptamer heat treatment: 100 pmol (picomole) nucleic acid aptamer was prepared in PD-L1 screening buffer solution to make 150 μL solution, heated at 95°C for 5 minutes, quenched on ice for 10 minutes, and kept at room temperature in the dark Leave for 10 minutes.

Step 2. Tissue Section Processing

Step 2.1. Soak the tissue section in p-xylene for 30 minutes, take out the tissue section at 15 minutes, and then soak the above solution in 95%, 90%, 85%, 80%, and 75% ethanol for two times respectively. times, soaking for 3 minutes each time.

Step 2.2. Put 0.01M sodium citrate solution (pH 6.0) in a clean beaker, and immerse the above-mentioned tissue slices in the solution, and heat in a microwave oven. When the solution starts to boil and bubbles appear, start timing immediately, pause for 30 seconds for every 1 minute of heating, and repeat this operation for 15 minutes. The beaker was then removed from the microwave and allowed to cool slowly to room temperature.

Step 2.3. Remove the tissue slice from the beaker and wash three times with 1×PBS/0.1M NaCl.

Step 2.4. Prepare blocking solution: 10 μL of salmon essence (10 mg/mL) per 100 μL of blocking solution; 10 μL of yeast tRNA (10 mg/mL); 50 μL of 10×PBS/0.1M NaCl; 20 μL of 5% bovine serum albumin (BSA ); 10 μL of Tween-20 (0.1%) was prepared.

Step 2.5. Sealing of tissue sections: Add blocking solution to the tissue sections (pay attention to the location of the tissue), 100 μL/section, block at 37°C for 1 hour, and then wash three times with 1×PBS/0.1M NaCl again.

Step 3. Add the heat-treated nucleic acid aptamer solution dropwise onto the tissue section, incubate at 37°C for 20 minutes, and wash three times with 1×PBS/0.1M NaCl.

Step 4. Use a fluorescence microscope to perform fluorescence imaging on the tissue section.

Description of drawings

Figure 1A and Figure 1B are Encoded-SELEX flowcharts, wherein Figure 1A shows the design of the DNA library used in Encoded-SELEX; Figure 1B shows the specific process of Encoded-SELEX.

Figures 2A-2F are monitoring diagrams of the library's resistance to enzyme digestion and affinity evolution during the Encoded-SELEX screening process, wherein Figures 2A-2D show the first to fourth rounds of screening in the library after the restriction enzyme reaction product (REP -1, REP-2, REP-3, REP-4), where "+" indicates the presence of PD-L1 or Alu I enzyme in the corresponding digestion solution, and "-" indicates the presence of the corresponding The digestion solution does not contain PD-L1 or Alu I enzyme, and the final concentration of Alu I enzyme in all digestion solutions is 0.7U/μL; Figure 2E shows the library and PD after each round of digestion reaction determined by qPCR method. -The binding rate of L1, where R0 is the initial library after three rounds of MCP-SELEX enrichment; RE2, RE3, and RE4 are the PCR amplification and single-strand preparation of REP-2, REP-3, and REP-4, respectively library, in which RE3 and RE4 combined with PD-L1 were negatively screened with a mixture of 0.06% serum and HSA (two final concentrations were 65nM and 50nM) before testing; Figure 2F shows A fiber optic sensor measures the dissociation constant of fluorescently labeled (Cy5.5) R4.

Figures 3A and 3B are the histograms of the affinity of the top 20 sequences (Figure 3A) and the other 20 sequences (Figure 3B) characterized by the Nano-Affi method, where Δd is the addition of PD-L1 or PD - Change value of gold nanometer (GNP) particle size after L1 and DNA sequence, wherein N-39 and N-60 are negative control sequences, the final concentration of DNA in all tests is 20nM (nanomole per liter), and the final concentration of PD-L1 The concentration is 40 nM.

Figure 4 is a gel electrophoresis image of the affinities of 5 candidate nucleic acid aptamers of different lengths determined by gel retardation assay (EMSA). The gray value (I) of each band was determined by Image Lab TM Software, and the data are listed in each lane Below, nucleic acid aptamer binding rate (%)=I(PD-L1-nucleic acid aptamer complex)/[I(PD-L1-nucleic acid aptamer complex)+I(unbound nucleic acid aptamer) ].

Figures 5A-5C are the affinity and specificity characterization diagrams of representative candidate nucleic acid aptamers: (Figure 5A) gel retardation assay (EMSA) to determine the selectivity of 8-60 to different proteins; (Figure 5B) using Nano- Affi determined the dissociation constant of 8-60, and the final concentration of PD-L1 was always 40nM; (Figure 5C) the secondary structure of 8-60 simulated by online software (www.idt.com), the arrow refers to Alu I recognition For single base mutations in the sequence, the original recognition sequence is AG^CT.

Figures 6A-6D are diagrams of the interaction between Cy5-8-60 and four kinds of cancer cells with different expression levels of PD-L1 using flow cytometry: (Figure 6A) COLO205 cells without PD-L1 expression (human colon cancer cells); (Fig. 6B) HCC70 cells with low PD-L1 expression (human breast ductal carcinoma cells); (Fig. 6C) ES-2 cells (human ovarian clear cell carcinoma cells) expressing PD-L1; (Fig. 6D ) BCPAP cells (human thyroid cancer papillary cells) with high expression of PD-L1, Cy5-A60 was used as negative control in each group of experiments.

Figure 7A and Figure 7B are schematic diagrams of measuring the dissociation constant of Cy5-8-60 on PD-L1 expressed on BCPAP cells based on flow cytometry, (Figure 7A) after different concentrations of Cy5-8-60 are incubated with BCPAP cells (Fig. 7B) The binding curve for the determination of the Cy5-8-60 dissociation constant drawn from the data in Fig. A, BCPAP cells are human thyroid cancer papillary cells with high expression of PD-L1, FL4-H The channel is the Cy5 fluorescence channel, and 200nM Cy5-A60 is used as a negative control.

Figures 8A-8C are fluorescence microscopy images of FAM-8-60 detecting the expression levels of PD-L1 in various tissue sections. Fluorescent imaging of low crypts, germinal centers and epithelial tissues; (Fig. 8B, Fig. 8C) Fluorescent imaging of non-small cell lung cancer (NSCLC) tissue sections with positive and negative PD-L1 expression, all organized with PD-L1 antibody The results of section immunohistochemistry were positive controls; FAM-383-33 and FAM-A60 were used as negative controls, and all types of tissue sections were serial sections of the same tissue.

Figures 9A and 9B are fluorescence imaging images of malignant melanoma tissue sections with positive (Figure 9A) and negative (Figure 9B) expression of FAM-8-60 on PD-L1, and the immunohistochemical results of PD-L1 antibody tissue sections are Positive control; with FAM-383-33 and FAM-A60 as negative controls, all types of tissue sections are serial sections of the same tissue.

Detailed ways

Specific embodiments of the present invention will be described in detail below for further understanding of the present invention. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

In general, the starting random DNA library of the method of the present invention comprises two regions of random sequences and a fixed region (ie, internal coding) in between that contains recognition sites for restriction enzymes. The internal coding region hybridizes with a short-strand complementary sequence (cDNA) to form a double-stranded DNA that can be cut by type II restriction endonucleases. The library was pre-enriched by magnetic cross-linking precipitation-SELEX (MCP-SELEX, Anal.Chem.2019, 91, 13383-13389), and then the DNA library was incubated with PD-L1, and then the type II restriction enzyme Alu I was added . The sequence that can bind to PD-L1, its internal coding structure is destroyed, so the sequence is not cut by restriction endonuclease, and can be retained by PCR amplification. In each round of screening, MCP-SELEX was used to specifically enrich for sequences that produced changes in the internal coding structure induced by target binding, but not restriction endonucleases.

The names, sequences and purposes of all DNAs used in the specific experimental steps of the present invention are shown in Table 1. Overall technical scheme of the present invention comprises following process:

1. Using PD-L1 as the target protein, three rounds of MCP-SELEX were used to prepare a pre-enrichment library (see Table 3 for the experimental parameters of each round of screening);

2. 4 rounds of Encoded-SELEX (see Table 4 for the experimental parameters of each round);

3. High-throughput sequencing of enriched libraries;

4. Affinity and specificity tests for selecting candidate nucleic acid aptamer sequences;

5. The application of the obtained nucleic acid aptamer in the imaging of normal human tonsil tissue slices and cancer tissue slices.

In the accompanying drawings, Fig. 1A and Fig. 1B are Encoded-SELEX flowcharts, wherein Fig. 1A shows the design of DNA library used in Encoded-SELEX; Fig. 1B shows the specific process of Encoded-SELEX. Figures 2A-2F are monitoring diagrams of the library's resistance to enzyme digestion and affinity evolution during the Encoded-SELEX screening process, wherein Figures 2A-2D show the first to fourth rounds of screening in the library after the restriction enzyme reaction product (REP -1, REP-2, REP-3, REP-4), where "+" indicates the presence of PD-L1 or Alu I enzyme in the corresponding digestion solution, and "-" indicates the presence of the corresponding The digestion solution does not contain PD-L1 or Alu I enzyme, and the final concentration of Alu I enzyme in all digestion solutions is 0.7U/μL; Figure 2E shows the library and PD after each round of digestion reaction determined by qPCR method. -The binding rate of L1, where R0 is the initial library after three rounds of MCP-SELEX enrichment; RE2, RE3, and RE4 are the PCR amplification and single-strand preparation of REP-2, REP-3, and REP-4, respectively library, in which RE3 and RE4 combined with PD-L1 were negatively screened with a mixture of 0.06% serum and HSA (two final concentrations were 65nM and 50nM) before testing; Figure 2F shows A fiber optic sensor measures the dissociation constant of fluorescently labeled (Cy5.5) R4. Figures 3A and 3B are the histograms of the affinity of the top 20 sequences (Figure 3A) and the other 20 sequences (Figure 3B) characterized by the Nano-Affi method, where Δd is the addition of PD-L1 or PD - Change value of gold nanometer (GNP) particle size after L1 and DNA sequence, wherein N-39 and N-60 are negative control sequences, the final concentration of DNA in all tests is 20nM (nanomole per liter), and the final concentration of PD-L1 The concentration is 40 nM. The sequence information of each candidate aptamer is shown in Table 5. Figure 4 is a gel electrophoresis image of the affinities of 5 candidate nucleic acid aptamers of different lengths determined by gel retardation assay (EMSA). The gray value (I) of each band was determined by Image Lab TM Software, and the data are listed in each lane Below, nucleic acid aptamer binding rate (%)=I(PD-L1-nucleic acid aptamer complex)/[I(PD-L1-nucleic acid aptamer complex)+I(unbound nucleic acid aptamer) ]. The specific experimental parameters are shown in Table 6. Figures 5A-5C are the affinity and specificity characterization diagrams of representative candidate nucleic acid aptamers: (Figure 5A) gel retardation assay (EMSA) to determine the selectivity of 8-60 to different proteins; (Figure 5B) using Nano- Affi determined the dissociation constant of 8-60, and the final concentration of PD-L1 was always 40nM; (Figure 5C) the secondary structure of 8-60 simulated by online software (www.idt.com), the arrow refers to Alu I recognition For single base mutations in the sequence, the original recognition sequence is AG^CT. Figures 6A-6D are diagrams of the interaction between Cy5-8-60 and four kinds of cancer cells with different expression levels of PD-L1 using flow cytometry: (Figure 6A) COLO205 cells without PD-L1 expression (human colon cancer cells); (Fig. 6B) HCC70 cells with low PD-L1 expression (human breast ductal carcinoma cells); (Fig. 6C) ES-2 cells (human ovarian clear cell carcinoma cells) expressing PD-L1; (Fig. 6D ) BCPAP cells (human thyroid cancer papillary cells) with high expression of PD-L1. Cy5-A60 was used as negative control in each group of experiments. Figure 7A and Figure 7B are schematic diagrams of measuring the dissociation constant of Cy5-8-60 on PD-L1 expressed on BCPAP cells based on flow cytometry, (Figure 7A) after different concentrations of Cy5-8-60 are incubated with BCPAP cells (Fig. 7B) The binding curve for the determination of the Cy5-8-60 dissociation constant drawn from the data in Fig. A, BCPAP cells are human thyroid cancer papillary cells with high expression of PD-L1, FL4-H The channel is the Cy5 fluorescent channel, and 200nMCy5-A60 is used as a negative control. Figures 8A-8C are fluorescence microscopy images of FAM-8-60 detecting the expression levels of PD-L1 in various tissue sections. Fluorescent imaging of low crypts, germinal centers and epithelial tissues; (Fig. 8B, Fig. 8C) Fluorescent imaging of non-small cell lung cancer (NSCLC) tissue sections with positive and negative PD-L1 expression, all organized with PD-L1 antibody The results of section immunohistochemistry were positive controls; FAM-383-33 and FAM-A60 were used as negative controls, all types of tissue sections were serial sections of the same tissue, and the areas with higher brightness in Figure 8A-8C were PD- Regions with higher levels of L1 expression. Figures 9A and 9B are fluorescence imaging images of malignant melanoma tissue sections with positive (Figure 9A) and negative (Figure 9B) expression of FAM-8-60 on PD-L1, and the immunohistochemical results of PD-L1 antibody tissue sections are Positive control; with FAM-383-33 and FAM-A60 as negative controls, all types of tissue sections are serial sections of the same tissue, and the areas with higher brightness in Figure 9A and 9B are those with higher expression levels of PD-L1 area.

Table 1. DNA used in the present invention

Table 2. Reagents used in the present invention

Table 3. Experimental parameters for 3 rounds of MCP-SELEX for library pre-enrichment

Each round of selection was performed in selection buffer: 50 mM HEPES, 100 mM NaCl, 1 mM MgCl ₂ , 5 mM KCl, 1 mM CaCl ₂ , pH 7.4.

Table 4. Experimental parameters of each round of Encoded-SELEX screening

1: Digestion buffer solution: 10mM Tris-HCl, 10mM MgCl ₂ , 1mM Dithiothreitol, pH 7.5

2: Screening buffer solution: 50mM HEPES, 100mM NaCl, 1mM MgCl ₂ , 5mM KCl, 1mM CaCl ₂ , pH7.4. All screening steps were performed at room temperature.

Table 5. 40 candidate nucleic acid aptamers obtained by high-throughput sequencing

1: The "serial number" in the name is the sequence number sorted according to the abundance of each sequence in high-throughput sequencing; the "base number" is the number of bases of each sequence except for the primer binding regions at both ends.

Table 6. Gel retardation assay (EMSA) to determine the binding rate of representative candidate nucleic acid aptamers to PD-L1

1: The "serial number" in the name of the nucleic acid aptamer is the sequence number sorted according to the abundance of each sequence in high-throughput sequencing; the "base number" is the number of bases in each sequence except for the primer-binding regions at both ends.

2: Utilize Image Lab™ Software to measure the gray value (I) of each band, and the gray value of the band containing only nucleic acid aptamer without PD-L1 is 1. Aptamer binding rate (%)=I (PD-L1-aptamer complex)/[I(PD-L1-aptamer complex)+I (unbound aptamer)].

Example 1. The process of screening PD-L1 nucleic acid aptamers using the method of the present invention

The chemically synthesized DNA random library (Fig. 1A, Table 1) consists of three major parts, namely the primer binding region at both ends, two random sequence regions and the middle fixed sequence region (ie, the internal coding region). Wherein, there is a restriction endonuclease recognition site in the middle fixed sequence. In addition, the intermediate fixed sequence hybridizes with the short-strand cDNA (Table 1) to form a double-strand structure that can be recognized by restriction endonucleases. At the same time, the formation of the double-stranded domain is conducive to reducing the interaction between sequences.

Firstly, three rounds of MCP-SELEX were used for pre-enrichment (patent application number: 201810589689.4) in order to reduce the enrichment efficiency of restriction endonuclease-SELEX (RE-SELEX) in Encoded-SELEX. The experimental parameters of each round are shown in Table 3. The specific experimental steps include:

1. DNA library heat treatment: DNA library was diluted in 500 μL of screening buffer solution (50 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 100 mM NaCl, 1 mM MgCl2, 5 mM KCl, 1 mM CaCl2, pH=7.4), 95 ° C Heated in a water bath for 10 minutes, quenched on ice for 10 minutes, and left at room temperature for 10 minutes.

2. Activation of carboxyl-coated magnetic beads:

2.1. Rotate and mix the carboxyl-coated magnetic beads at room temperature for 15 minutes.

2.2. Pipette 10 μL of magnetic beads and 100 μL of 25 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH=5.0) to mix thoroughly for 10 min, discard the supernatant, and then wash twice with 100 μL of 25 mM MES solution.

2.3. Use 25mM MES solution to freshly prepare 50mg/mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution and 50mg/mL N-hydroxybutyrate imide (NHS) solution.

2.4. Add 100 μL of the above EDC solution and 100 μL of the above NHS solution to the magnetic beads washed in 2.2, mix thoroughly, and shake at low speed for 30 minutes at room temperature.

2.5. Place the centrifuge tube containing EDC/NHS/magnetic beads on a strong magnet for 1 minute, remove the supernatant and wash twice with 100 μL of 25mM MES solution.

2.6. Add 10 μL of screening buffer solution to the above-mentioned activated magnetic beads to disperse the magnetic beads evenly in the solution.

3. Negative screening with activated magnetic beads: Add the heat-treated DNA library in step 1 to the activated magnetic beads, with a total volume of 500 μL, and incubate for 30 minutes at room temperature with rotation and mixing. After incubation, discard the magnetic beads and keep the supernatant.

4. Incubation of the library with the target protein PD-L1 after negative screening: add PD-L1 to the supernatant collected in 3, and incubate for 30 minutes at room temperature with rotation and mixing. After incubation, discard the supernatant and keep the magnetic beads. Add 200 μL of screening buffer solution to the magnetic beads, incubate for 5 minutes at room temperature, place on a strong magnet for 1 minute, and repeat washing 4 times. Finally, 120 μL of screening buffer solution was added, heated with shaking at 95° C. for 20 minutes, magnetically separated for 1 minute, and the supernatant (eluate) was collected.

5. Perform polymerase chain reaction (PCR) to amplify the DNA sequence in the eluate, using a biotin-labeled reverse primer.

6. Preparation of single-stranded DNA:

6.1. Prepare the column: place the streptavidin sepharose beads on a rotator at room temperature for 15 minutes, add 60 μl streptavidin sepharose beads to a 200 μl anti-aerosol tip, and use 200 μl containing The column was washed with 1M NaCl in phosphate buffered saline (1×PBS/1M NaCl).

6.2. Pass through the column: Add the PCR mixture to the column in batches with a pipette gun (100 μL each time), and use the pipette gun to apply pressure to fully mix the PCR mixture with the streptavidin sepharose beads for 3 minutes Finally, press the pipette gun to discharge the mixture in the column from the tip of the pipette, and then wash the column with 200 μL 1×PBS/1M NaCl. Repeat this operation until all the PCR solution has been added to the column.

6.3. NaOH elution: After washing the column twice with 200μL 1×PBS/1M NaCl, add 100μL 30mM NaOH to the column with a pipette gun, and after 5 minutes, use a pipette gun to pressurize the NaOH solution out of the column and collect a clean centrifuge tube, i.e. single-stranded DNA library.

6.4. Library quantification: The above-mentioned single-stranded DNA library was neutralized with an equal amount of HCl, and the concentration was quantified by measuring the ultraviolet-visible absorption intensity at 260 nm.

This was followed by 4 rounds of Encoded-SELEX (Fig. 1B). Except that the first round only includes RE-SELEX, the 2-4 rounds of Encoded-SELEX all include two steps of RE-SELEX and MCP-SELEX. The experimental parameters of each round are shown in Table 4, and the specific operation steps are as follows:

1. Restriction enzyme-SELEX (RE-SELEX)

1.1. DNA heat treatment and complementary hybridization: The DNA library and the complementary short-strand DNA (cDNA) of the fixed sequence in the middle are prepared in the digestion buffer (10mM Tris-HCl, 10mM MgCl2, 1mM dithiothreitol, pH 7.5) The final concentration was 0.4 μM library DNA solution. The above solution was heated in a water bath at 95°C for 10 minutes, and then slowly cooled to room temperature.

1.2. Incubate the library with PD-L1: add PD-L1 to the above system at a final concentration of 0.2 μM, and incubate at room temperature for 1 hour. The specific experimental parameters are shown in Table 3.

1.3. Enzyme digestion reaction: Alu I enzyme solution with a final concentration of 0.7 U/μL was prepared in the enzyme digestion buffer solution. Add 50 μL of newly prepared Alu I enzyme solution to the above 150 μL DNA solution, divide into two 100 μL PCR small tubes, set the heat treatment program of the PCR instrument as 37°C for 30 minutes; 80°C for 15 minutes; 4°C for 10 minutes, and finally get The digestion solution was stored in a 4°C refrigerator for later use.

1.4. Gel electrophoresis experiment: take 6 μL of enzyme digestion solution for gel electrophoresis experiment, see gel electrophoresis experiment for detailed steps.

1.5. PCR amplification.

1.6 Preparation of single-strand DNA: For detailed steps, see Preparation of Single-Strand DNA in MCP-SELEX.

2.MCP-SELEX: See MCP-SELEX for detailed steps. The enriched library enters the next round of Encoded-SELEX. The first round of Encoded-SELEX did not perform this step.

As shown in Figure 1B, during the RE-SELEX digestion reaction, two proteins, the target protein PD-L1 and the restriction endonuclease Alu I, competed for the binding of the nucleic acid sequence. If the DNA sequence has no/low affinity with PD-L1, the sequence is more likely to be digested; on the contrary, if there is a high affinity between the two, it is not easy to be digested. At the same time, each nucleic acid sequence will evolve to different degrees at the restriction site and the surrounding area, that is, deletion or mutation, to resist degradation by restriction endonucleases, and the degree of evolution is closely related to the affinity strength of the sequence and the target. After the digestion reaction, those nucleic acid aptamers that can bind to the target enter the MCP-SELEX process, capture with activated magnetic beads, and then amplify and prepare ssDNA by PCR, so as to be put into the next round of screening; while those that use evolutionary escape The sequences that cannot be degraded by restriction endonucleases and cannot bind to the target are not captured by activated magnetic beads and cannot enter the next round of screening. The role of MCP-SELEX can not only further screen and enrich nucleic acid aptamers with strong affinity as soon as possible, but also eliminate some non-target binding sequences that are not easy to be digested due to evolution or the formation of special secondary structures during the digestion process.

After the above steps are completed, the enriched library obtained is subjected to commercial high-throughput sequencing. Then, affinity and specificity tests were performed on the candidate nucleic acid aptamers, and the optimal nucleic acid aptamers were finally screened out for testing the expression level of PD-L1 in human cancer tissue sections.

Example 2. Monitoring of the evolution process of Encoded-SELEX each round of library resistance to enzyme digestion and affinity

We first monitored the evolution of the resistance to enzyme cleavage of the library induced by each round of Encoded-SELEX. 2A-D are gel electrophoresis images of Alu I cleavage reaction products (REP-1, REP-2, REP-3 and REP-4) in each round of screening.

Gel electrophoresis experiment, the specific operation steps are as follows:

1. Preparation of 12% denatured glue: Clean glass plates, beakers, syringes, combs and other equipment that are in direct contact with the glue with deionized water, and prepare according to the order of the formula in brackets (9.6g urea, 12mL deionized water, 2mL 10 ×TBE, 20 μL tetramethylethylenediamine, 6 mL 40% acrylamide, 200 μL 10% ammonium persulfate), and the prepared gel was allowed to stand at room temperature for 4 hours.

2. Preparation of Maker solution (total 12 μL): 6 μL 2×RNA gel loading buffer solution, 1.2 μL 20bp DNA Ladder, 4.8 μL nuclease-free water.

3. Preparation of sample solution (total 12 μL): 6 μL 2×RNA gel loading buffer solution, 6 μL enzyme cutting solution.

4. Add 500 μL of 1×TBE solution (electrophoresis solution) to the electrophoresis tank, put the static denatured gel into the electrophoresis tank, soak the gel in the electrophoresis solution, take out the comb, and let it stand for 30 minutes.

5. Heat treatment: Heat Maker and sample solution at 95°C for 10 minutes, then quench on ice for 5 minutes.

6. Pre-electrophoresis: At the same time of heat treatment, perform pre-electrophoresis on the static denatured gel at 170V (volt) for 20 minutes.

7. Add 10 μL of Maker and sample solution to the denaturing gel, and perform electrophoresis at 170V for 45 minutes.

8. Staining: Take a clean Petri dish, put it into 30mL 1x TBE solution, and take 3μL 1x Mix the Gold nucleic acid dye evenly, put the denatured gel after electrophoresis into a petri dish, and let it stand for 5 minutes.

9. Illumination gel: Take out the denatured gel and put it into the Molecular Gel Doc TM XR+ (BIO-RAD) was used to illuminate the gel, and Image Lab TM Software was used for analysis.

As shown in Figures 2A-2D, the ratio of sequences cleaved by Alu I to sequences that were not cleaved from the first round to the fourth round decreased continuously, that is, the DNA sequences in the library that were not cleaved by Alu I increased. The first round of Chinese library was almost completely cut by Alu I, and the band of REP-1 at the original long position of the library was almost invisible, indicating that R0 contained a large number of sequences that could not resist restriction endonuclease cutting; while the fourth round Almost none of the round library was cut by Alu I, and the band of REP-4 at the position of the cut short chain was hardly visible. It shows that the library has evolved to be able to resist the sequence of Alu I enzyme cleavage reaction.

We also monitored the evolution of the library's affinity for the target resulting from each round of Encoded-SELEX. The digested products of the above rounds (REP-2, REP-3 and REP-4) were amplified by PCR and prepared with single-stranded DNA to obtain libraries RE2, RE3 and RE4, respectively. Real-time quantitative PCR (qPCR) was used to measure the amount of DNA in each library that was captured by the activated magnetic beads and bound to the target, and then the percentage of the library bound to the input PD-L1 was calculated, that is, the binding rate. The specific operation steps of qPCR experiment are as follows:

1. Preparation of initial library standards and samples to be tested: prepare the following 7 different concentrations of initial library Pool0 solutions with nuclease-free water as standards, and the DNA concentrations are 1000pM (picomoles per liter), 100pM, 10pM, and 1pM respectively , 0.1pM, 0.01pM, 0. The sample can be diluted in different multiples according to the actual situation, so that the final concentration is within the range of the above-mentioned standard substance concentration.

2. Preparation of qPCR solution (three parallel groups for each solution): Each 100 μL qPCR solution contains 10 μL 2×Premix Taq hot start enzyme, 2 μL FP (10 μM), 2 μL RP (10 μM), 3 μL nuclease-free water, 1 μL 20 ×Eva Green nucleic acid dye, 2μL sample or standard formula for preparation.

3. Set the program of qPCR instrument: the first step is to preheat at 95°C for 1min; the second step is to heat at 95°C for 30s; the third step is to anneal at 51°C for 30s (seconds); the fourth step is to extend to 72°C for 30s; times, the fluorescence signal is collected in the fourth step each time.

4. Put the qPCR solution into the sample pool, and set the solution name and fluorescence type at the corresponding position.

5. The qPCR instrument starts to work. After the end, the working curve is drawn according to the data of the collected standard samples, and the amount of DNA contained in the unknown sample is obtained according to the working curve and the Ct value of the sample to be tested.

The binding rate of the target to the library (R0) obtained after 3 rounds of pre-enrichment was 3.3%, and the binding rate to RE2 was 1.46%. It shows that the 2 rounds of screening did not improve the affinity of the library to the PD-L1 target. This should be caused by two reasons. (1) A large number of DNA sequences with low affinity in the library cannot cause structural changes in the recognition site of the restriction endonuclease, so they will be cut off by the restriction endonuclease. In this way, the proportion of sequences with affinity in the total sequences is significantly reduced, resulting in a decrease in the binding rate. (2) The Alu I cleavage reaction will not only cut the sequence with a restriction endonuclease recognition site (sequence that does not have or have low affinity with the target), but also cause mutations in certain sequences to make it difficult to digest. Not sheared during the reaction. Most of these mutated sequences do not have affinity with the target. This was confirmed by RE2's low binding rate (1.46%) and high resistance to enzymatic cleavage (almost half could not be degraded, Figure 2B). However, the MCP-SELEX step in each round of screening can exclude non-specific sequences brought about by (2) from the enriched library.

The binding rate of RE3 (0.04%) was lower than that of RE-2, which should be due to the negative screening before the library bound to the target was captured with activated magnetic beads in the third round, which eliminated the human serum and human serum albumin in the library. A large number of sequences bound by protein (HSA). The ability of RE3 to resist enzymatic cleavage was comparable to that of RE2 (Fig. 2B and 2C), indicating that the MCP-SELEX step was effective in excluding non-specific sequences (about 98.6%) of sequence mutations from the enriched library. Otherwise, almost all of the library will not be cut in the third round of digestion. The binding rate of RE4 was 35.9%, which was nearly 1000 times higher than that of RE3, and RE4 could completely resist the cleavage of Alu I enzyme (Figure 2D), indicating that the proportion of PD-L1 high-affinity sequences in the RE4 library was already very high. We eluted RE4 captured by magnetic beads, PCR amplified and prepared single-stranded DNA to obtain enriched library R4.

We used the evanescent wave fiber optic sensor to measure the dissociation constant (KD) of Cy5.5 fluorophore-modified R4 (Figure 2F), and performed nonlinear fitting according to the 1:1 binding, and the KD was 36±21nM.

Example 3. High-throughput sequencing of the R4 library

We subjected the above R4 library to commercial high-throughput sequencing. After the R4 library was amplified by PCR, the purified amplified product was subjected to high-throughput sequencing. The PCR product was purified using Sangon (Shanghai) UNIQ-10 Column Oligonucleotide Purification Kit. The specific operation steps are as follows:

1. Add 10 times the volume of binding buffer to the PCR solution and vortex to mix.

2. Take four adsorption columns with collection tubes, add 600 μL of the mixed solution to each adsorption column, let stand at room temperature for 2 minutes, and then centrifuge at 8000 rpm (rotation per minute) for 2 minutes. Pour off the liquid in the collection tube and repeat the above steps again until the mixture is used up.

3. Add 500 μL of washing buffer (the correct amount of absolute ethanol has been added) to the adsorption column, centrifuge at 10,000 rpm for 1 minute, and discard the waste liquid in the collection tube.

4. Repeat step 3 once.

5. In order to volatilize the residual ethanol, avoid affecting the follow-up experiments. The adsorption column was emptied once, and centrifuged at 10,000 rpm for 2 minutes.

6. Put the adsorption column into a clean 1.5mL centrifuge tube (it is best to cut off the cap of the centrifuge tube), and add 50 μL of nuclease-free water to the center of the adsorption membrane (in order to further improve the yield, it is necessary to preheat to 60°C in advance ), after standing at room temperature for 5 minutes, centrifuge at 12000rpm for 1 minute. The resulting DNA solution was stored at -20°C.

Of the 10,000 sequences obtained after high-throughput sequencing, the lengths of the parts other than the primer-binding region vary from 33 to 60 bases. The results showed that all the intermediate fixed sequences (internal coding) of the library were mutated or deleted: mutations basically occurred in the Alu I recognition sequence AGCT; base deletions basically covered the recognition sequence, and some entire internal coding regions no longer existed. This severe base deletion has not been reported in the existing SELEX technology. We speculate that the occurrence of this situation may be related to two factors. One is that the DNA library will produce editing mutations at the recognition site in order to avoid Alu I digestion; The structure of the Dicer recognition site changes, which inhibits enzyme cleavage, and then inhibits sequence variation to varying degrees.

Affinity Characterization of Example 4.40 Representative Candidate Nucleic Aptamers

In order to explore whether there is a correlation between the number of bases of each candidate sequence and its affinity, we randomly selected 40 DNA sequences with different base numbers from the top 1000 sequences in the high-throughput library for affinity testing (Table 5). Each sequence is named with "serial number-base number", where "serial number" is the sequence number sorted according to the abundance of each sequence in high-throughput sequencing; Outer bases.

We used the colloidal gold-based Nano-Affi method (Analyst, 2020, 145, 4276–4282) to characterize the affinity of each candidate nucleic acid aptamer. The specific operation steps are as follows:

1. Preparation of gold nanoparticles: According to the method reported in the literature, gold nanoparticles (GNP, 8.3nM, pH 6.5) with a diameter of 13nm were synthesized. Then the pH of the gold nanoparticles was adjusted to 5.0 with HCl.

2. Prepare the sample: add 4 pmol PD-L1 and 2 pmol DNA (candidate nucleic acid aptamer or negative control strand) to 4 μL screening buffer solution and mix. The final concentrations of PD-L1 and DNA were 1 μM and 0.5 μM, respectively.

3. Incubation: Incubate the above samples at room temperature for 10 minutes.

4. After the incubation, add 96 μL of GNP to the mixture and mix slowly with a pipette 4 times. At this time, the final concentrations of PD-L1 and DNA were 40 nM and 20 nM, respectively.

5. Use a dynamic light scattering instrument (DLS) to measure the particle size of GNP, the detection time is 10 minutes, and use a sample that does not contain aptamer, that is, only PD-L1, as a negative control.

As shown in Figures 3A and 3B, according to the detection principle of the Nano-Affi method, the higher the Δd value of the GNP particle size change, the worse the aptamer affinity. To further confirm that the length of the sequence has no effect on the Δd value, we used three negative control sequences of different lengths as negative controls: thrombin (N-29), phthalate derivatives (N-39) and pan- Nucleic acid aptamers of prime (N-60), the number of bases are 29, 39 and 60 respectively. The Δd values of gold nanoparticles caused by the three negative control sequences were all smaller than those caused by PD-L1. Except for sequences 383-33 and 258-36, the Δd values caused by the other 38 candidate nucleic acid aptamers were all smaller than those caused by the three negative control sequences. It shows that the affinity of these 38 candidate nucleic acid aptamers is higher than that of the negative control sequence. Interestingly, we found that the longer the length of these 40 candidate aptamers, the higher the affinity for PD-L1. This rule is applicable not only to the top 20 sequences in high-throughput sequencing (Figure 3A), but also to the lower sequences (Figure 3B).

In order to further verify the accuracy of this rule, we used the gel retardation assay (EMSA) to test the affinity of five representative candidate nucleic acid aptamers with different base numbers.

The specific operation steps are as follows:

1. DNA heat treatment: Add 50 pmol candidate nucleic acid aptamers to the screening buffer solution, heat at 95°C for 10 minutes, and slowly cool down to room temperature.

2. 20 μL of 0.1% Tween-80 was thoroughly mixed with the above DNA solution, and after centrifugation to remove foam, mixed with 100 pmol PD-L1, the final volume was 200 μL, and incubated for 1 hour at room temperature with rotation.

3. Gel electrophoresis experiment: After the incubation, 1.2 μL of sample was taken for gel electrophoresis experiment. See the gel electrophoresis experiment in Example 2 for detailed steps. Quantitative analysis of band brightness was carried out with Image Lab TM Software (Table 6).

The results are shown in Figures 5A-C and Table 6, which also show that the longer the sequence, the higher its affinity for PD-L1. This result shows that the laws we found have objective existence, repeatability and accuracy.

Example 5. Specificity, dissociation constant and secondary structure of PD-L1 nucleic acid aptamer 8-60

The preferred nucleic acid aptamers 8-60 of PD-L1 were characterized for their specificity, dissociation constant and secondary structure (Fig. 5A-C). We first use the gel retardation (EMSA) experiment to determine the specificity of 8-60, and the specific operation steps are as follows:

1. DNA heat treatment: Add 50pmol 8-60 to the digestion buffer, heat at 95°C for 10min, and slowly cool down to room temperature.

2. Mix 20 μL 0.1% Tween-80 with the above DNA solution thoroughly, centrifuge to remove the foam, and then slowly mix with 100 pmol PD-L1, SA, polypeptide, β-casein and ovalbumin respectively, the final volume is 200 μL, and do not use Samples containing protein, that is, only 8-60, were used as negative controls, and incubated with rotation for 1 hour at room temperature.

3. Gel electrophoresis experiment: After the incubation, 1.2 μL of sample was taken for gel electrophoresis experiment. See the gel electrophoresis experiment in Example 2 for detailed steps.

The results are shown in Figure 5A. In the gel retardation experiment, only the samples incubated with PD-L1 and 8-60 showed obvious complex bands, and no complex bands were observed on the gel after incubation of 8-60 with other proteins. bring. This result indicates that 8-60 has high specificity for PD-L1.

We next determined the dissociation constant of 8-60 using the Nano-Affi method, and the measured dissociation constant was 6.2±2.7nM ( FIG. 5B ). Specific steps are as follows:

1. Preparation of nano-gold: According to the method reported in the literature, the nano-gold colloid (GNP, 8.3nm, pH 6.5) was synthesized, and the pH of the nano-gold was adjusted to 5.0 with HCl.

2. Prepare the sample: mix PD-L1 with different concentrations of 8-60 in the screening buffer solution, and the final volume is 4 μL. The final concentrations of 8-60 are 0, 125nM, 250nM, 375nM, 500nM, 1250nM, 1875nM, 2500nM, respectively, and each concentration solution contains 4pmol PD-L1.

3. Incubation: Incubate the above samples at room temperature for 10 minutes.

4. After the incubation, add 96 μL of GNP to the mixture, mix slowly with a pipette 4 times, and incubate for 10 minutes. The final concentrations of 8-60 are 0, 5nM, 10nM, 15nM, 20nM, 50nM, 75nM, 100nM respectively; the final concentration of PD-L1 is 40nM.

5. Using dynamic light scattering (DLS) to measure the particle size of GNP.

The secondary structure of 8-60 was fitted using the online secondary structure simulation software of Integrated DNA Technologies (IDT) (Fig. 5C). There is no deletion in the middle fixed sequence (internal coding) of 8-60, but a single base mutation occurs in the Alu I recognition sequence AG^CT, that is, G→A, and the mutated sequence forms a double-stranded complementary structure with the random sequence.

Example 6. Recognition ability of PD-L1 nucleic acid aptamer 8-60 to PD-L1 expressed on the cell surface

We measured the ability of 8-60 to recognize PD-L1 expressed on the cell surface by flow cytometry. In this experiment, cells with different levels of PD-L1 expression, which are commonly used in the performance test of PD-L1 antibodies, were used for identification. According to the instructions of Abcam’s PD-L1 antibody, four kinds of cells were purchased, namely COLO205 cells (human colon cancer cells) without PD-L1 expression, HCC70 cells (human breast ductal carcinoma cells) with low PD-L1 expression, and PD-L1 ES-2 cells (human ovarian clear cell carcinoma cells) expressing in ES-2 cells, and BCPAP cells (human thyroid cancer papillary cells) expressing PD-L1 highly. Cy5-A60 (Cy5 fluorophore modified A60 chain, Table 1) which has no affinity for PD-L1 was used as a negative control in all tests.

The specific operation steps are as follows:

1. DNA heat treatment: 2.4 pmol Cy5-8-60 (8-60 modified by Cy5 fluorophore, Table 1) or 2.4 pmol Cy5-A60 were respectively prepared in the screening buffer solution to a final concentration of 200 nM. Heating at 95°C for 5 minutes, quenching on ice for 10 minutes, and standing at room temperature for 30 minutes (avoid light as much as possible).

2. Cell Processing

2.1. HCC70 cells, ES-2 cells, and BCPAP cells were digested with 1mL 5mM EDTA in the incubator for 5-10 minutes, and then transferred to 1.5mL clean centrifuge tubes, while COLO205 cells were directly transferred to 1.5mL clean centrifuge tube.

2.2. Centrifuge the four types of cells at 1000 rpm for 3 minutes, discard the supernatant and keep the bottom cells.

2.3. Prepare blocking solution. Prepare according to the following ratio: 800 μL blocking solution contains 16 μL salmon essence (10 mg/mL); 80 μL 10% BSA; 704 μL screening buffer solution.

2.4. Add 200 μL of blocking solution to each centrifuge tube containing cells, and vortex to mix.

3. Divide the two heat-treated DNA solutions into 4 tubes, 8 tubes in total.

4. Incubation: Add cell solution containing blocking solution to each DNA solution, 100 μL per tube, and vortex to mix. All mixtures were incubated in a dark place away from light for 30 minutes and vortexed every 10 minutes.

5. After the incubation, centrifuge for 3 minutes at 1000rpm, discard the supernatant again, and keep the bottom cells.

6. Wash the cells: Add 300 μL of screening buffer solution to the cells, vortex to mix, and centrifuge for 3 minutes at 1000 rpm.

7. After repeated washing once, dilute to volume with 300 μL screening buffer solution, and filter cells with 400 mesh sieve.

8. Measured using a flow cytometer (FACScalibur, Becton Dickinson, USA). 10,000 cells were measured each time, using Cy5 channel (FL4-H), and using FlowJo software (Treestar, San Carlos, USA) for data analysis.

The results are shown in Figures 6A-6D, cells with higher expression levels of PD-L1, the greater the increase in fluorescence intensity after incubation with Cy5-8-60, indicating that 8-60 can recognize PD-L1 expressed on the cell surface. However, the fluorescence intensity of Cy5-A60 as a negative control did not increase significantly after incubation with the four types of cells, indicating that 8-60 specifically recognized PD-L1 on the cell surface.

We next measured the dissociation constant of Cy5-8-60 for PD-L1 expressed on BCPAP cells by flow cytometry. The specific operation steps are as follows:

1. Cy5-8-60 heat treatment: 3 μL of Cy5-8-60 solutions with final concentrations of 5nM, 10nM, 20nM, 50nM, 75nM, 100nM and 200nM were prepared in the screening buffer solution. And heated at 95°C for 5 minutes, quenched on ice for 10 minutes, and stood at room temperature for 30 minutes (avoid light as much as possible).

2. Cell Processing

2.1.BCPAP cells were digested with 1mL 5mM EDTA for 5-10 minutes in the incubator and then transferred to

1.5mL clean centrifuge tube.

2.2. Centrifuge for 3 minutes at 1000 rpm, discard the supernatant and keep the bottom cells.

2.3. Prepare blocking solution. According to the following recipe configuration: 700 μL blocking solution contains 14 μL salmon essence (10 mg/mL); 70 μL 10% BSA; 616 μL screening buffer solution.

2.4. Add blocking solution to the centrifuge tube containing BCPAP cells, and vortex to mix.

3. Incubation: Add the above-mentioned BCPAP cell solution containing blocking solution to the 7 different concentrations of Cy5-8-60 solutions that have been heat-treated, 100 μL per tube, and vortex to mix. All mixtures were incubated in a dark place away from light for 30 minutes and vortexed every 10 minutes.

4. After the incubation, centrifuge for 3 minutes at 1000rpm, discard the supernatant again, and keep the bottom cells.

5. Wash the cells: Add 300 μL of screening buffer solution to the cells, vortex to mix, and centrifuge for 3 minutes at 1000 rpm.

6. After repeated washing once, dilute to volume with 300 μL screening buffer solution, and filter cells with 400 mesh sieve.

7. Measured using a flow cytometer (FACScalibur, Becton Dickinson, USA). 10,000 cells were measured each time, using Cy5 channel (FL4-H), and using FlowJo software (Treestar, San Carlos, USA) for data analysis.

Results As shown in Figure 7A, the fluorescence intensity of BCPAP cells increased with the increase of Cy5-8-60 concentration. The fluorescence intensity of BCPAP cells incubated with 200nM Cy5-A60 was close to that of Cy5-8-60 at 5nM. According to the fluorescence intensity in Figure 7A, the binding curve (Figure 7B) for the determination of the dissociation constant was plotted. According to the 1:1 binding mode, the dissociation constant obtained by nonlinear fitting was 82.5±25.5nM. It shows that 8-60 has high affinity to PD-L1 expressed on the surface of cancer cells.

Example 7. The ability of PD-L1 nucleic acid aptamer 8-60 to recognize the expression level of PD-L1 on tissue sections

We used FAM-8-60 (FAM fluorophore-modified 8-60) to perform fluorescence imaging on three different tissue sections, namely normal human tonsil sections, non-small cell lung cancer tissue sections and malignant melanoma tissue sections . All types of consistent tissue sections are serial sections of the same tissue. Among them, normal human tonsil tissue sections are the gold standard for detecting the performance of PD-L1 antibodies, while non-small cell lung cancer and malignant melanoma are tumor tissues with high expression of PD-L1. We first performed PD-L1 antibody immunohistochemical experiments on the same batch of tissue sections, and the experimental results were used as positive controls. At the same time, we used FAM-383-33 and FAM-A60 (FAM fluorophore modification) as negative controls.

The specific operation steps are as follows:

1. Nucleic acid aptamer heat treatment: 100 pmol nucleic acid aptamer was prepared into 150 μL solution in PD-L1 screening buffer solution, heated at 95°C for 5 minutes, quenched on ice for 10 minutes, and kept at room temperature in the dark for 10 minutes.

2. Tissue section processing

2.1. Soak the tissue section in p-xylene for 30 minutes, take out the tissue section at 15 minutes, and soak the above solution in 95%, 90%, 85%, 80%, and 75% ethanol twice , Soak for 3 minutes each time.

2.2. Put 0.01M sodium citrate solution (pH 6.0) in a clean beaker, and immerse the above-mentioned tissue slices in the solution, and heat in a microwave oven. When the solution starts to boil and bubbles appear, start timing immediately, pause for 30 seconds for every 1 minute of heating, and repeat this operation for 15 minutes. The beaker was then removed from the microwave and allowed to cool slowly to room temperature.

2.3. Take out the tissue section from the beaker and wash it three times with 1×PBS/0.1M NaCl.

2.4. Prepare blocking solution: 10 μL salmon essence (10 mg/mL) per 100 μL blocking solution; 10 μL yeast tRNA (10 mg/mL); 50 μL 10×PBS/0.1M NaCl; 20 μL 5% bovine serum albumin (BSA) ; 10 μL Tween-20 (0.1%) formula for preparation.

2.5. Sealing of tissue sections: Add the blocking solution onto the tissue sections (pay attention to the location of the tissue), 100 μL/section, block at 37°C for 1 hour, and then wash with 1×PBS/0.1M NaCl three times again.

3. Add the heat-treated aptamer solution dropwise to the tissue section, incubate at 37°C for 20 minutes, and wash three times with 1×PBS/0.1M NaCl.

4. Use a fluorescence microscope to perform fluorescence imaging on tissue sections.

The results are shown in Figure 8A, which are completely consistent with the corresponding immunohistochemical results of the PD-L1 antibody (Figure 8A, left). High expression of L1), germinal center (low fluorescence intensity, low expression of PD-L1) (Figure 8A, right). However, the fluorescence intensities of tissue sections incubated with the negative control sequences FAM-383-33 and FAM-A60 were very low. From the results of non-small cell lung cancer tissue sections (Figure 8B and 8C), it can be seen that 8-60 can very effectively distinguish NSCLC sections with positive and negative expression of PD-L1, and the distribution of fluorescence intensity is basically the same as that of PD-L1 antibody immunohistochemistry. The results were consistent. The fluorescence intensities of tissue sections incubated with the negative control sequences FAM-383-33 and FAM-A60 were also very low. Similarly, as shown in Figures 9A and 9B, 8-60 can very effectively distinguish malignant melanoma tissue sections with positive and negative expression of PD-L1, and the fluorescence intensity distribution is basically consistent with the immunohistochemical results of PD-L1 antibody. The fluorescence intensities of tissue sections incubated with the negative control sequences FAM-383-33 and FAM-A60 were also very low.

In summary, 8-60 can be used to identify the expression level of PD-L1 in tissue sections, and the effect is comparable to that of PD-L1 antibodies.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention.

Sequence List

<110> Capital Normal University

<120> A method for in vitro screening of DNA aptamers of PD-L1 and its application in cancer diagnosis

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

<213> Artificial sequence

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<212>DNA

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<212>DNA

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<212>DNA

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<220>

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<212>DNA

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<212>DNA

<213> Artificial sequence

<220>

<223> Candidate aptamer

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

<213> Artificial sequence

<220>

<223> Candidate aptamer

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<211> 60

<212>DNA

<213> Artificial sequence

<220>

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<212>DNA

<213> Artificial sequence

<220>

<223> Candidate aptamer

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<211> 60

<212>DNA

<213> Artificial sequence

<220>

<223> Candidate aptamer

<400> 50

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<210> 51

<211> 60

<212>DNA

<213> Artificial sequence

<220>

<223> Candidate aptamer

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cgcgacccat cctgcctact acgagacgaa cttatgcgta aactctcttc caccgctcgt 60

Claims (3)

1. A method for in vitro screening of a DNA aptamer for PD-L1, characterized in that the DNA sequence in the initial random DNA library of the method comprises a primer binding region at both ends, two random sequence regions and a A fixed region containing a restriction endonuclease recognition site between two random sequence regions, said fixed region can complementarily hybridize with a short cDNA sequence to form a double-stranded DNA that can be cut by a type II restriction endonuclease; The steps of the method for in vitro screening of DNA aptamers of PD-L1 include: firstly prepare a pre-enriched library through 3 rounds of magnetic cross-linking precipitation-SELEX, and then perform 4 rounds of internal encoding-SELEX to obtain an enriched library; The three rounds of magnetic cross-linking precipitation-SELEX preparation pre-enrichment library includes the following steps: Step 1. DNA library heat treatment: random DNA library was diluted in 500 μL of screening buffer solution, heated in a water bath at 95°C for 10 minutes, quenched on ice for 10 minutes, and left at room temperature for 10 minutes. The screening buffer solution contained 50 mM 4-hydroxyethyl Piperazineethanesulfonic acid, 100mM NaCl, 1mM MgCl ₂ , 5mM KCl, 1mM CaCl ₂ , the pH value of the screening buffer solution is 7.4; Step 2. Activation of carboxyl-coated magnetic beads: Step 2.1. Rotate and mix the carboxyl-coated magnetic beads at room temperature for 15 minutes; Step 2.2. Pipette 10 μL of magnetic beads and 100 μL of 25 mM MES solution with a pH value of 5.0 to mix thoroughly for 10 minutes, discard the supernatant, and then wash twice with 100 μL of 25 mM MES solution; Step 2.3. Freshly prepare 50mg/mL EDC solution and 50mg/mL NHS solution with 25mM MES solution; Step 2.4. Add 100 μL of EDC solution and 100 μL of NHS solution to the magnetic beads washed in step 2.2, mix thoroughly, and shake at low speed for 30 minutes at room temperature; Step 2.5. Place the centrifuge tube containing EDC, NHS, and magnetic beads on a strong magnet for 1 minute, remove the supernatant and wash twice with 100 μL of 25 mM MES solution to obtain activated magnetic beads; Step 2.6. Add 10 μL of screening buffer solution to the activated magnetic beads to evenly disperse the magnetic beads in the solution; Step 3. Negative screening with activated magnetic beads: add the heat-treated DNA library in step 1 to the activated magnetic beads, the total volume is 500 μL, and incubate for 30 minutes at room temperature with rotation and mixing; discard the magnetic beads after the incubation, Keep the supernatant; Step 4. Incubation of the DNA library after negative screening and the target protein PD-L1: add PD-L1 to the supernatant collected in step 3, rotate and mix at room temperature for 30 minutes; add activated magnetic For the beads, discard the supernatant and keep the magnetic beads; add 200 μL screening buffer solution to the magnetic beads, incubate for 5 minutes at room temperature, place on a strong magnet for 1 minute, and repeat washing 4 times; finally, add 120 μL screening buffer solution , shake and heat at 95°C for 20 minutes, magnetically separate for 1 minute, and collect the supernatant; Step 5. Carry out polymerase chain reaction (PCR) to amplify the DNA sequence in the supernatant collected in step 4, using a biotin-labeled reverse primer; Step 6. prepare DNA single strand; Step 6.1. Prepare the column: place the streptavidin sepharose beads on a rotator at room temperature for 15 minutes, add 60 μl streptavidin sepharose beads to a 200 μl anti-aerosol tip, and use 200 μl Wash the column with a phosphate buffer solution containing 1M NaCl, which is a 1×PBS solution containing 1M NaCl; Step 6.2. Passing the column: add the PCR-amplified DNA sequence to the column in batches with a pipette gun, 100 μL each time, and use the pipette gun to apply pressure to make the PCR-amplified DNA sequence and streptavidin agarose gel The beads were mixed thoroughly, and after 3 minutes, pressurize the pipette gun to discharge the mixture in the column, and then wash the column with 200 μL of the phosphate buffer solution; repeat this operation until all the DNA sequences amplified by PCR are added. column; Step 6.3. NaOH elution: After washing the column twice with 200 μL of the phosphate buffer solution, add 100 μL of 30 mM NaOH solution into the column with a pipette gun, and discharge the NaOH solution out of the column with a pipette gun after 5 minutes. Collect into a clean centrifuge tube to obtain a single-stranded DNA library; Step 6.4. Library quantification: neutralize the above-mentioned single-stranded DNA library with an equal amount of HCl, and perform concentration quantification by measuring the ultraviolet-visible absorption intensity at 260 nm; The 4 rounds of coding-SELEX include the following steps: Step 7. Restriction enzyme-SELEX; Step 7.1. DNA heat treatment and complementary hybridization: prepare the DNA library and the short cDNA sequence in step 6.3 in an enzyme digestion buffer containing 10mM Tris-HCl, 10mMMgCl2, and 1mM dithiothreitol to a final concentration of 0.4 μM library DNA Solution, the pH value of the digestion buffer is 7.5; the above solution is heated in a water bath at 95°C for 10 minutes, and slowly cooled to room temperature; Step 7.2. Incubate the library with PD-L1: add 0.2 μM PD-L1 to the heat-treated library DNA solution, and incubate at room temperature for 1 hour; Step 7.3. Enzyme digestion reaction: Prepare Alu I enzyme solution with a final concentration of 0.7U/μL in the enzyme digestion buffer solution; add 50 μL of AluI enzyme solution prepared newly to 150 μL of the library DNA solution incubated at room temperature in step 7.2, and divide Put in two 100μL PCR small tubes, set the heat treatment program of the PCR instrument as 37°C for 30 minutes; 80°C for 15 minutes; Step 7.4. Gel electrophoresis experiment: Take 6 μL of enzyme cutting solution for gel electrophoresis experiment; Step 7.5.PCR amplification; Step 7.6 prepares DNA single strand, the method is the same as step 6; Step 8. Repeat the above magnetic cross-linking precipitation-SELEX; the enrichment library enters the next round of internal coding-SELEX; The first round of the 4 rounds of internal coding-SELEX only includes restriction endonuclease-SELEX, and the internal coding-SELEX of the second to fourth rounds all include restriction endonuclease-SELEX and magnetic cross-linking precipitation-SELEX. 2. The method according to claim 1, further comprising high-throughput sequencing of the enriched library. 3. The method according to claim 2, further comprising affinity and specificity tests for selecting candidate nucleic acid aptamer sequences.