Method for screening targeted thrombin non-natural nucleic acid aptamer based on streptavidin magnetic beads and application

Abstract

The invention discloses a method for screening a non-natural nucleic acid aptamer of targeted thrombin based on streptavidin magnetic beads and application, and belongs to the technical field of biology. According to the method, an initial screening library is efficiently constructed through a primer extension technology, and a chemical modification SELEX technical process is simplified; a streptavidin magnetic bead separation technology is introduced, so that the screening specificity and efficiency are improved, the screening period is remarkably shortened, and the cost is reduced; by selecting non-natural nucleic acid modification sites and adopting a strategy of combining reverse screening and forward screening, high specificity and affinity of the aptamer are ensured, and limitation of an existing screening method is overcome; the screened non-natural nucleic acid aptamer shows high affinity and excellent stability, the aptamer with a specific structure and function is successfully obtained by optimizing the proportion of nucleoside triphosphate monomers and extension reaction conditions, and a wide application prospect is provided for the fields of drug development, biosensor preparation and the like. A new path is opened up for research and application of the nucleic acid aptamer.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method and application of screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads. Background Art

[0002] Aptamers are a class of single-stranded DNA or RNA molecules with specific molecular recognition capabilities obtained by systematic evolution of ligands by exponential enrichment (SELEX). Compared with traditional antibodies, aptamers have the advantages of easy synthesis, easy modification, high stability, and non-immunogenicity. Therefore, they have broad application prospects in biomedical testing, disease diagnosis, drug delivery and other fields. However, natural aptamers have some limitations in practical applications. First, natural DNA or RNA is easily degraded by nucleases in organisms, resulting in a short half-life in the body, which limits their application in vivo; secondly, the stability and affinity of natural aptamers may not be sufficient to meet the needs of certain high-sensitivity detection; in order to solve these problems, researchers usually chemically modify aptamers to improve their stability and affinity. However, chemical modification may change the structure of aptamers, thereby affecting their ability to bind to target molecules.

[0003] In recent years, the introduction of non-natural nucleic acids (such as chemically modified nucleotides) has provided new ideas for aptamer screening. Non-natural nucleic acids have better biological stability and chemical diversity, and can significantly improve the stability and affinity of aptamers. Therefore, direct screening of aptamers containing non-natural nucleic acids has become an effective method, especially in application scenarios that require high stability and high affinity. Thrombin is a serine protease that plays a key role in blood coagulation. In addition, thrombin is also an important extracellular signaling molecule that participates in a variety of pathological processes, such as the occurrence and development of acute leukemia, by activating thrombin receptors. Acute leukemia is a common malignant tumor of the blood system. The disease progresses rapidly and may be life-threatening in the short term if not treated in time. At present, bone marrow cell morphology examination is mainly used in clinical practice to evaluate the progression and treatment effect of acute leukemia, but this method is an invasive examination and may cause secondary infection in patients. Therefore, the development of a highly sensitive and non-invasive thrombin detection method is of great significance for the early diagnosis and treatment of acute leukemia. Traditional thrombin detection methods usually rely on antibodies, but antibodies have high preparation costs, poor stability, and may have immunogenicity issues. In contrast, nucleic acid aptamers have the advantages of easy synthesis, easy modification, and high stability, and can be used as an ideal substitute for thrombin detection. However, the stability and short action time of natural nucleic acids in the body limit their clinical application. Non-natural nucleic acids are obtained by modifying the bases or backbones on nucleic acid molecules. They are synthetic substitutes for natural DNA and RNA. Compared with natural DNA or RNA aptamers, non-natural nucleic acid aptamers have stronger stability and resistance to nuclease degradation. Therefore, they have higher stability and wider applicability in actual application scenarios. Summary of the invention

[0004] In view of the problems that the existing non-natural nucleic acid aptamer screening methods are not easily recognized by naturally occurring polymerases during the preparation of libraries containing non-natural nucleic acids, and the time-consuming and costly directed evolution of non-natural nucleases is required, the present invention aims to provide a method and application of screening non-natural nucleic acid aptamers based on streptavidin magnetic beads.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads, comprising: S1, synthesis of initial random library of DNA molecules and primers; S2, based on the initial DNA molecule random library, using polymerase to perform primer extension reaction with four nucleoside triphosphate monomers of dT, dC, dG, and A* as substrates to obtain a single-stranded non-natural nucleic acid library; S3, based on the single-stranded non-natural nucleic acid library, incubating with carboxyl magnetic beads that are not bound to thrombin, performing reverse screening, separating the non-natural nucleic acid library that is not bound to the magnetic beads, and obtaining the non-natural nucleic acid library after reverse screening; S4, based on the non-natural nucleic acid library after reverse screening, incubate with streptavidin magnetic beads coupled with thrombin, separate the nucleic acid solution bound to the magnetic beads, reverse transcribe, PCR amplify, separate single-stranded DNA to obtain the first round of screening enrichment library; S5, based on the first round of enriched library, repeat S1 to S4 for multiple rounds of screening, sequence the enriched library in the last round of screening, obtain the sequence information of non-natural nucleic acid molecules, and prepare single-stranded non-natural nucleic acid molecules, which are potential non-natural nucleic acid aptamers.

[0006] The synthesized initial DNA molecule random library and primers, and the DNA template sequence for synthesizing the initial DNA molecule random library are shown in SEQ ID NO.1.

[0007] Furthermore, the nucleic acid sequence of the initial DNA molecule random library is: 5'-biotin--CTATAGCAATGGTACGGTACTTCC- N40 -CAAAAGTGCACGCTACTTTGCTAA-3' (as shown in SEQ ID NO.1), wherein N40 It stands for random nucleic acid sequence and is used to generate a diverse DNA molecule library. Both ends of the sequence are fixed primer binding regions for subsequent PCR amplification, and the 5' end is labeled with biotin.

[0008] The primers include an upstream primer and a downstream primer, and the sequences are shown in SEQ ID NO.2 to SEQ ID NO.3.

[0009] Furthermore, the upstream primer nucleic acid sequence: 5'-biotin-CTATAGCAATGGTACGGTACTTCC-3' (as shown in SEQ ID NO.2) is complementary to the 5'-end fixed region of the DNA template sequence and is used to guide PCR amplification; the downstream primer nucleic acid sequence: 5'-FAM-TTAGCAAAGTAGCGTGCACTTTTG-3' (as shown in SEQ ID NO.3) is complementary to the 3'-end fixed region of the DNA template sequence and is used for the other chain of PCR amplification, and the 5' end is labeled with FAM (fluorescein).

[0010] The primer extension reaction conditions are as follows: the reaction buffer is a Therminator enzyme reaction buffer, the reaction temperature is 36-38 °C, and the reaction time is 1-2 h.

[0011] Furthermore, the primer extension reaction conditions are: reaction temperature is 37 ° C, and reaction time is 2 h.

[0012] The ratio of the four nucleoside triphosphate monomers of dT, dC, dG and A* is 1:1:1:30-20.

[0013] Furthermore, the ratio of the four nucleoside triphosphate monomers of dT, dC, dG, and A* is 1:1:1:10.

[0014] In S3, the incubation conditions are: incubation at room temperature for 1 to 2 h.

[0015] In S4, the streptavidin magnetic beads coupled with thrombin are obtained by the following steps: using 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N -Hydroxysuccinimide activated magnetic beads; incubated the activated magnetic beads with thrombin solution to couple thrombin; and used ethanolamine to block unreacted active sites.

[0016] Furthermore, the incubation conditions of the activated magnetic beads and the thrombin solution are: incubation at room temperature for 1 to 2 hours.

[0017] In S4, the PCR amplification conditions are: pre-denaturation at 95 °C for 5 minutes; denaturation at 95 °C for 10 seconds, annealing at 55 °C for 10 seconds, extension at 72 °C for 30 seconds, extension at 70 °C for 5 minutes, and 25 to 35 cycles.

[0018] In S5, the multiple rounds of screening are 5 to 10 rounds of screening.

[0019] The above-mentioned method for screening non-natural nucleic acid aptamers based on streptavidin magnetic beads also includes testing the non-natural nucleic acid molecules and target The affinity step of the protein is used to predict the secondary structure of non-natural nucleic acid aptamers and verify their affinity step through experiments.

[0020] The non-natural nucleic acid aptamer targeting thrombin is obtained by the above method.

[0021] Furthermore, the sequence of the non-natural nucleic acid aptamer targeting thrombin is shown in SEQ ID NO.4.

[0022] The above method is used in screening non-natural nucleic acid aptamers for targeting proteins, wherein the targeting protein is any one of SARS-CoV-2 nucleocapsid protein, tumor necrosis factor-related OX40 protein, and LAG-3 immune checkpoint protein CD223.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads. The method uses polymerase to use four nucleoside triphosphate monomers, dT, dC, dG, and A* (where A* represents a non-natural nucleoside triphosphate monomer), as substrates, to generate a large number of single-stranded non-natural nucleic acid molecules on the basis of an initial DNA molecule random library, and introduces the non-natural nucleoside triphosphate monomer A*. The primer extension reaction can introduce non-natural elements into the generated nucleic acid molecules. The non-natural elements give the nucleic acid molecules new chemical and biological properties, thereby increasing their potential and specificity as aptamers, and providing abundant candidate molecules for subsequent screening. The high specificity of thrombin is achieved by combining streptavidin magnetic beads with biotinylated thrombin. Specific identification improves the specificity and efficiency of screening, shortens the screening cycle, reduces the number of operating steps, and reduces the screening cost; through multiple rounds of forward and reverse screening, the method continuously enriches nucleic acid aptamers with high affinity to thrombin, which can effectively bind to thrombin at lower concentrations, thereby improving the sensitivity of detection; the screened non-natural nucleic acid aptamers have good affinity and stability, overcoming the defects of natural nucleic acid aptamers that are easily degraded by nucleases in organisms and have poor biological stability, which is conducive to improving the application effect of aptamers in the biomedical field; it can be used for high-sensitivity detection of thrombin, which is of great significance for medical clinical diagnosis, and is conducive to improving the accuracy of early diagnosis and prognosis of diseases such as malignant tumors.

[0024] Furthermore, by introducing random sequence regions, a large number of DNA molecules with different sequences are generated. This diversity provides abundant candidate molecules for subsequent nucleic acid aptamer screening, increasing the possibility of screening high-specificity and high-affinity aptamers. By adjusting the length of the random sequence region and the primer sequence in the DNA template sequence, a random library of DNA molecules with specific properties can be generated; by introducing non-natural nucleic acid residues, the design space of aptamers is broadened, so that the screened aptamers have better biological stability and affinity with the target molecule while maintaining high specificity; by combining reverse screening and forward screening with streptavidin magnetic beads, non-natural nucleic acid aptamers with high affinity for the target thrombin protein can be efficiently screened out from the library containing non-natural nucleic acid residues; multiple rounds of screening further improve the accuracy and efficiency of screening, ensuring that the aptamers finally obtained have high specificity and affinity.

[0025] The present invention obtains a non-natural nucleic acid aptamer targeting thrombin by screening with streptavidin magnetic beads. The screened non-natural nucleic acid aptamer has a high affinity binding ability with thrombin and can achieve effective binding at a lower concentration, providing a more sensitive and efficient tool for the detection and application of thrombin; it can effectively distinguish thrombin from other similar or related proteins, reduce non-specific binding, and improve the accuracy and reliability of detection; it has better stability, can resist the influence of adverse factors such as enzymatic hydrolysis and thermal denaturation, and extend its service life and application range. Compared with traditional nucleic acid aptamers, non-natural nucleic acid aptamers improve their ability to resist nuclease degradation by introducing non-natural nucleosides or chemical modifications; it has broader application prospects, such as playing a more important role in the fields of biosensing, drug delivery, disease diagnosis, etc.

[0026] The method provided by the present invention is used in screening non-natural nucleic acid aptamers targeting proteins. The method can significantly improve the stability, affinity and other functional properties of nucleic acid aptamers by introducing non-natural nucleosides. The method is based on in vitro screening technology and has the advantages of high efficiency, rapidity and repeatability. It is not only applicable to small molecule targets such as thrombin, but also to other types of targeting proteins, including macromolecular proteins, enzymes, receptors, etc. The screened non-natural nucleic acid aptamers have broad application prospects in the fields of biosensing, disease diagnosis, drug development, etc., and are of great significance for the development of nucleic acid aptamer drugs with practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the structural formula of the non-natural nucleoside triphosphate used in the primer extension reaction of the present invention; Figure 2 is the high-throughput sequencing result of the candidate aptamers of the present invention; Figure 3 Prediction of the secondary structure of the candidate aptamers of the present invention; Figure 4 This is an affinity test for the aptamer of the present invention. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings: Example 1 This embodiment provides a method for screening non-natural nucleic acid aptamers based on streptavidin magnetic beads, which specifically includes: S1, synthesis of initial random library of DNA molecules and primers; (1) DNA template sequence design DNA molecule random library nucleic acid sequence: 5'-biotin--CTATAGCAATGGTACGGTACTTCC- N40 -CAAAAGTGCACGCTACTTTGCTAA-3' (as shown in SEQ ID NO.1), wherein N40 Represents a random nucleic acid sequence, which is used to generate a diverse DNA molecule library. Both ends of the sequence are fixed primer binding regions for subsequent PCR amplification. The 5' end is labeled with biotin to facilitate subsequent magnetic bead separation or purification steps. The ratio of the four nucleoside triphosphate monomers used is: T:C:G:A*=1:1:10; it is used to introduce more A* monomers into the random sequence to increase the diversity and specific properties of the library; the structure of the A* monomer is as follows Figure 1 shown.

[0030] (2) Primer design The upstream primer nucleic acid sequence: 5'-biotin-CTATAGCAATGGTACGGTACTTCC-3' (as shown in SEQ ID NO.2), which is complementary to the 5'-end fixed region of the DNA template sequence and is used to guide PCR amplification; the downstream primer nucleic acid sequence: 5'-FAM-TTAGCAAAGTAGCGTGCACTTTTG-3' (as shown in SEQ ID NO.3), which is complementary to the 3'-end fixed region of the DNA template sequence and is used for the other chain of PCR amplification, and the 5' end is labeled with FAM (fluorescein) to facilitate subsequent fluorescence detection or analysis.

[0031] (3) Preparation of initial DNA random library by PCR amplification Mix the DNA template sequence containing 20 random nucleotides with the primers, add appropriate amount of PCR buffer, MgCl2, deoxyribonucleoside triphosphates (dNTPs) (in the ratio of A*:T:C:G=10:1:1:1), DNA polymerase and sterile water; preheat at 95 °C for 5 minutes, cycle amplification at 95 °C for 10 seconds (denaturation); anneal at 55 °C for 10 seconds; extend at 72 °C for 30 seconds, repeat 25 to 35 times, and finally extend at 72 °C for 5 minutes, keep at 4 °C or room temperature until taken out; use a PCR product purification kit or magnetic bead separation method to remove impurities such as unreacted primers, dNTPs and DNA polymerase, and the purified PCR product is the initial DNA molecule random library.

[0032] S2, based on the initial DNA molecule random library, using polymerase to perform primer extension reaction with four nucleoside triphosphate monomers of dT, dC, dG, and A* as substrates to obtain a single-stranded non-natural nucleic acid library; (1) Perform primer extension reaction (PEX) on the initial random library of DNA molecules to obtain a library containing non-natural nucleic acid residues The specific steps are as follows: take 3 μM DNA molecule random library and add it to 1X standard Therminator enzyme reaction buffer, then add 3 μM downstream primers, 10 mM NTPs (dTTP / dCTP / dGTP / A*TP), where A*TP represents non-natural or modified adenosine triphosphate, used to introduce non-natural nucleic acid residues into the DNA chain, dTTP, dCTP, dGTP are standard deoxynucleoside triphosphates, 10 mM MnCl2, 2U Therminator enzyme for reaction, reaction conditions are 37 °C, reaction time is 2h; the primer extension reaction product solution obtained above is subjected to chain separation to obtain a single-stranded non-natural nucleic acid library.

[0033] (2) Activation of magnetic beads and protein coupling Magnetic bead washing: Take 1 mL of streptavidin magnetic beads and wash them with ultrapure water at least three times, remove the supernatant and keep the magnetic beads; Activation of magnetic beads: Mix 500 μL 0.4 mM EDC and 500 μL 0.1 mM NHS in advance, add to a 1.5 mL tube containing magnetic beads and activate for 20 minutes; Protein coupling: After washing with ultrapure water for at least three times, remove the supernatant, add 500 μL of 2% thrombin protein sodium acetate solution, place in a rotary mixer (SCJLOGEX, SCI-RD-E), and incubate at room temperature for 1 h; Blocking and washing: After incubation, use 1 mL of ultrapure water to repeatedly blow and mix, let it stand, and aspirate the supernatant. Repeat this step 5 times, add 1 mL of 1 mM ethanolamine, mix well, and let it stand for 10 minutes to block unreacted carboxyl groups; use 1 mL of ultrapure water to repeatedly blow and mix, let it stand, and aspirate the supernatant. Repeat this step 3 times; after aspirating the supernatant, add 1 mL of phosphate buffered saline (PBS) and mix well for use.

[0034] S3, based on the single-stranded non-natural nucleic acid library, incubating with carboxyl magnetic beads that are not bound to the target protein, performing reverse screening, separating the non-natural nucleic acid library that is not bound to the magnetic beads, and obtaining the non-natural nucleic acid library after reverse screening; Reverse screening using carboxyl magnetic beads: Use carboxyl magnetic beads without binding proteins and incubate with non-natural nucleic acid aptamer library for reverse screening to separate the library bound to the magnetic beads from the library not bound; Library extension and incubation: Take 100 μL of non-natural nucleic acid aptamer and fully extend it at 37 °C for 2 h. Incubate it with carboxyl magnetic beads at room temperature for 1 h and then wash it repeatedly three times. Aspirate the supernatant and discard the magnetic beads.

[0035] S4, based on the non-natural nucleic acid library after reverse screening, incubate with streptavidin magnetic beads coupled with target protein, separate the nucleic acid solution bound to the magnetic beads, reverse transcribe, PCR amplify, separate single-stranded DNA for forward screening to obtain the first round of screening enrichment library; (1) Forward screening: Take 100 μL of magnetic beads bound to thrombin protein, add the supernatant retained in the previous step and incubate at room temperature for 1 h; Washing and reverse transcription: After repeated washing with ultrapure water, remove the supernatant, incubate at 95 °C for 5 minutes, remove the supernatant, and use Bst 2.0 polymerase for reverse transcription reaction to reverse transcribe the RNA template into cDNA.

[0036] (2) Preparation and amplification of cDNA PCR reaction system The supernatant obtained by the above reverse transcription was added to 5X Bst 2.0 reaction buffer, and 50 nM upstream primer, 0.5 nM dNTPs, 3 nM MnCl2, and 2.5 mM Bst 2.0 polymerase were added for reaction; the reaction mixture was incubated at 55 °C for 3 h for PCR amplification.

[0037] (3) PCR amplification and single-strand separation and enrichment library preparation PCR amplification and single-strand separation: Take the first round of non-natural single-stranded DNA template (i.e., the cDNA obtained by the above PCR amplification) in a 100 μL PCR reaction system, add it to 1X standard DNA polymerase reaction buffer, add 10 μL upstream and downstream primers, 10 μM dNTPs, and 5 μL DNA polymerases for PCR amplification, separate the single-stranded DNA, and obtain the enriched library of this round of screening, which can be used for the next round of screening.

[0038] The PCR amplification conditions were: 95 °C, 5 min; (95 °C, 10 sec; 55 °C, 10 sec; 72 °C, 30 sec) x 25-35; 70 °C, 5 min.

[0039] S5, based on the first round of enriched library, repeat S1 to S4 for multiple rounds of screening, sequence the enriched library in the last round of screening, obtain the sequence information of non-natural nucleic acid molecules, and prepare single-stranded non-natural nucleic acid molecules, which are potential non-natural nucleic acid aptamers.

[0040] Sequencing: After 5 rounds of screening, the library obtained after the last round of screening was sequenced. The results are as follows Figure 2 shown.

[0041] Sequence analysis and affinity testing: The sequence information of the non-natural nucleic acid molecule is inferred based on the sequencing results, and the affinity of the non-natural nucleic acid molecule binding to the target protein (Thrombin) is analyzed by the gel electrophoresis mobility method to obtain the non-natural nucleic acid aptamer that can bind to the Thrombin protein.

[0042] Secondary structure prediction and affinity testing: The secondary structure of the non-natural nucleic acid aptamers with higher affinity was predicted, and the aptamer structure was predicted using DNA fold online software. The results are as follows: Figure 3 shown.

[0043] The binding force between protein and small molecule was detected by microscale thermophoresis (MST). The binding force between protein and small molecule was detected by RED-NHS protein labeling kit (NanoTemper, MO-L011) and the affinity and specificity of non-natural nucleic acid aptamers were verified. The specific steps are as follows: A column treatment and protein sample preparation: open the A column, remove the lower plastic seal, and put a 1.5 mL centrifuge tube on the bottom, add 300 µL Labeling Buffer to the filler, centrifuge at 3000 rpm for 30 seconds using a handheld centrifuge (SCJLOGEX, S1010), and filter the liquid; repeat the above steps 3 times; take another clean 1.5 mL centrifuge tube and put it on the outside of the A column, add the protein sample (40-100 μL) to the center of the filler, transfer it gently to the centrifuge (eppendorf, 5425), centrifuge at 3000 rpm for 60 seconds, and keep the filtered protein solution for later use; use Labeling Buffer to adjust the volume of the protein to be labeled to 100 μL, and the final concentration should be 2-20 μ2, which is recorded as C1.

[0044] Dye preparation: Directly add commercial solid dyes (Alexa Fluor TM 488 (Thermo Fisher Scientific, A12379)) was added with 30 μL of 100% DMSO and thoroughly shaken to dissolve to obtain the dye stock solution (concentration of 470 μM). An appropriate amount of the dye stock solution was taken and diluted to 100 μL with Labeling Buffer. The final concentration was 2 to 3 times C1 to ensure that the dye was in excess and the protein could be fully labeled.

[0045] Protein labeling: Evenly mix 100 μL of the protein to be labeled (C1) and 100 μL of dye solution (concentration is 2~3 times that of C1), and incubate the reaction solution in the dark at room temperature for 30 minutes.

[0046] Treatment and elution of column B: open column B, remove the lower stopper, and put a gasket and a 15 mL centrifuge tube on the bottom in sequence; add 3 mL of Assay Buffer containing 0.05% Tween-20 to the filler, and repeat 3 times; add 200 μL of the incubated reaction solution to column B, add 300 μL of Assay Buffer, and wait until all the solution flows out; add 600 μL Assay Buffer to the molecular sieve, collect all the eluate, about 600 μL, and the final concentration is 1 / 6 C1 as the stock solution.

[0047] MST experiment: small molecules were used as ligands and proteins were used as targets. The proteins were labeled with the RED-NHS protein labeling kit. The original proteins and proteins after saturation incubation with proteins and antibodies were labeled respectively. The small molecules were serially diluted in 16 gradients in the reaction buffer (50 mM HEPES buffer [pH= 7.4], containing 0.05% Tween-20), and the mixtures were mixed in equal amounts and placed at room temperature for 20 minutes. The mixture samples were loaded into a capillary (Monolith NT.115 Capillary), and the thermophoresis signal was measured using Monolith NT.115.

[0048] Data analysis: Nano Temper analysis software was used to analyze the changes in Kd values. The results are shown in Figure 4 As shown, the affinity and specificity of the non-natural nucleic acid aptamers were verified.

[0049] By the attached Figure 4 The data show that the Kd value is 0.55±0.06 μM, indicating that the aptamer has a higher affinity. Compared with the non-natural nucleic acid aptamers screened by other methods with an affinity of 0.36~0.44±0.06 µM, the method of the present invention can achieve the same level of affinity as other methods incorporating multiple non-natural nucleic acids by incorporating only one non-natural nucleic acid.

[0050] Non-natural nucleic acid aptamer sequence: The non-natural nucleic acid aptamer sequence is: 5'-TGGTGGA*A*TGGA*TTGGGTGGTTTGGTCGA*CGCGTTA*CA*CG-3' (as shown in SEQ ID NO.4).

[0051] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads, characterized in that: include: S1, synthesis of initial DNA random library and primers; S2, based on the initial DNA molecule random library, using polymerase to perform primer extension reaction with four nucleoside triphosphate monomers of dT, dC, dG, and A* as substrates to obtain a single-stranded non-natural nucleic acid library; S3, based on the single-stranded non-natural nucleic acid library, incubating with carboxyl magnetic beads that are not bound to thrombin, performing reverse screening, separating the non-natural nucleic acid library that is not bound to the magnetic beads, and obtaining the non-natural nucleic acid library after reverse screening; S4, based on the non-natural nucleic acid library after reverse screening, incubate with streptavidin magnetic beads coupled with thrombin, separate the nucleic acid solution bound to the magnetic beads, reverse transcribe, PCR amplify, separate single-stranded DNA for forward screening to obtain the first round of screening enrichment library; S5, based on the first round of enriched library, repeat S1 to S4 for multiple rounds of screening, sequence the enriched library in the last round of screening, obtain the sequence information of non-natural nucleic acid molecules, and prepare single-stranded non-natural nucleic acid molecules, which are potential non-natural nucleic acid aptamers.

2. The method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads according to claim 1, characterized in that: The synthesized initial DNA molecule random library and primers, the DNA template sequence for synthesizing the initial DNA molecule random library is shown as SEQ ID NO.1, and the primers include upstream primers and downstream primers, and the sequences are shown as SEQ ID NO.2 to SEQ ID NO.

3.

3. The method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads according to claim 1, characterized in that: The primer extension reaction conditions are as follows: the reaction buffer is a Therminator enzyme reaction buffer, the reaction temperature is 36-38 °C, and the reaction time is 1-2 h.

4. The method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads according to claim 1, characterized in that: The ratio of the four nucleoside triphosphate monomers of dT, dC, dG and A* is 1:1:1:3-20.

5. The method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads according to claim 1, characterized in that: In S3, the incubation conditions with the carboxyl magnetic beads that are not bound to thrombin are: incubation at room temperature for 1 to 2 hours.

6. The method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads according to claim 1, characterized in that: In S4, the streptavidin magnetic beads coupled with thrombin are obtained by the following steps: using 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N -Hydroxysuccinimide activated magnetic beads; incubated the activated magnetic beads with thrombin solution to couple thrombin; and used ethanolamine to block unreacted active sites.

7. The method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads according to claim 1, characterized in that: In S4, the PCR amplification reaction conditions are: pre-denaturation at 95 °C for 5 minutes; denaturation at 95 °C for 10 seconds, annealing at 55 °C for 10 seconds, extension at 72 °C for 30 seconds, extension at 70 °C for 5 minutes, and 25 to 35 cycles.

8. The method for screening non-natural nucleic acid aptamers targeting thrombin based on streptavidin magnetic beads according to any one of claims 1 to 7, characterized in that: It also includes testing non-natural nucleic acid molecules and target The affinity step of the protein is used to predict the secondary structure of non-natural nucleic acid aptamers and verify their affinity step through experiments.

9. A non-natural nucleic acid aptamer targeting thrombin obtained by the method according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 in screening non-natural nucleic acid aptamers targeting proteins, characterized in that: The targeting protein is any one of SARS-CoV-2 nucleocapsid protein, tumor necrosis factor-related OX40 protein, and LAG-3 immune checkpoint protein.

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