CN114127293B - Method for screening PD-L1 DNA nucleic acid aptamer in vitro and application of method 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

Method for screening PD-L1 DNA nucleic acid aptamer in vitro and application of method in cancer diagnosis

Technical Field

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

Background

Programmed cell death receptor 1 (PD-1) is a cell surface receptor, a 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 lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells). PD-1 binds to 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 will react to foreign antigens that accumulate in the lymph nodes or spleen, promoting antigen-specific T cell proliferation. And the PD-1 and the PD-L1 are combined, so that an inhibitory signal can be transmitted, the activation and proliferation of T cells are reduced, and tumor cells can obtain immune escape. PD-1 and PD-L1 can be used as targets, and antibodies thereof have been proved to enhance the anti-tumor, anti-infection, anti-autoimmune disease and organ transplantation survival rate. Currently, antibody drugs on the market that are based on blocking PD-L1/PD-1 binding for cancer treatment 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 clinically interesting issue. PD-L1 immunohistochemical detection is a simple and effective method for predicting the curative effect of the PD-1/PD-L1 inhibitor. Currently, indications for PD-1/PD-L1 inhibitors that have been approved by the FDA/NMPA include lung cancer, melanoma, and urothelial cancer, among others. As a predictive marker of the efficacy of PD-1/PD-L1 immune checkpoint inhibitor drugs, PD-L1 detection has been approved by the FDA as a concomitant or complementary diagnosis of immunotherapy. Currently, there are five main types of PD-L1 immunohistochemical detection kit/antibody: 22C3, 28-8, SP263, SP142 and 73-10, were tested on two immunohistochemical platforms Dako and Ventana, respectively. Currently, the PD-L1 immunohistochemical detection technique faces the following main detection problems. (1) different antibodies require the use of different detection platforms. For example, 22C3 and 28-8 antibody assays use the DAKO AutoStainer Link platform, while SP142 and SP263 antibody assays use the Ventana Benchmark Ultra platform. (2) The interpretation standards of different antibody detection results are different, and the interpretation of PD-L1 can be ensured by a great deal of training of a specialized pathologist. (3) there is a difference in the detection results of different antibodies. For example, although the consistency of the detection results of 28-8, 22C3 and SP263 has been proved to be high by the prior studies, the comparison is only performed on specific tumor types, and the detection results of different antibody types still have not small differences among individual tumor types. In addition, the antibody has the defects of high price, poor stability, easy inactivation, large batch-to-batch performance difference and the like.

Nucleic acid aptamers (aptamers) are a class of short-chain nucleotide sequences (RNA or DNA) that can specifically bind to a variety of target molecules, and are commonly obtained by an in vitro screening technique called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) (Science, 1990,249,505-510). SELEX is an in vitro screening technique that screens nucleic acid aptamers with high affinity and specificity from a chemically synthesized random nucleic acid library through multiple rounds of affinity enrichment for target molecules. Compared with the antibody, the aptamer has the advantages of long shelf life, high stability, small batch-to-batch variation, low or no immunogenicity, low cost, convenient chemical modification and the like (Biotechnol.Adv.2018, 37,28-50), and has good application prospect in the field of tumor diagnosis. However, no related literature report on the detection of PD-L1 expression level of human cancer tissue sections based on a PD-L1 detection kit of a nucleic acid aptamer exists at present.

Traditional SELEX techniques utilize cellulose acetate membranes to separate nucleic acid aptamers that bind to protein targets. In order to improve the screening efficiency and performance of nucleic acid aptamers, various improved SELEX techniques 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 requires a fixed library, and the enrichment effect is poor due to unavoidable self-dissociation of the library, so that multiple rounds of screening are required; capillary electrophoresis-SELEX screens nucleic acid aptamer under high voltage conditions, and often fails to identify target proteins in a natural state; MB-SELEX and Microfluidic-SELEX need to fix target proteins on magnetic beads, so that the secondary structure of the proteins is partially changed, and interface steric hindrance and interface adsorption exist, so that the affinity and the specificity of the aptamer are limited; cell-SELEX and Tissue-SELEX use Cell or Tissue section to carry on screening of aptamer separately, because Cell or Tissue section is comparatively complicated, so easy to appear in the screening process to have enrichment of the sequence of affinity to the non-target, cause the disadvantage such as being not high of the enrichment efficiency.

U.S. Pat. No. 2010 discloses (patent publication No. US 2010/0152056A 1) a nuclease-based SELEX technology. The method does not require solid phase immobilization of the library or target, does not require complicated equipment, but requires the use of multiple nucleases. Screening of nucleic acid aptamers is achieved by using exonucleases (exonucleases I, III, T, or T7 exonuclease I) to degrade sequences that cannot bind to a target, and sequences that can bind to a target are not degraded. The DNA library used in the method has a random sequence region of 5 to 1000 bases in length and a fixed sequence (in 5 'or 3' or 5 'and 3'). It is necessary to add a sequence of 5 to 30 bases to the 3' of the sequence using a terminal transferase after each round of nuclease degradation reaction in order to perform PCR amplification of the sequence. Each nuclease has different requirements on the structure of the nucleic acid, for example, exonuclease I can degrade single-stranded DNA only from the 3 'end, while exonuclease III can degrade double-stranded DNA only in the 3' to 5 'direction, and can only blunt ends or 3' recessed ends. Since nucleic acid sequences that do not bind to a target can form a wide variety of secondary structures, such as double-stranded secondary structures that are often single-stranded and complementary at the ends, the use of multiple exonucleases simultaneously is required to adequately degrade sequences that do not bind to a target. The simultaneous use of multiple nucleases presents great difficulties for optimizing the nuclease reaction, since the structural selectivity of nucleases is closely related to the structure, composition and concentration of the substrate, which cannot be predicted during the screening process. In addition, the method has the advantage that the sequence for PCR amplification is added at the 3' end of the sequence after nuclease reaction, so that the operation is complicated.

The US patent issued in 2014 (US patent number: US8680017B 2) overcomes the above-described disadvantage of requiring the addition of sequences for PCR amplification at the 3' end of the sequence after the nuclease reaction by using a library design with complementary ends. The DNA library used in the method has a region of intermediate random sequence and two fixed sequences on either side, which are complementary in sequence. The complementary double-stranded region may or may not contain a recognition site for a restriction enzyme, and the sequence that does not bind to the target is degraded by the restriction enzyme or by one or more endonucleases or exonucleases. Recent studies have shown that binding of the target to sequences with the above-described structure does not disrupt the double-stranded structure at the ends of the sequence. Indeed, the design of the terminal double-stranded structure is often used to stabilize the structure of the aptamer and to enhance the affinity of the target for the aptamer, such as cocaine aptamer and thrombin aptamer TBA29, and the like. Thus the method degrades a large number of high affinity sequences. Moreover, even if the ends of the library are designed as complementary sequences, it is not ensured that most of the sequences in the library form end-complementary double-stranded DNA structures due to the diversity of the secondary structure of the library and the hybridization between the library sequences, and that a large number of sequences not bound to the target cannot be degraded by double-stranded structure-specific nucleases. To solve this problem, there is still a need to use a plurality of nucleases simultaneously, and not only the reaction conditions are difficult to optimize, but also the sequence capable of binding to the target is lost.

Therefore, a new screening technology which has the advantages of simple operation, high enrichment efficiency, strong specificity, good compatibility with real samples and the like is needed at present. In addition, all the existing SELEX techniques need to perform affinity test on candidate sequences to select the aptamer with the highest affinity, which is a most time-consuming and expensive technical step in the existing SELEX techniques.

Currently, reported DNA Nucleic acid aptamers of human PD-L1 are screened by using traditional cellulose acetate membrane-based SELEX (Molecular Therapy-Nucleic Acids,2016,5, e 397) or target immobilized-based MB-SELEX technology (microchip Acta 2017,184,4029-4035), respectively, but are not applied to detection of PD-L1 expression levels of human cancer tissue sections. The 2018 american scholars reported that DNA aptamer of PD-L1 screened based on library-modified microbeads was applied to fluorescence imaging of human prostate cancer tissue sections using a chemically modified library, but lacked a negative control, and sequence information and chemical modification information of the aptamer were not disclosed (Biochimie 2018,145,125-130).

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a novel method for screening DNA nucleic acid aptamer with high affinity and specificity of a broad-spectrum tumor marker apoptosis receptor 1-ligand 1 (PD-L1), namely, an in-vivo encoding-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 restriction enzyme recognition site located therebetween. Wherein the inner coding region hybridizes to a short-chain complementary sequence (cDNA) to form a double-stranded DNA that is cleavable by a type II restriction enzyme. The library was pre-enriched using magnetic cross-linked precipitation-SELEX (MCP-SELEX, anal.Chem.2019,91, 13383-13389), followed by incubation of the DNA library with PD-L1, followed by addition of the type ii restriction enzyme Alu I. The sequence capable of binding to PD-L1, wherein the internal coding structure is disrupted, so that the sequence is not cleaved by a restriction enzyme, and is retained by PCR amplification. The MCP-SELEX specific enrichment in each round of screening was induced by target binding, but not by restriction endonucleases, resulting in sequences with altered internal coding structure. The method avoids the fixation of the library and the target on a solid phase, can realize the separation of the sequence combined with the target by only one restriction enzyme, and has simple operation and easy optimization of experimental conditions. The candidate aptamer obtained by the invention has the characteristic that the longer the inner coding length is, the higher the affinity is, and the selection of the high-affinity aptamer is greatly accelerated, which is not possessed by all the current SELEX technology. In addition, the method of the invention utilizes MCP-SELEX to enrich the library for 3 rounds, greatly reduces the diversity of the library, is beneficial to reducing the non-specific interaction between sequences and promotes the formation of a recognition structure of restriction enzyme in the sequences, thereby greatly screening efficiency. The method also utilizes MCP-SELEX to efficiently capture the sequence combined with the target after each round of restriction enzyme reaction, eliminates the sequence mutation induced by the restriction enzyme, and thus, eliminates the nonspecific sequence escaped from the restriction enzyme reaction. In the prior art, the sequence which is resistant to nuclease degradation is not cleared from the enriched library due to the mutation induced by the nuclease, so that the screening efficiency is low. The preferred fluorescent-labeled aptamer is successfully used for fluorescent imaging of PD-L1 expression levels of various tumor tissue sections, has the performance equivalent to that of an antibody, is extremely simple to operate, and has great potential for the concomitant diagnosis or supplementary diagnosis of PD-1/PD-L1 immunotherapy. The superior performance of the selected nucleic acid aptamer fully demonstrates the feasibility of the method of the invention. The prior art (US 2010/01520567A 1; US8680017B 2) gives only a description of the method, none of which has proven its feasibility experimentally.

Compared with the existing nuclease-based SELEX technology (US 8680017B 2), the method has the following advantages:

1) The method of the invention can conveniently and efficiently separate the sequence combined with the target from the sequence not combined with the target by only using one type II restriction enzyme. Whereas the prior art requires a plurality of nucleases, condition optimization is difficult.

2) Library design of the method of the invention is more advantageous for binding of targets to sequences. The library design of the method of the invention comprises two random sequence regions and an immobilization region (i.e., an inner coding) containing a recognition site for a type II restriction enzyme located therebetween. Wherein the inner coding region hybridizes to a short-chain complementary sequence (cDNA) to form a double-stranded DNA that is cleavable by a type II restriction enzyme. In the prior art, libraries with complementary double chains formed at the tail ends are adopted, so that hybridization between sequences is unavoidable, so that cross-linked products of the sequences are easy to form in a system, the combination between a target and the sequences is blocked, and certain sequences with affinity are degraded; in the method, excessive short-chain cDNA is hybridized with the inner coding region of the library to form a recognition structure of the type II restriction endonuclease, so that the hybridization efficiency is high, the formation of the inner coding/cDNA double-chain structure can reduce the interaction between sequences, and the effective combination of a target and a single sequence is facilitated.

3) The library design of the method is more favorable for inducing the recognition structure of the type II restriction enzyme to generate large secondary structure change when the target is combined with the sequence, so that the efficiency of screening the sequence with affinity is higher. The random region is positioned at two sides of the internal code in the library design, the length is only 20 bases, and the combination of the target and one or two random region sequences can cause great change of the internal code/cDNA double-stranded DNA structure, so that the sequence is not degraded in the restriction enzyme reaction. The random region in the prior art is positioned at the position of a loop in the sequence 'stem-loop structure', and the combination of a target and the random region often does not cause the change of a double-chain 'stem' structure, and even prolongs the length of the 'stem', so that the sequence is still degraded in a restriction enzyme reaction, and the sequence with affinity is lost.

4) The method of the invention utilizes MCP-SELEX to efficiently capture the sequence combined with the target after each round of restriction enzyme reaction, eliminates the mutant sequence generated by the induction of the restriction enzyme, and thus eliminates the non-specific sequence escaping from the restriction enzyme reaction, thus the screening specificity is good. In the prior art, the sequence resistant to nuclease degradation is not cleared from the enriched library due to nuclease-induced mutation.

5) The nucleic acid aptamer obtained by the method has the characteristic of higher affinity as the length of the internal code is longer, and can be conveniently selected through the length of the nucleic acid aptamer without carrying out affinity test on a large number of candidate sequences one by one. All SELEX techniques currently do not possess this feature.

6) The optimal aptamer screened by the method is used for specifically identifying PD-L1 in various tumor tissue sections for the first time, reaches the level equivalent to that of a PD-L1 antibody, is extremely simple and quick to operate, and has great potential for the concomitant diagnosis or supplementary diagnosis of PD-1/PD-L1 immunotherapy.

The specific experimental steps of the invention are as follows: the names, sequences and uses of all the DNAs used are shown in Table 1. 1. Preparation of Pre-enriched library Using PD-L1 as target protein Using 3 rounds of MCP-SELEX

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

Step 1. Heat treatment of DNA library: the DNA library was diluted in 500 μl (microliter) of screening buffer (50 mM (millimoles per liter) of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 100mM NaCl,1mM MgCl2,5mM KCl,1mM CaCl2,pH =7.4), heated in a 95 ℃ (celsius) water bath for 10 minutes, quenched on ice for 10 minutes, and left at room temperature for 10 minutes.

Step 2, activating carboxyl coated magnetic beads:

and 2.1, rotating and uniformly mixing the carboxyl coated magnetic beads for 15 minutes at room temperature.

Step 2.2. Mu.L of beads were pipetted and mixed thoroughly with 100. Mu.L of 25mM 2- (N-morpholino) ethanesulfonic acid (MES, pH=5.0) for 10min (min), the supernatant was discarded and washed twice with 100. Mu.L of 25mM MES solution.

Step 2.3. Fresh 50mg/mL (mg/mL) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution and 50mg/mL of N-hydroxysuccinimide (NHS) solution were prepared with 25mM MES solution, respectively.

Step 2.4. 100. Mu.L of the EDC solution and 100. Mu.L of the NHS solution were added to the 2.2 washed beads, and the mixture was thoroughly mixed and shaken at a low speed for 30 minutes at room temperature.

Step 2.5. Centrifuge tubes containing EDC/NHS/magnetic beads were placed on a strong magnet for 1 min, after which the supernatant was removed and washed 2 times with 100. Mu.L of 25mM MES solution.

Step 2.6. Add 10. Mu.L of screening buffer solution to the activated beads to uniformly disperse the beads in the solution.

Step 3, negative screening with activated magnetic beads: the DNA library heat-treated in step 1 was added to the activated magnetic beads in a total volume of 500. Mu.L and incubated for 30 minutes at room temperature with spin mixing. After the incubation, the beads were discarded and the supernatant was left.

Step 4, incubation of library with target protein PD-L1 after negative screening: PD-L1 was added to the supernatant collected in step 3, and the mixture was spun and incubated at room temperature for 30 minutes. After the incubation, the supernatant was discarded and the beads were left. 200. Mu.L of screening buffer solution was added to the beads, and the mixture was incubated for 5 minutes at room temperature with spin mixing, placed on a strong magnet for 1 minute, and washed repeatedly 4 times. Finally, 120. Mu.L of the screening buffer was added, and the mixture was heated with shaking at 95℃for 20 minutes, magnetically separated for 1 minute, and the supernatant (eluent) was collected.

Step 5. The DNA sequence in the eluate is subjected to Polymerase Chain Reaction (PCR) to amplify the DNA sequence using a reverse primer labeled with biotin.

Step 6, preparing a DNA single strand:

step 6.1. Preparing the column: the streptavidin sepharose beads were placed on a rotator for 15 min at room temperature, 60. Mu.L of streptavidin sepharose beads were added to a 200. Mu.L anti-aerosol gun head, and the column was washed with 200. Mu.L of phosphate buffer solution (1 XPBS/1M NaCl) containing 1M NaCl.

Step 6.2. Column passing: the PCR mixture was added to the column (100. Mu.L each) in portions by a pipette, the PCR mixture was thoroughly mixed with streptavidin agarose gel beads by applying pressure by the pipette, the mixture in the column was discharged from the tip after 3 minutes by applying pressure by the pipette, and then the column was washed with 200. Mu.L of 1 XPBS/1M NaCl. This operation was repeated until the PCR solution was completely added to the column.

Step 6.3.Naoh elution: after washing the column twice with 200. Mu.L of 1 XPBS/1M NaCl, 100. Mu.L of 30mM NaOH was added to the column with a pipette, after 5 minutes the NaOH solution was drained out of the column with a pipette pressure and collected in a clean centrifuge tube, i.e., a single-stranded DNA library.

Step 6.4. Library quantification: the single-stranded DNA library was neutralized with an equivalent amount of HCl and concentration was quantified by measuring the UV-visible absorbance at 260nm (nanometers).

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 endonuclease-SELEX (RE-SELEX)

Step 1.1.DNA heat treatment and complementary hybridization: the DNA library was prepared with complementary short-chain DNA (cDNA) of the intermediate fixed sequence in an enzyme digestion buffer (10 mM Tris-HCl,10mM MgCl2,1mM dithiothreitol, pH 7.5) to a final concentration of 0.4. Mu.M (micromoles per liter) of library DNA solution. The solution was heated in a 95℃water bath for 10 minutes and allowed to slowly cool to room temperature.

Step 1.2. Incubation of library with PD-L1: PD-L1 was added to the above system at a final concentration of 0.2. Mu.M and incubated for 1 hour at room temperature. The specific experimental parameters are shown in Table 3.

Step 1.3, enzyme digestion reaction: an Alu I enzyme solution was prepared in a final concentration of 0.7U/. Mu.L (activity units/. Mu.L) in the digestion buffer. Adding newly prepared 50 mu L Alu I enzyme solution into the 150 mu L DNA solution, subpackaging into two 100 mu L PCR small tubes, and setting a PCR instrument heat treatment program to 37 ℃ for 30 minutes; 15 minutes at 80 ℃; and (3) preserving the obtained enzyme cutting solution in a refrigerator at the temperature of 4 ℃ for 10 minutes for later use.

Step 1.4. Gel electrophoresis experiments: gel electrophoresis experiments were performed on 6. Mu.L of the enzyme-digested solution, and the detailed procedures were shown in the gel electrophoresis experiments.

Step 1.5.PCR amplification.

Step 1.6 preparation of DNA single strand: the detailed procedure is described in MCP-SELEX for single strand DNA production.

mcp-SELEX: see MCP-SELEX for detailed steps. The enriched library goes to 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. And selecting the affinity and specificity test of the candidate nucleic acid aptamer sequence.

5. The obtained aptamer is applied to normal human tonsil tissue section and cancer tissue section imaging.

The application experiment of the tissue section uses paraffin embedded PD-L1 high expression and non-expression non-small cell lung cancer (NSCLC) tissue section, PD-L1 high expression and non-expression malignant melanoma tissue section and normal human tonsil tissue section, and the specific operation steps are as follows:

step 1, aptamer heat treatment: 100pmol (picomolar) of aptamer was prepared as 150. Mu.L solution in PD-L1 screening buffer, heated at 95℃for 5 min, quenched on ice for 10 min, and allowed to stand at room temperature in the dark for 10 min.

Step 2, tissue section treatment

Step 2.1, immersing the tissue slice in paraxylene for 30 minutes, taking out the tissue slice at 15 minutes, immersing the solution in 95%, 90%, 85%, 80% and 75% ethanol twice in turn, each for 3 minutes.

Step 2.2. 0.01M sodium citrate solution (pH 6.0) was placed in a clean beaker and the tissue sections were immersed in the solution and heated in a microwave oven. When the solution started to boil, timing was immediately performed when bubbles appeared, and the operation was repeated for 15 minutes every 1 minute of heating, and was suspended for 30 seconds. The beaker was then removed from the microwave oven and slowly cooled to room temperature.

Step 2.3 tissue sections were removed from the beaker and washed three times with 1 XPBS/0.1M NaCl.

Step 2.4, preparing a sealing liquid: 10. Mu.L salmon sperm (10 mg/mL) was contained per 100. Mu.L of the blocking solution; 10. Mu.L yeast tRNA (10 mg/mL); 50 μL of 10 XPBS/0.1M NaCl;20 μL of 5% Bovine Serum Albumin (BSA); formulation of 10. Mu.L Tween-20 (0.1%) was prepared.

Step 2.5. Blocking of tissue sections: the block was applied dropwise to tissue sections (note tissue location), 100. Mu.L/plate, blocked for 1 hour at 37℃and then washed three more times with 1 XPBS/0.1M NaCl.

And 3, dripping the heat-treated aptamer solution onto a tissue slice, incubating for 20 minutes at 37 ℃, and then washing three times by using 1 XPBS/0.1M NaCl.

And 4, performing fluorescence imaging shooting on the tissue slice by using a fluorescence microscope.

Drawings

FIGS. 1A and 1B are flowcharts of Encoded-SELEX, wherein FIG. 1A shows the design of a DNA library used by Encoded-SELEX; FIG. 1B shows the Encoded-SELEX specific process.

FIGS. 2A-2F are graphs showing monitoring of library cleavage resistance and affinity evolution during the Encodified-SELEX screening, wherein FIGS. 2A-2D show gel electrophoresis of the products (REP-1, REP-2, REP-3, REP-4) of the library after restriction enzyme reaction in 1 st to 4 th rounds of screening, wherein "+" indicates the presence of PD-L1 or Alu I enzyme in the corresponding cleavage solution, and "-" indicates the absence of PD-L1 or Alu I enzyme in the corresponding cleavage solution, and the final concentration of Alu I enzyme in all cleavage solutions is 0.7U/. Mu.L; FIG. 2E shows the binding rate of the library to PD-L1 after each round of cleavage reaction determined by qPCR method, wherein R0 is the initial library after three rounds of MCP-SELEX enrichment; RE2, RE3, RE4 are libraries obtained after PCR amplification and single strand preparation of REP-2, REP-3, REP-4, respectively, wherein the binding rates of RE3 and RE4 to PD-L1 were negatively screened with a mixture of 0.06% serum and HSA (final concentrations of 65nM and 50nM, respectively) before testing; FIG. 2F shows the dissociation constant of R4 for a fluorescent label (Cy5.5) using an evanescent wave fiber sensor.

FIGS. 3A and 3B are bar graphs depicting affinity of 20 sequences (FIG. 3A) and 20 other sequences (FIG. 3B) prior to high throughput sequencing using the Nano-Affi method, where Δd is the change in particle size of the Nano-Gold (GNP) after addition of PD-L1 alone or PD-L1 and DNA sequences, where N-39 and N-60 are negative control sequences, with final DNA concentration of 20nM (nanomole per liter) and final PD-L1 concentration of 40nM in all assays.

FIG. 4 is a gel electrophoresis chart of gel blocking assay (EMSA) for determining the affinity of 5 candidate nucleic acid aptamers of different lengths, using Image Lab TM Software to determine the gray scale value (I) for each band, the data is shown below each lane, aptamer binding (%) = I (PD-L1-aptamer complex)/[ I (PD-L1-aptamer complex) +I (unbound aptamer) ].

FIGS. 5A-5C are graphs depicting affinity and specificity of representative candidate nucleic acid aptamers: (FIG. 5A) gel blocking assay (EMSA) to determine the selectivity of 8-60 for different proteins; (FIG. 5B) the dissociation constant of 8-60 was determined using Nano-Affi, with a final PD-L1 concentration of 40nM at all times; (FIG. 5C) the 8-60 secondary structure (www.idt.com) was simulated using software on the network, the arrow indicates the single base mutation in the Alu I recognition sequence, the original recognition sequence being AG CT.

FIGS. 6A-6D are graphs showing interactions of Cy5-8-60 with four cancer cells with different levels of PD-L1 expression using flow cytometry: (FIG. 6A) COLO205 cells (human colon cancer cells) that are not expressed by PD-L1; (FIG. 6B) HCC70 cells with low expression of PD-L1 (human ductal breast cancer cells); (FIG. 6C) ES-2 cells expressed in PD-L1 (human ovarian clear cell carcinoma cells); (FIG. 6D) BCPAP cells (human thyroid carcinoma papillary cells) with high PD-L1 expression, each group of experiments was treated with Cy5-A60 as a negative control.

FIGS. 7A and 7B are graphs showing fluorescence distribution data of Cy5-8-60 at different concentrations after incubation with BCPAP cells, based on flow cytometry to determine dissociation constants of Cy5-8-60 for PD-L1 expressed on BCPAP cells (FIG. 7A); (FIG. 7B) binding graphs for Cy5-8-60 dissociation constant assay plotted from the data of panel A, BCPAP cells were human thyroid carcinoma papillary cells with high PD-L1 expression, FL4-H channel was Cy5 fluorescent channel, with 200nM Cy5-A60 as negative control.

FIGS. 8A-8C are fluorescence microscopy imaging diagrams of FAM-8-60 for detecting PD-L1 expression levels in various tissue sections, (FIG. 8A) normal human tonsil tissue sections, fluorescence imaging of crypt, germinal center and epithelial tissue with PD-L1 expression levels from high to low, respectively; (FIGS. 8B, 8C) fluorescence imaging of PD-L1 positive and negative expressed non-small cell lung cancer (NSCLC) tissue sections, both positively controlled by PD-L1 antibody tissue section immunohistochemical results; using FAM-383-33 and FAM-A60 as negative controls, all types of tissue sections were serial sections of the same tissue.

FIGS. 9A and 9B are fluorescence imaging diagrams of FAM-8-60 on malignant melanoma tissue sections expressed positively (FIG. 9A) and negatively (FIG. 9B) for PD-L1, both positively controlled by the result of immunohistochemical treatment of PD-L1 antibody tissue sections; using FAM-383-33 and FAM-A60 as negative controls, all types of tissue sections were serial sections of the same tissue.

Detailed Description

The following detailed description of specific embodiments of the invention is provided to facilitate a further understanding of the invention. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

In general, the starting random DNA library of the method of the invention comprises two random sequence regions and an immobilization region (i.e.an internal coding) containing a restriction enzyme recognition site located therebetween. Wherein the inner coding region hybridizes to a short-chain complementary sequence (cDNA) to form a double-stranded DNA that is cleavable by a type II restriction enzyme. The library was pre-enriched using magnetic cross-linked precipitation-SELEX (MCP-SELEX, anal.Chem.2019,91, 13383-13389), followed by incubation of the DNA library with PD-L1, followed by addition of the type ii restriction enzyme Alu I. The sequence capable of binding to PD-L1, wherein the internal coding structure is disrupted, so that the sequence is not cleaved by a restriction enzyme, and is retained by PCR amplification. The MCP-SELEX specific enrichment in each round of screening was induced by target binding, but not by restriction endonucleases, resulting in sequences with altered internal coding structure.

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

1. preparation of a pre-enriched library using 3 rounds of MCP-SELEX with PD-L1 as target protein (experimental parameters for each round of screening are shown in Table 3);

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

3. high throughput sequencing of enriched libraries;

4. selecting an affinity and specificity test of candidate nucleic acid aptamer sequences;

5. the obtained aptamer is applied to normal human tonsil tissue section and cancer tissue section imaging.

In the drawings, FIGS. 1A and 1B are flowcharts of Encoded-SELEX, wherein FIG. 1A shows the design of a DNA library used by Encoded-SELEX; FIG. 1B shows the Encoded-SELEX specific process. FIGS. 2A-2F are graphs showing monitoring of library cleavage resistance and affinity evolution during the Encodified-SELEX screening, wherein FIGS. 2A-2D show gel electrophoresis of the products (REP-1, REP-2, REP-3, REP-4) of the library after restriction enzyme reaction in 1 st to 4 th rounds of screening, wherein "+" indicates the presence of PD-L1 or Alu I enzyme in the corresponding cleavage solution, and "-" indicates the absence of PD-L1 or Alu I enzyme in the corresponding cleavage solution, and the final concentration of Alu I enzyme in all cleavage solutions is 0.7U/. Mu.L; FIG. 2E shows the binding rate of the library to PD-L1 after each round of cleavage reaction determined by qPCR method, wherein R0 is the initial library after three rounds of MCP-SELEX enrichment; RE2, RE3, RE4 are libraries obtained after PCR amplification and single strand preparation of REP-2, REP-3, REP-4, respectively, wherein the binding rates of RE3 and RE4 to PD-L1 were negatively screened with a mixture of 0.06% serum and HSA (final concentrations of 65nM and 50nM, respectively) before testing; FIG. 2F shows the dissociation constant of R4 for a fluorescent label (Cy5.5) using an evanescent wave fiber sensor. FIGS. 3A and 3B are bar graphs depicting affinity of 20 sequences (FIG. 3A) and 20 other sequences (FIG. 3B) prior to high throughput sequencing using the Nano-Affi method, where Δd is the change in particle size of the Nano-Gold (GNP) after addition of PD-L1 alone or PD-L1 and DNA sequences, where N-39 and N-60 are negative control sequences, with final DNA concentration of 20nM (nanomole per liter) and final PD-L1 concentration of 40nM in all assays. The sequence information for each candidate aptamer is shown in table 5. FIG. 4 is a gel electrophoresis chart of gel blocking assay (EMSA) for determining the affinity of 5 candidate nucleic acid aptamers of different lengths, using Image Lab TM Software to determine the gray scale value (I) for each band, the data is shown below each lane, aptamer binding (%) = I (PD-L1-aptamer complex)/[ I (PD-L1-aptamer complex) +I (unbound aptamer) ]. The specific experimental parameters are shown in Table 6. FIGS. 5A-5C are graphs depicting affinity and specificity of representative candidate nucleic acid aptamers: (FIG. 5A) gel blocking assay (EMSA) to determine the selectivity of 8-60 for different proteins; (FIG. 5B) the dissociation constant of 8-60 was determined using Nano-Affi, with a final PD-L1 concentration of 40nM at all times; (FIG. 5C) the 8-60 secondary structure (www.idt.com) was simulated using software on the network, the arrow indicates the single base mutation in the Alu I recognition sequence, the original recognition sequence being AG CT. FIGS. 6A-6D are graphs showing interactions of Cy5-8-60 with four cancer cells with different levels of PD-L1 expression using flow cytometry: (FIG. 6A) COLO205 cells (human colon cancer cells) that are not expressed by PD-L1; (FIG. 6B) HCC70 cells with low expression of PD-L1 (human ductal breast cancer cells); (FIG. 6C) ES-2 cells expressed in PD-L1 (human ovarian clear cell carcinoma cells); (FIG. 6D) BCPAP cells (human thyroid carcinoma papillary cells) with high PD-L1 expression. Each set of experiments was run with Cy5-A60 as a negative control. FIGS. 7A and 7B are graphs showing fluorescence distribution data of Cy5-8-60 at different concentrations after incubation with BCPAP cells, based on flow cytometry to determine dissociation constants of Cy5-8-60 for PD-L1 expressed on BCPAP cells (FIG. 7A); (FIG. 7B) binding graphs for Cy5-8-60 dissociation constant assay plotted from the data of panel A, BCPAP cells were human thyroid carcinoma papillary cells with high PD-L1 expression, FL4-H channel was Cy5 fluorescent channel, with 200nM Cy5-A60 as negative control. FIGS. 8A-8C are fluorescence microscopy imaging diagrams of FAM-8-60 for detecting PD-L1 expression levels in various tissue sections, (FIG. 8A) normal human tonsil tissue sections, fluorescence imaging of crypt, germinal center and epithelial tissue with PD-L1 expression levels from high to low, respectively; (FIGS. 8B, 8C) fluorescence imaging of PD-L1 positive and negative expressed non-small cell lung cancer (NSCLC) tissue sections, both positively controlled by PD-L1 antibody tissue section immunohistochemical results; using FAM-383-33 and FAM-A60 as negative controls, all types of tissue sections were serial sections of the same tissue, and the areas of higher brightness in FIGS. 8A-8C were areas of higher PD-L1 expression levels. FIGS. 9A and 9B are fluorescence imaging diagrams of FAM-8-60 on malignant melanoma tissue sections expressed positively (FIG. 9A) and negatively (FIG. 9B) for PD-L1, both positively controlled by the result of immunohistochemical treatment of PD-L1 antibody tissue sections; using FAM-383-33 and FAM-A60 as negative controls, all types of tissue sections were serial sections of the same tissue, and the areas of higher brightness in FIGS. 9A and 9B were areas of higher PD-L1 expression levels.

TABLE 1 DNA used in the present invention

TABLE 2 reagents used in the invention

TABLE 3 Experimental parameters for 3 rounds of MCP-SELEX for library preconcentration

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

TABLE 4 Experimental parameters for the screening of Encoded-SELEX rounds

1, enzyme 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 pH 7.4. 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 serial number which is sequenced according to the abundance of each sequence in high-throughput sequencing; "number of bases" is the number of bases of each sequence excluding the primer binding regions at both ends.

TABLE 6 gel blocking assay (EMSA) determination of binding Rate of representative candidate nucleic acid aptamers to PD-L1

1: the sequence number in the aptamer name is the sequence number which is sequenced according to the abundance of each sequence in high-throughput sequencing; "number of bases" is the number of bases of each sequence excluding the primer binding regions at both ends.

2 the gray value (I) of each band was determined by Image Lab TM Software, and the gray value was 1 for the band containing no PD-L1 but only the aptamer. Aptamer binding rate (%) =i (PD-L1-aptamer complex)/[ I (PD-L1-aptamer complex) +i (unbound aptamer) ].

Example 1 Process for screening PD-L1 nucleic acid aptamers using the method of the invention

The chemically synthesized DNA random library (FIG. 1A, table 1) consists of three major parts, a primer binding region at each end, two random sequence regions and a middle fixed sequence region (i.e., an internal coding region). Wherein a recognition site for the restriction enzyme is present within the intermediate fixing sequence. In addition, the intermediate fixed sequences were hybridized and complemented with short-chain cDNAs (Table 1) to form double-stranded structures that can be recognized by restriction enzymes. At the same time, the formation of the double-stranded domain is advantageous in reducing interactions between sequences.

Pre-enrichment was first performed over 3 rounds of MCP-SELEX (patent application number 201810589689.4) to reduce the enrichment efficiency of restriction endonuclease-SELEX (RE-SELEX) in the Encoded-SELEX. The experimental parameters of each round are shown in table 3, and the specific experimental steps comprise:

heat treatment of dna library: the DNA library was diluted in 500 μl of screening buffer (50 mM 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), 100mM NaCl,1mM MgCl2,5mM KCl,1mM CaCl2,pH =7.4), heated in a 95 ℃ 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. the carboxyl coated magnetic beads were spun and mixed for 15 minutes at room temperature.

2.2. mu.L of the beads were pipetted and mixed thoroughly with 100. Mu.L of 25mM 2- (N-morpholino) ethanesulfonic acid (MES, pH=5.0) for 10min, the supernatant was discarded and washed twice with 100. Mu.L of 25mM MES solution.

2.3. 50mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution and 50mg/mL of N-hydroxysuccinimide (NHS) solution were freshly prepared with 25mM MES solution, respectively.

2.4. 100. Mu.L of the EDC solution and 100. Mu.L of the NHS solution were added to the 2.2-washed beads, and the mixture was thoroughly mixed and shaken at a low speed for 30 minutes at room temperature.

2.5. Centrifuge tubes containing EDC/NHS/beads were placed on a strong magnet for 1 min, and after removal of the supernatant, washed 2 times with 100. Mu.L of 25mM MES solution.

2.6. 10 mu L of screening buffer solution was added to the activated beads to uniformly disperse the beads in the solution.

3. Negative selection with activated magnetic beads: the DNA library after heat treatment in 1 was added to the activated magnetic beads in a total volume of 500. Mu.L and incubated for 30 minutes at room temperature with spin mixing. After the incubation, the beads were discarded and the supernatant was left.

4. Incubation of library with target protein PD-L1 after negative screening: PD-L1 was added to the supernatant collected in 3 and incubated for 30 minutes at room temperature with spin mixing. After the incubation, the supernatant was discarded and the beads were left. 200. Mu.L of screening buffer solution was added to the beads, and the mixture was incubated for 5 minutes at room temperature with spin mixing, placed on a strong magnet for 1 minute, and washed repeatedly 4 times. Finally, 120. Mu.L of the screening buffer was added, and the mixture was heated with shaking at 95℃for 20 minutes, magnetically separated for 1 minute, and the supernatant (eluent) was collected.

5. The DNA sequence in the eluate was subjected to Polymerase Chain Reaction (PCR) to amplify the DNA sequence using biotin-labeled reverse primer.

6. Preparing a DNA single strand:

6.1. preparing a column: the streptavidin sepharose beads were placed on a rotator for 15 min at room temperature, 60. Mu.L of streptavidin sepharose beads were added to a 200. Mu.L anti-aerosol gun head, and the column was washed with 200. Mu.L of phosphate buffer solution containing 1M NaCl (1 XPBS/1M NaCl).

6.2. Passing through a column: the PCR mixture was added to the column (100. Mu.L each) in portions by a pipette, the PCR mixture was thoroughly mixed with streptavidin agarose gel beads by applying pressure by the pipette, the mixture in the column was discharged from the tip after 3 minutes by applying pressure by the pipette, and then the column was washed with 200. Mu.L of 1 XPBS/1M NaCl. This operation was repeated until the PCR solution was completely added to the column.

Naoh elution: after washing the column twice with 200. Mu.L of 1 XPBS/1M NaCl, 100. Mu.L of 30mM NaOH was added to the column with a pipette, after 5 minutes the NaOH solution was drained out of the column with a pipette pressure and collected in a clean centrifuge tube, i.e., a single-stranded DNA library.

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

4 rounds of Encoded-SELEX are then performed (FIG. 1B). Except that the first round contains only RE-SELEX, the Encoded-SELEX of the 2-4 rounds each contain two steps, 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 endonuclease-SELEX (RE-SELEX)

Dna heat treatment and complementary hybridization: the DNA library was prepared with complementary short-chain DNA (cDNA) of the intermediate fixed sequence in an enzyme digestion buffer (10 mM Tris-HCl,10mM MgCl2,1mM dithiothreitol, pH 7.5) to give a final concentration of 0.4. Mu.M library DNA solution. The solution was heated in a 95℃water bath for 10 minutes and allowed to slowly cool to room temperature.

1.2. Library incubation with PD-L1: PD-L1 was added to the above system at a final concentration of 0.2. Mu.M and incubated for 1 hour at room temperature. The specific experimental parameters are shown in Table 3.

1.3. Enzyme digestion reaction: an Alu I enzyme solution was prepared in a final concentration of 0.7U/. Mu.L in the cleavage buffer solution. Adding newly prepared 50 mu L Alu I enzyme solution into the 150 mu L DNA solution, subpackaging into two 100 mu L PCR small tubes, and setting a PCR instrument heat treatment program to 37 ℃ for 30 minutes; 15 minutes at 80 ℃; and (3) preserving the obtained enzyme cutting solution in a refrigerator at the temperature of 4 ℃ for 10 minutes for later use.

1.4. Gel electrophoresis experiment: gel electrophoresis experiments were performed on 6. Mu.L of the enzyme-digested solution, and the detailed procedures were shown in the gel electrophoresis experiments.

PCR amplification.

1.6 preparation of DNA Single strands: the detailed procedure is described in MCP-SELEX for single strand DNA production.

mcp-SELEX: see MCP-SELEX for detailed steps. The enriched library goes to the next round of Encoded-SELEX. The first round of Encoded-SELEX did not perform this step.

As shown in FIG. 1B, during the cleavage reaction of RE-SELEX, the target protein PD-L1 and the restriction enzyme Alu I are simultaneously present in the system to compete for binding with the nucleic acid sequence. If the DNA sequence has no/low affinity to PD-L1, the sequence is more susceptible to cleavage; on the contrary, if there is high affinity between the two, the two are not easy to be cut by enzyme. Meanwhile, each nucleic acid sequence can evolve to different degrees, namely, deletion or mutation, at enzyme cutting sites and nearby areas so as to resist degradation by restriction enzymes, and the degree of evolution is closely related to the affinity strength of the sequence and a target. After the enzyme digestion reaction, the aptamer capable of binding the target enters an MCP-SELEX process, is captured by using activated magnetic beads, and is amplified and prepared into ssDNA by PCR, so that the ssDNA is put into the next round of screening; those sequences which escape restriction enzyme degradation by evolution and cannot bind to the target are not captured by the activated magnetic beads and cannot enter the next round of screening. The MCP-SELEX can further screen and quickly enrich nucleic acid aptamer with strong affinity, and can exclude some non-target binding sequences which are not easy to be digested due to evolution or formation of special secondary structures in the digestion process.

The enriched library obtained after completion of the above steps was subjected to commercial high throughput sequencing. And then carrying out affinity and specificity tests on the candidate aptamer, and finally screening out the optimal aptamer for testing the PD-L1 expression level of the human cancer tissue section.

EXAMPLE 2 monitoring of the evolution process of the cleavage resistance and affinity of the Encoded-SELEX round libraries

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

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

1.12% denatured glue: the equipment such as glass plate, beaker, syringe, comb, etc. which would be in direct contact with the glue was rinsed with deionized water and formulated in the order of formulation in brackets (9.6 g urea, 12mL deionized water, 2mL 10 XTBE, 20. Mu.l tetramethyl ethylenediamine, 6mL40% acrylamide, 200. Mu.l 10% ammonium persulfate) and the formulated glue was allowed to stand at room temperature for 4 hours.

Maker solution (12 μl total) formulation: 6. Mu.L of 2 XRNA gel loading buffer, 1.2. Mu.L 20bp DNA Ladder,4.8. Mu.L of nuclease-free water.

3. Sample solution (12 μl total) was prepared: 6. Mu.L of 2 XRNA gel loading buffer, 6. Mu.L of enzyme cutting solution.

4. 500. Mu.L of 1 XTBE solution (i.e., an electrophoresis solution) was placed in the electrophoresis tank, and the gel was immersed in the electrophoresis solution, taken out of the comb, and allowed to stand for 30 minutes.

5. And (3) heat treatment: the Maker and sample solutions were heated at 95 ℃ for 10 minutes and quenched on ice for 5 minutes.

6. Pre-electrophoresis: while heat-treating, the well-standing denatured gel was subjected to pre-electrophoresis at 170V (volts) for 20 minutes.

7. mu.L of Maker and the sample solution were added to the denaturing gel, respectively, and electrophoresis was performed at 170V for 45 minutes.

8. Dyeing: a clean petri dish was taken, 30mL of 1 XTBE solution was placed in it, and 3. Mu.L of 1X was takenGold nucleic acid dye is evenly mixed, and the denatured glue after electrophoresis is put into a culture dish and kept stand for 5 minutes.

9. And (3) photoresist: taking out the denatured gel, and adding MolecularGel was applied in Gel Doc (TM) XR+ (BIO-RAD) and analyzed with Image Lab TM Software.

As shown in FIGS. 2A-2D, the ratio of sequences cleaved by Alu I to those not cleaved from the first round to the fourth round was continuously decreasing, i.e., the DNA sequences in the library not cleaved by Alu I were increased. The first round of Chinese library was almost entirely sheared by Alu I, and REP-1 bands at the original length of the library were almost invisible, indicating that R0 contains a large number of sequences that cannot resist cleavage by restriction enzymes; while in the fourth round the library was almost entirely not sheared by Alu I, the bands of REP-4 at the short chain positions after shearing were almost invisible. The library has been described as being capable of withstanding the Alu I enzymatic cleavage reaction.

We also monitored the evolution of the affinity of the library for the target caused by each round of Encoded-SELEX. The cleavage products (REP-2, REP-3 and REP-4) of each round were amplified by PCR and single-stranded DNA was prepared to obtain libraries RE2, RE3 and RE4, respectively. The amount of DNA in each library that bound to the target captured by the activated magnetic beads was determined using the real-time quantitative PCR technique (qPCR), and the percentage of the library bound in the charged PD-L1, i.e., the binding rate, was calculated. The specific operating steps of the qPCR experiment are as follows:

1. preparing an initial library standard substance and a sample to be tested: the following 7 initial library Pool0 solutions of different concentrations were prepared with nuclease-free water as standard, and the DNA concentrations were 1000pM (picomoles per liter), 100pM, 10pM, 1pM, 0.1pM, 0.01pM, 0, respectively. The sample can be diluted by different times according to actual conditions, so that the final concentration of the sample is within the range of the standard concentration.

Qpcr solution formulation (three groups per solution parallel): the preparation was carried out in a formulation containing 10. Mu.L of 2 XPremix Taq hot-start enzyme, 2. Mu.L of FP (10. Mu.M), 2. Mu.L of RP (10. Mu.M), 3. Mu.L of nuclease-free water, 1. Mu.L of 20 XEva Green nucleic acid dye, 2. Mu.L of sample or standard per 100. Mu.L of qPCR solution.

3. Setting a qPCR instrument program: firstly, preheating at 95 ℃ for 1min; the second step is heating at 95 ℃ for 30s; thirdly, annealing at 51 ℃ for 30s; the fourth step is prolonged at 72 ℃ for 30 seconds, the second to fourth steps are circulated for 34 times, and fluorescent signals are collected in each fourth step.

4. And putting the qPCR solution into a sample cell, and setting the solution name and fluorescence type of the corresponding position.

And 5. The qPCR instrument starts to work, a working curve is drawn according to the acquired data of the standard sample after the qPCR instrument is ended, and the quantity of DNA contained in the unknown sample is obtained according to the working curve and the Ct value of the sample to be detected.

The binding rate of the target to the library (R0) obtained by 3 rounds of pre-enrichment was 3.3% and to RE2 was 1.46%. It was demonstrated that 2 rounds of screening did not increase the affinity of the library for PD-L1 targets. This should be due to two reasons. (1) The large number of DNA sequences in the library with low affinity cannot cause structural changes in the recognition site of the restriction enzyme and thus will be cleaved by the restriction enzyme. The proportion of sequences with affinity in the total sequence is thus significantly reduced, so that the binding rate is reduced. (2) Alu I cleavage reactions not only cleave sequences that have a recognition site for a restriction endonuclease (sequences that do not have or have low affinity for the target), but also cause mutations in certain sequences that do not cleave during the cleavage reaction. While most of these sequences that undergo sequence mutations have no affinity for the target. This was confirmed by the low binding rate of RE2 (1.46%) and high cleavage resistance (almost half cannot be degraded, fig. 2B). However, the MCP-SELEX step in each round of screening can remove the non-specific sequences from the enriched library resulting from (2).

The binding rate of RE3 (0.04%) was lower than that of RE-2, which should be due to the negative screening performed before capturing the library bound to the target with activated magnetic beads in the third round, eliminating a large number of sequences in the library that bind to human serum and Human Serum Albumin (HSA). The resistance to cleavage by RE3 was comparable to RE2 (FIGS. 2B and 2C), demonstrating that the removal of sequence mutated non-specific sequences (about 98.6%) from the enriched library using the MCP-SELEX step was effective. Otherwise the library will be almost not sheared in the third round of cleavage reaction. The binding rate of RE4 was 35.9%, which was approximately 1000-fold higher than RE3, and RE4 was completely resistant to cleavage by Alu I enzyme (FIG. 2D), indicating that the proportion of PD-L1 high affinity sequences in the RE4 library was already high. We performed elution, PCR amplification and single-stranded DNA preparation of RE4 captured by magnetic beads to obtain enriched library R4.

We performed dissociation constant (KD) measurements (fig. 2F) of cy5.5 fluorophore-modified R4 using evanescent wave fiber sensors, non-linear fits were performed with 1:1 binding, KD of 36±21nM.

Example 3 high throughput sequencing of R4 library

We performed commercial high throughput sequencing of the R4 library described above. After the R4 library is amplified by PCR, the purified amplified product is subjected to high throughput sequencing. The PCR product is purified by using a UNIQ-10 column type oligonucleotide purification kit of the manufacturer (Shanghai), and the specific operation steps are as follows:

1. 10 volumes of binding buffer were added to the PCR solution and vortexed.

2. Four adsorption columns with collection tubes were taken, 600. Mu.L of the mixture was added to each column, and after 2 minutes at room temperature, centrifugation was carried out at 8000rpm for 2 minutes. The liquid in the collection tube is poured out, and the steps are repeated again until the mixed liquid is used up.

3. To the column 500. Mu.L of wash buffer (the correct amount of absolute ethanol had been added) was added and centrifuged at 10000rpm for 1 min, and the waste liquid in the collection tube was decanted.

4. The step 3 is repeated once.

5. In order to volatilize residual ethanol, the influence on subsequent experiments is avoided. The column was idle once and centrifuged at 10000rpm for 2 minutes.

6. The column was placed in a clean 1.5mL centrifuge tube (preferably, the lid of the tube was cut off), 50. Mu.L of non-ribozyme water was added to the center of the adsorption membrane (to further increase the yield, pre-heating to 60 ℃ C. Was required), and after 5 minutes of standing at room temperature, it was centrifuged at 12000rpm for 1 minute. The resulting DNA solution was stored at-20 ℃.

10000 sequences obtained after high throughput sequencing, the portions other than the primer binding region have lengths varying from 33 to 60 bases. The results indicated that all intermediate fixed sequences (internal coding) of the library were mutated or deleted: mutations occur essentially in Alu I recognition sequence AGCT; the base deleted portion substantially encompasses the recognition sequence, and some of the entire internal coding region is not already present. This severe base deletion is not reported in the current SELEX technique. We speculate that this may be related to two factors, one is that DNA libraries may produce cut-off variations at recognition sites in order to avoid Alu I cleavage; secondly, the combination of PD-L1 and the sequence can affect the structure of the recognition site of the restriction enzyme to change in different degrees, thereby inhibiting the enzyme digestion and further inhibiting the variation of the sequence to different degrees.

Example 4.40 affinity characterization of representative candidate nucleic acid aptamers

To investigate whether the number of bases of each candidate sequence correlated with its affinity, we randomly selected 40 DNA sequences with different numbers of bases from the top 1000 sequences of the high throughput library number for affinity testing (table 5). Each sequence is named as a sequence number-base number, wherein the sequence number is a sequence number which is sequenced according to the abundance of each sequence in high-throughput sequencing; "number of bases" is the number of bases of each sequence excluding the primer binding regions at both ends.

We characterized the affinity of each candidate aptamer using the colloidal gold-based Nano-Affi method (analysis, 2020,145,4276-4282). The specific operation steps are as follows:

1. preparing nano gold: according to the method reported in the literature, gold nanoparticles (GNP, 8.3nM,pH 6.5) with a diameter of 13nm were synthesized. The pH of the nanogold was then adjusted to 5.0 using HCl.

2. Preparing a sample: 4pmol PD-L1 and 2pmol DNA (candidate aptamer or negative control strand) were added to and mixed in 4. Mu.L of screening buffer. The final concentrations of PD-L1 and DNA were 1. Mu.M and 0.5. Mu.M, respectively.

3. Incubation: the samples were incubated at room temperature for 10 minutes.

4. After incubation was completed, 96 μl GNP was added to the mixture and mixed slowly 4 times with a pipette. The final concentrations of PD-L1 and DNA were 40nM and 20nM, respectively.

5. GNP particle size determination was performed using Dynamic Light Scattering (DLS) for 10min and samples without aptamer, i.e. only PD-L1, were used as negative controls.

As shown in FIGS. 3A and 3B, according to the detection principle of the Nano-Affi method, the higher the Δd value of the change of the GNP particle size, 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: the nucleic acid aptamer of thrombin (N-29), phthalate derivative (N-39) and ubiquitin (N-60) has the base numbers of 29, 39 and 60 respectively. The gold nanoparticles Δd values caused by the three negative control sequences are smaller than the particle size increase caused by PD-L1. Except for the 383-33 and 258-36 sequences, the Δd values caused by the remaining 38 candidate nucleic acid aptamers were all smaller than those caused by the three negative control sequences. Indicating that the affinity of all 38 candidate nucleic acid aptamers was higher than that of the negative control sequences. Interestingly, we found that the longer the 40 candidate nucleic acid aptamers were, the higher the affinity for PD-L1. This rule applies to sequences of the first 20 in high throughput sequencing (FIG. 3A) and to sequences of the later sequences (FIG. 3B).

To further verify the accuracy of this rule, we performed affinity tests on 5 representative candidate nucleic acid aptamers with different base numbers using gel blocking Experiments (EMSA).

The specific operation steps are as follows:

DNA heat treatment: 50pmol of candidate aptamer was added to the screening buffer, heated at 95℃for 10 minutes, and cooled slowly to room temperature.

2.20. Mu.L of 0.1% Tween-80 was thoroughly mixed with the above DNA solution, centrifuged to remove foam, and then mixed with 100pmol of PD-L1 to a final volume of 200. Mu.L, and incubated at room temperature for 1 hour with rotation.

3. Gel electrophoresis experiment: after the incubation, 1.2. Mu.L of the sample was taken for gel electrophoresis experiments, and the detailed procedure is given in example 2. Quantitative analysis of band brightness was performed with Image Lab TM Software (table 6).

The results are shown in FIGS. 5A-C and Table 6, which also show that the longer the sequence length, the higher its affinity for PD-L1. This result suggests that our discovered laws are objectively present, repeatable and accurate.

Example 5 specificity, dissociation constant and secondary Structure of PD-L1 aptamer 8-60

The preferred PD-L1 aptamer 8-60 is characterized for its specificity, dissociation constant and secondary structure (FIGS. 5A-C). We first used gel blocking (EMSA) experiments to determine the specificity of 8-60, and the specific procedure was as follows:

DNA heat treatment: 50pmol 8-60 was added to the cleavage buffer, heated at 95℃for 10min, and cooled slowly to room temperature.

2.20. Mu.L of 0.1% Tween-80 was thoroughly mixed with the above DNA solution, centrifuged to remove foam, and then slowly mixed with 100pmol of PD-L1, SA, polypeptide, beta-casein and ovalbumin, respectively, to a final volume of 200. Mu.L, and incubated with a protein-free sample, i.e., only 8-60 samples, as a negative control at room temperature for 1 hour with rotation.

3. Gel electrophoresis experiment: after the incubation, 1.2. Mu.L of the sample was taken for gel electrophoresis experiments, and the detailed procedure is given in example 2.

As a result, as shown in FIG. 5A, only the samples incubated with PD-L1 and 8-60 showed distinct complex bands in the gel blocking experiment, and no complex bands were observed on the gel after incubation of 8-60 with other proteins. This result shows that 8-60 has high specificity for PD-L1.

We next determined dissociation constants of 8-60 using the Nano-Affi method, with a dissociation constant of 6.2+ -2.7 nM (FIG. 5B). The method comprises the following specific steps:

1. preparing nano gold: according to the method reported in the literature, gold nanomaterials (GNP, 8.3nm, pH 6.5) were synthesized and the pH of the gold nanomaterials was adjusted to 5.0 with HCl.

2. Preparing a sample: PD-L1 was mixed with 8-60 at different concentrations in screening buffer solution to a final volume of 4. Mu.L. The final concentrations of 8-60 were 0, 125nM, 250nM, 375nM, 500nM, 1250nM, 1875nM, 2500nM, respectively, where the solutions at each concentration contained 4pmol PD-L1.

3. Incubation: the samples were incubated at room temperature for 10 minutes.

4. After incubation was completed, 96 μl GNP was added to the mixture and slowly mixed 4 times with a pipette and incubated 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 was 40nM.

5. GNP particle size measurements were performed using Dynamic Light Scattering (DLS).

The 8-60 secondary structure was fitted using online secondary structure simulation software from Integrated DNA Technologies (IDT) (fig. 5C). 8-60 intermediate fixed sequences (internal coding) have no deletion, but Alu I recognition sequence AG≡CT has single base mutation, namely G- & gt A, and the mutated sequences and random sequences form double-chain complementary structures.

Example 6 recognition ability of PD-L1 nucleic acid aptamer 8-60 on cell surface expressed PD-L1

We measured the ability of 8-60 to recognize cell surface expressed PD-L1 using a flow cytometer. In this experiment, cells with different degrees of PD-L1 expression were identified using the protocol commonly used for performance testing of PD-L1 antibodies. Four relevant cells were purchased by referring to Abcam company PD-L1 antibody instructions, namely COLO205 cells (human colon cancer cells) which do not express PD-L1, HCC70 cells (human breast ductal carcinoma cells) which do not express PD-L1, ES-2 cells (human ovarian clear cell carcinoma cells) which express PD-L1, and BCPAP cells (human thyroid cancer papillary cells) which do not express PD-L1. Cy5-A60 (Cy 5 fluorophore modified A60 strand, table 1) which had no affinity for PD-L1 in all tests served as a negative control.

The specific operation steps are as follows:

DNA heat treatment: 2.4pmol of Cy5-8-60 (Cy5 fluorophore modified 8-60, table 1) or 2.4pmol of Cy5-A60 were each prepared in screening buffer solutions to a final concentration of 200 nM. Heating at 95deg.C for 5 min, quenching on ice for 10 min, and standing at room temperature for 30 min (in the absence of light as much as possible).

2. Cell treatment

HCC70 cells, ES-2 cells, BCPAP cells were digested with 1mL of 5mM EDTA in an incubator for 5-10 min, then transferred to 1.5mL clean centrifuge tubes, respectively, while COLO205 cells were pipetted directly into 1.5mL clean centrifuge tubes.

2.2. The four cells were each centrifuged at 1000rpm for 3 minutes, the supernatant was discarded, and the bottom cells were retained.

2.3. Preparing a sealing liquid. The preparation method is characterized by comprising the following steps: 800. Mu.L of the blocking solution contained 16. Mu.L of salmon sperm (10 mg/mL); 80 μL of 10% BSA; 704. Mu.L of screening buffer.

2.4. 200. Mu.L of blocking solution was added to each cell-loaded centrifuge tube and vortexed.

3. The two DNA solutions after heat treatment were equally divided into 4 tubes of solutions, 8 tubes in total.

4. Incubation: to each DNA solution, 100. Mu.L of a cell solution containing a blocking solution was added to each tube, and the mixture was vortexed and homogenized. All the mixtures were incubated in dark for 30 minutes and vortexed once every 10 minutes.

5. After incubation was completed, centrifugation was carried out for 3 minutes at 1000rpm, the supernatant was discarded again, and the bottom cells were retained.

6. Washing the cells: mu.L of the screening buffer was added to the cells, vortexed, and centrifuged for 3 minutes at 1000rpm.

7. After repeated washing, the volume was fixed with 300. Mu.L of the screening buffer and the cells were filtered with a 400 mesh screen.

8. The measurement was performed using a flow cytometer (FACScalibur, becton Dickinson, USA). 10000 cells were assayed each, using the Cy5 channel (FL 4-H) and data analysis was performed with FlowJo software (Treestar, san Caros, USA).

As shown in FIGS. 6A-6D, the higher the PD-L1 expression level, the more the fluorescence intensity increases after incubation with Cy5-8-60, indicating that 8-60 is able to recognize PD-L1 expressed on the cell surface. The fluorescence intensity of Cy5-A60, which was negative control, was not significantly increased after incubation with the four cells, indicating that 8-60 was specifically recognized by PD-L1 on the cell surface.

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

cy5-8-60 heat treatment: 3 μL Cy5-8-60 solutions were prepared in the screening buffer at final concentrations of 5nM, 10nM, 20nM, 50nM, 75nM, 100nM and 200nM, respectively. And heated at 95℃for 5 minutes, quenched on ice for 10 minutes, and allowed to stand at room temperature for 30 minutes (protected from light as much as possible).

2. Cell treatment

BCPAP cells were transferred to an incubator after digestion with 1mL of 5mM EDTA for 5-10 min

1.5mL of clean centrifuge tube.

2.2. Centrifugation was performed for 3 minutes at 1000rpm, the supernatant was discarded, and the bottom cells were retained.

2.3. Preparing a sealing liquid. The preparation method comprises the following steps: 700. Mu.L of the blocking solution contained 14. Mu.L salmon sperm (10 mg/mL); 70 μL 10% BSA;616 uL of screening buffer.

2.4. Adding the sealing liquid into a centrifuge tube filled with BCPAP cells, and mixing by vortex.

3. Incubation: the above-mentioned BCPAP cell solution containing the blocking solution was added to the heat-treated 7 kinds of Cy5-8-60 solutions of different concentrations, 100. Mu.L per tube, and vortexed and mixed well. All the mixtures were incubated in dark for 30 minutes and vortexed once every 10 minutes.

4. After incubation was completed, centrifugation was carried out for 3 minutes at 1000rpm, the supernatant was discarded again, and the bottom cells were retained.

5. Washing the cells: mu.L of the screening buffer was added to the cells, vortexed, and centrifuged for 3 minutes at 1000rpm.

6. After repeated washing, the volume was fixed with 300. Mu.L of the screening buffer and the cells were filtered with a 400 mesh screen.

7. The measurement was performed using a flow cytometer (FACScalibur, becton Dickinson, USA). 10000 cells were assayed each, using the Cy5 channel (FL 4-H) and data analysis was performed with FlowJo software (Treestar, san Caros, USA).

As a result, as shown in FIG. 7A, the fluorescence intensity of BCPAP cells increased with increasing concentration of Cy 5-8-60. The fluorescence intensity of BCPAP cells incubated with 200nM Cy5-A60 was similar to that of Cy5-8-60 at 5nM. The binding curve for the dissociation constant measurement was plotted according to the fluorescence intensity of FIG. 7A (FIG. 7B), and the dissociation constant obtained by nonlinear fitting was 82.5.+ -. 25.5nM according to the 1:1 binding mode. 8-60 is shown to have high affinity for PD-L1 expressed on the surface of cancer cells.

Example 7 ability of PD-L1 nucleic acid aptamer 8-60 to recognize the expression level of PD-L1 on a tissue section

The FAM-8-60 (FAM fluorescent group modified 8-60) is used for carrying out fluorescence imaging on three groups of different tissue slices, namely normal human tonsil slices, non-small cell lung cancer tissue slices and malignant melanoma tissue slices. All tissue sections of the same type are consecutive sections of the same tissue. Wherein, normal human tonsil tissue section is the gold standard for detecting PD-L1 antibody performance, and non-small cell lung cancer and malignant melanoma are tumor tissues with higher PD-L1 expression. We first performed PD-L1 antibody immunohistochemical experiments on the same batch of tissue sections, with experimental results as positive control. At the same time, we used FAM-383-33 and FAM-A60 (FAM fluorophore modified) as negative controls.

The specific operation steps are as follows:

1. aptamer heat treatment: 100pmol of aptamer was prepared as 150. Mu.L solution in PD-L1 screening buffer, heated at 95℃for 5 min, quenched on ice for 10 min, and allowed to stand at room temperature in the dark for 10 min.

2. Tissue slice processing

2.1. Tissue sections were soaked in p-xylene for 30 min, 15 min, the tissue sections were removed, the above solutions were then sequentially soaked in 95%, 90%, 85%, 80%, 75% ethanol twice for 3 min each.

2.2. A0.01M sodium citrate solution (pH 6.0) was placed in a clean beaker and the tissue sections were immersed in the solution and heated in a microwave oven. When the solution started to boil, timing was immediately performed when bubbles appeared, and the operation was repeated for 15 minutes every 1 minute of heating, and was suspended for 30 seconds. The beaker was then removed from the microwave oven and slowly cooled to room temperature.

2.3. Tissue sections were removed from the beaker and washed three times with 1 XPBS/0.1M NaCl.

2.4. Preparing a sealing liquid: 10. Mu.L salmon sperm (10 mg/mL) was contained per 100. Mu.L of the blocking solution; 10. Mu.L yeast tRNA (10 mg/mL); 50 μL of 10 XPBS/0.1M NaCl;20 μL of 5% Bovine Serum Albumin (BSA); formulation of 10. Mu.L Tween-20 (0.1%) was prepared.

2.5. Closure of tissue sections: the block was applied dropwise to tissue sections (note tissue location), 100. Mu.L/plate, blocked for 1 hour at 37℃and then washed three more times with 1 XPBS/0.1M NaCl.

3. The heat treated aptamer solution was added dropwise to the tissue sections, incubated at 37℃for 20 minutes, and washed three times with 1 XPBS/0.1M NaCl.

4. The tissue sections were imaged by fluorescence microscopy.

As shown in FIG. 8A, the FAM-8-60 was able to very accurately distinguish between normal human tonsil tissue slice crypt (high fluorescence intensity, high PD-L1 expression) and center of development (low fluorescence intensity, low PD-L1 expression) in complete agreement with the corresponding immunohistochemical results of PD-L1 antibody (FIG. 8A, left) (FIG. 8A, right). The fluorescence intensity of the tissue sections incubated with the negative control sequences FAM-383-33 and FAM-A60 was very low. As can be seen from the non-small cell lung cancer tissue section results (FIGS. 8B and 8C), 8-60 is very effective in distinguishing between PD-L1 positive and negative expressed NSCLC sections, and the fluorescence intensity distribution is substantially consistent with PD-L1 antibody immunohistochemical results. The fluorescence intensity of tissue sections also incubated with the negative control sequences FAM-383-33 and FAM-A60 was very low. Similarly, as shown in FIGS. 9A and 9B, 8-60 was able to very effectively distinguish malignant melanoma tissue sections with positive and negative expression of PD-L1, and the fluorescence intensity distribution was substantially consistent with the PD-L1 antibody immunohistochemical results. The fluorescence intensity of tissue sections also incubated with the negative control sequences FAM-383-33 and FAM-A60 was very low.

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

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention.

Sequence list

<110> university of capital and education

<120> a method for screening DNA aptamer of PD-L1 in vitro and application thereof in cancer diagnosis

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<170> PatentIn version 3.3

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Claims (3)

1. A method for in vitro screening of DNA nucleic acid aptamers to PD-L1, wherein the DNA sequences in the starting random DNA library of the method comprise primer binding regions at both ends, two random sequence regions and an immobilization region comprising a recognition site for a restriction enzyme located between the two random sequence regions, said immobilization region being capable of complementary hybridization with a cDNA short sequence to form double stranded DNA cleavable by a type II restriction enzyme; the method for screening the DNA aptamer of the PD-L1 in vitro comprises the following steps: firstly preparing a pre-enrichment library through 3 rounds of magnetic cross-linked precipitation-SELEX, and then carrying out 4 rounds of intra-coding-SELEX to obtain an enrichment library;

The preparation of the pre-enriched library by 3 rounds of magnetic cross-linked precipitation-SELEX comprises the following steps:

step 1. Heat treatment of DNA library: the random DNA library was diluted in 500. Mu.L of screening buffer containing 50mM 4-hydroxyethylpiperazine ethanesulfonic acid, 100mM NaCl, 1mM MgCl, heated in a 95℃water bath for 10 minutes, quenched on ice for 10 minutes, and left at room temperature for 10 minutes ₂ 、5mM KCl、1mM CaCl ₂ The pH value of the screening buffer solution is 7.4;

step 2, activating carboxyl coated magnetic beads:

step 2.1, rotating and uniformly mixing the carboxyl coated magnetic beads for 15 minutes at room temperature;

step 2.2. 10. Mu.L of beads were pipetted and mixed thoroughly with 100. Mu.L of 25mM MES solution at pH 5.0 for 10 min, the supernatant was discarded and washed twice with 100. Mu.L of 25mM MES solution;

step 2.3. Fresh preparation of 50mg/mL EDC solution and 50mg/mL NHS solution with 25mM MES solution, respectively;

step 2.4, adding 100 mu L of EDC solution and 100 mu L of NHS solution into the magnetic beads washed in the step 2.2, fully and uniformly mixing, and shaking at a low speed for 30 minutes at room temperature;

step 2.5. Placing a centrifuge tube containing EDC, NHS and magnetic beads on a powerful magnet for 1 minute, removing supernatant, and washing with 100 mu L of 25mM MES solution for 2 times to obtain activated magnetic beads;

Step 2.6, adding 10 mu L of screening buffer solution into the activated magnetic beads to uniformly disperse the magnetic beads in the solution;

step 3, negative screening with activated magnetic beads: adding the DNA library subjected to the heat treatment in the step 1 into activated magnetic beads, wherein the total volume is 500 mu L, and performing rotary mixing and incubation for 30 minutes at room temperature; discarding the magnetic beads after incubation is finished, and leaving supernatant;

step 4, incubation of the DNA library after negative screening with target protein PD-L1: adding PD-L1 into the supernatant collected in the step 3, and incubating for 30 minutes at room temperature by rotating and mixing; adding activated magnetic beads after incubation, discarding supernatant, and retaining the magnetic beads; adding 200 mu L of screening buffer solution into the magnetic beads, rotating and uniformly mixing at room temperature, incubating for 5 minutes, placing the mixture on a powerful magnet for 1 minute, and repeatedly cleaning for 4 times; finally, 120 mu L of screening buffer solution is added, vibration heating is carried out for 20 minutes at 95 ℃, magnetic separation is carried out for 1 minute, and supernatant fluid is collected;

step 5, performing Polymerase Chain Reaction (PCR) on the DNA sequence in the supernatant collected in the step 4 to amplify the DNA sequence, and marking a reverse primer by using biotin;

step 6, preparing a DNA single strand;

step 6.1. Preparing the column: the streptavidin agarose gel beads were placed on a rotator for 15 minutes at room temperature, 60. Mu.L of streptavidin agarose gel beads were added to 200. Mu.L of anti-aerosol gun head, and the column was washed with 200. Mu.L of 1M NaCl-containing phosphate buffer solution, which was 1 XPBS solution;

Step 6.2. Column passing: adding the PCR amplified DNA sequence into the column in batches by using a pipette gun, applying pressure to each time by 100 mu L, fully mixing the PCR amplified DNA sequence with streptavidin agarose gel beads by using the pipette gun, applying pressure to discharge the mixed solution in the column from the gun head after 3 minutes by using the pipette gun, and then washing the column by using 200 mu L of the phosphate buffer solution; repeating the operation until all the DNA sequences amplified by PCR are added into the column;

step 6.3.Naoh elution: washing the column twice with 200 mu L of the phosphate buffer solution, adding 100 mu L of 30mM NaOH solution into the column by using a pipette, pressing the NaOH solution out of the column by using the pipette after 5 minutes, and collecting the NaOH solution into a clean centrifuge tube to obtain a single-stranded DNA library;

step 6.4. Library quantification: neutralizing the single-stranded DNA library with equal amount of HCl, and quantifying the concentration by measuring the ultraviolet visible absorption intensity at 260 nm;

the 4-round in-coding-SELEX comprises the following steps:

step 7, restriction enzyme-SELEX;

step 7.1.DNA heat treatment and complementary hybridization: preparing the DNA library of the step 6.3 and the cDNA short sequence into a library DNA solution with the final concentration of 0.4 mu M in an enzyme digestion buffer containing 10mM Tris-HCl, 10mM MgCl2 and 1mM dithiothreitol, wherein the pH value of the enzyme digestion buffer is 7.5; heating the solution in a water bath at 95 ℃ for 10 minutes, and slowly cooling to room temperature;

Step 7.2. Incubation of library with PD-L1: adding 0.2 mu M PD-L1 to the heat treated library DNA solution and incubating for 1 hour at room temperature;

step 7.3, enzyme digestion reaction: preparing Alu I enzyme solution with the final concentration of 0.7U/. Mu.L in enzyme digestion buffer solution; adding newly prepared 50 mu L of AluI enzyme solution into 150 mu L of library DNA solution incubated at room temperature in the step 7.2, subpackaging into two 100 mu LPCR small tubes, and setting a PCR instrument heat treatment program to 37 ℃ for 30 minutes; 15 minutes at 80 ℃; the enzyme cutting liquid obtained finally is stored in a refrigerator at the temperature of 4 ℃ for standby after 10 minutes at the temperature of 4 ℃;

step 7.4. Gel electrophoresis experiments: taking 6 mu L of enzyme cutting liquid for gel electrophoresis experiments;

step 7.5.PCR amplification;

step 7.6, preparing a DNA single strand, which is the same as the method of step 6;

step 8, repeating the magnetic crosslinked precipitation-SELEX; enriching the library to enter the next round of in-coding-SELEX;

round 1 of the 4-round inner coding-SELEX contains only restriction enzyme-SELEX, and rounds 2-4 of inner coding-SELEX each contain restriction enzyme-SELEX and magnetic cross-linked precipitation-SELEX.

2. The method of claim 1, further comprising high throughput sequencing of the enriched library.

3. The method of claim 2, further comprising selecting a candidate nucleic acid aptamer sequence for an affinity and specificity test.