Nucleic acid aptamer specifically recognizing soluble ST2 protein and use thereof

Abstract

The present invention discloses a nucleic acid aptamer that specifically recognizes soluble ST2 protein and its use, which belongs to the field of mutation or genetic engineering. The technical problem to be solved is how to specifically detect sST2 protein. To solve this technical problem, a nucleic acid aptamer of single-stranded DNA having nucleotide sequences of SEQ ID No. 1 to 10 is provided. By using SELEX technology and combining high-throughput sequencing technology and bioinformatics analysis, the number of screenings is reduced, candidate nucleic acid aptamers are obtained, and their affinity and specificity are further analyzed to obtain a nucleic acid aptamer that specifically recognizes sST2 protein. The nucleic acid aptamer has characteristics such as high specificity, high stability, easy synthesis, and easy labeling of functional groups, and can be used for the detection of sST2 protein, biosensors, and the manufacture of products for the diagnosis and prognosis of cardiovascular disease.

Description

[Technical field]

[0001] The present invention relates to a nucleic acid aptamer that specifically recognizes a soluble ST2 protein and its use in the field of mutation or genetic engineering. [Background technology]

[0002] A nucleic acid aptamer is a single-stranded DNA (ssDNA) or RNA that can specifically bind to a target molecule, selected from a random deoxyoligonucleotide library by the Systematic evolution of ligands by exponential enrichment (SELEX) method, also known as an in vitro selection method. Conventional antigen-antibody reactions have excellent sensitivity and specificity, and enzyme immunoreactions play a vital role in the detection of various biomolecules, but proteins as probe molecules are easily denatured by environmental factors such as pH and temperature, and the synthesis costs are high. A nucleic acid aptamer itself is a series of single-stranded DNA or RNA molecules that fold into a specific three-dimensional structure and bind to a biological target with high affinity and specificity. It has the advantages of small molecular weight, chemical synthesis, structural modification and labeling, good stability, reversible denaturation and regeneration, storage and transportation at room temperature, and no immunogenicity or toxicity, and therefore shows unparalleled advantages over conventional immunological and chemical molecular recognition in many aspects. In the field of biomedical technology, nucleic acid aptamers can be used in various fields such as clinical diagnosis, drug delivery, and disease treatment drugs. For example, nucleic acid aptamers have all the properties required for an inducer, and the molecules can be bound to radionuclides and delivered to target tissues, and also have the function of directly inhibiting target proteins, making them very promising for in vivo imaging and therapeutic drugs. In the research and development of new drugs, nucleic acid aptamers can be used to identify drug targets as drug molecules or lead molecules, or to guide targeted therapy as biological missiles. In addition, nucleic acid aptamers can bind to various target substances with high specificity and selectivity, and often cause structural changes when binding to target molecules, which is advantageous for the construction of sensing analysis methods, and the binding strength between labeled nucleic acid aptamers and target molecules generally does not change, which has led to great attention being paid to biochemical detection technology using nucleic acid aptamers. Currently, nucleic acid aptamers are widely used in various sensors such as electrochemical sensors, optical sensors, and acoustic sensors.

[0003] Growth stimulation expressed gene 2 (ST2) protein is a member of the interleukin 1 receptor / Toll-like receptor superfamily. There are two types of ST2 protein: soluble (sST2) and transmembrane receptor type (ST2L), and the functional ligand of ST2 is interleukin-33 (IL-33). Recent studies have shown that ST2 is not only involved in inflammatory responses, but also closely related to cardiovascular disease, and soluble ST2 (sST2) is a decoy receptor for IL-33, which competitively binds to IL-33, inhibiting the binding of IL-33 to ST2L and weakening the cardiovascular protective effect of the IL-33 / ST2 signal pathway. A series of experimental and clinical studies have shown that an increase in sST2 level is associated with the severity of heart failure, can predict the occurrence of sudden cardiac death, and is an excellent marker for evaluating the risk stratification of heart failure. sST2 is currently one of the most specific indicators for heart failure detection, and is hardly affected by age, sex, BMI, causes of heart failure, atrial fibrillation, anemia, and renal function, has low biological variability, and is highly stable, and can be used in clinical applications such as diagnosing heart failure, predicting the prognosis of heart failure, improving clinical stratification of heart failure, and predicting the probability of death of patients. In addition, recent studies have found that sST2 can differentiate and diagnose patients with aortic aneurysm / dissection, and can be used as a diagnostic marker for acute aortic dissection. However, clinical use is limited because the current clinical detection of sST2 is based on antigen-antibody detection and the detection kit is expensive. Therefore, there is still a need for a more accurate, stable, convenient, and affordable sST2 detection method. Since nucleic acid aptamers can specifically bind to targets, they are expected to be used in clinical detection of sST2 and disease treatment. Summary of the Invention

[0004] The object of the present invention is to provide a nucleic acid aptamer that specifically recognizes sST2 protein, which has high specificity, high stability, is easy to synthesize, and / or is easy to label with a functional group, and / or the use of this nucleic acid aptamer in detecting sST2 protein. The technical problems to be solved are not limited to the technical problems described above, and a person skilled in the art can clearly understand other technical problems not described in this specification from the following explanation.

[0005] In order to achieve the above object, the present invention provides a nucleic acid aptamer that specifically recognizes sST2 protein. A1) any single stranded DNA having the nucleotide sequence of SEQ ID No. 4, 6, 5, 1, 2, 3, 7, 8, 9 or 10; A2) Any single-stranded DNA having 75% or more identity with the nucleotide sequence defined in A1) and specifically recognizing the sST2 protein; A3) Any single-stranded RNA transcribed from the single-stranded DNA shown in A1) and specifically recognizing the sST2 protein; A4) It may be any single-stranded DNA or single-stranded RNA that hybridizes under stringent conditions with the nucleotide sequence defined in A1) or A3) and specifically recognizes the sST2 protein.

[0006] The sST2 protein is the soluble growth STimulation expressed gene2 protein.

[0007] 75% or more identity may be 80%, 85%, 90%, or 95% or more identity.

[0008] In this case, 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0009] Additionally, the nucleic acid aptamer may be bound to a functional group or molecule.

[0010] Furthermore, the 5' or 3' end of the nucleic acid aptamer is modified with a functional group or molecule.

[0011] Additionally, the functional group or molecule may be an isotope, a fluorescent label, a chemiluminescent label, a bioluminescent label, an enzyme label, a magnetic agent, biotin, an affinity ligand, a sulfhydryl group, and / or a therapeutic agent.

[0012] The functional groups or molecules are used to enhance the stability of the nucleic acid aptamer, to provide a detectable signal, or to conjugate the nucleic acid aptamer with other substances to form compositions.

[0013] The present invention further provides a probe. The probe may be any of the above-mentioned nucleic acid aptamers labeled with a label.

[0014] A label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and that can be attached to a nucleic acid. Labels include dyes, 32These include, but are not limited to, radiolabels such as P, conjugated binding groups such as biotin, haptens such as digoxigenin (DIG), chemiluminescent, phosphorescent or fluorescent moieties, and fluorescent dyes alone or in combination with a moiety that can suppress or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label may provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, quantum dots, electrochemistry, X-ray diffraction or absorption, magnetism, immunoenzymatic reactions, filter paper-based immunoassays, affinity precipitation, affinity chromatography, enzyme activity, microscopic projection or scanning imaging, super-resolution imaging, cell tracking, animal or human biological nanoparticle tracking imaging, nanoflow cytometry, tunable resistive pulse sensing, fluorescence correlation spectroscopy, surface plasmon resonance, fluorescence polarization, surface-enhanced Raman spectroscopy, electrochemical sensing, microfluidics or microfluidics, chip analysis, proteomics, genomics, metabolomics, microbiomics, RNA (mRNA, InRNA, snRNA), miRNA, etc. The label may be a charged moiety (positively or negatively charged) or, optionally, neutral in charge. The label may comprise or consist of a nucleic acid or protein sequence, so long as the sequence comprising the label is detectable. In some embodiments, the nucleic acids are detected directly without labeling (e.g., the sequence is read directly). Labeling can also be used for targeted drug delivery.

[0015] In some embodiments, the labels are fluorophores, colorimetric labels, quantum dots, biotin, and other labeling molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cycloolefins for click reactions, initiating groups for polymer labeling), and may be selected from polypeptide / protein molecules, LNA / PNA, non-natural amino acids and their analogs (such as pseudopeptides), non-natural nucleic acids and their analogs (such as pseudonucleotides), and nanostructures (such as inorganic nanoparticles, NV-centers, aggregation / aggregation induced luminescence molecules, rare earth ion ligand molecules, multi-metal oxygen clusters, etc.).

[0016] In some embodiments, the fluorophore may be selected from fluorescein-based dyes, rhodamine-based dyes, and cyanine dyes.

[0017] In some embodiments, fluorescein dyes include standard fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), cy5, cy3, Quasar 670, Alexa Fluor 488 / 647, and the like.

[0018] In some embodiments, rhodamine dyes include R101, tetraethylrhodamine (RB200), carboxytetramethylrhodamine (TAMRA), and the like.

[0019] In some embodiments, the cyanine dyes are selected from two main types: one is thiazole orange (TO), oxazole orange (YO) series and their dimeric dyes, and the other is polymethine series cyanine dyes.

[0020] In some embodiments, the fluorophores can be selected from dyes such as stilbenes, naphthalimides, coumarins, acridines, pyrenes, and the like.

[0021] Fluorophores are typically labeled at the 5' end of the probe sequence, but can also be placed at the 3' end by altering the modified bond (eg, an --OH or --NH bond).

[0022] In one embodiment of the present invention, the 5'-end of the single-stranded DNA (ssDNA) aptamer shown in SEQ ID Nos. 1 to 10 is labeled with biotin.

[0023] The present invention also provides a sensor comprising any of the nucleic acid aptamers or any of the probes.

[0024] The present invention also provides a reagent or kit for detecting sST2 protein, comprising any of the nucleic acid aptamers or any of the probes described herein.

[0025] The kit contains Taq DNA polymerase, dNTPs, PCR buffer, and Mg 2+ It further includes one or more of the following:

[0026] The various reagent components of the kit may be contained in separate containers or may be precombined in whole or in part into a reagent mixture.

[0027] The present invention also provides a pharmaceutical for preventing, ameliorating, or treating an sST2-associated disease, comprising any of the nucleic acid aptamers or any of the probes described herein.

[0028] In addition, the pharmaceutical agent further comprises one or more pharma- ceutically acceptable carriers.

[0029] The pharma- ceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant, or lubricant.

[0030] The present invention also provides a drug delivery system that specifically targets the sST2 protein, comprising any of the nucleic acid aptamers or any of the probes described herein.

[0031] The drug delivery system may be a liposomal drug delivery system, a polymeric micelle drug delivery system, a polymeric disk drug delivery system, or a nanoparticle drug delivery system.

[0032] Drug delivery systems are used for the targeted delivery and / or site-specific release of drugs.

[0033] The present invention also provides any of the following uses of any of the nucleic acid aptamers or any of the probes herein: B1) Use in the manufacture of medicines or products for preventing, improving or treating sST2-related diseases; B2) Use in detecting sST2 protein or in manufacturing a product for detecting sST2 protein, B3) Use in the manufacture of a product for binding sST2 protein; B4) Use of sST2 protein in the manufacture of products for in vivo imaging; B5) Use in the manufacture of products for screening, diagnosing or assisting in the diagnosis of sST2-related diseases; B6) Use in the manufacture of products for the prognosis evaluation of sST2-related diseases; B7) Use in the manufacture of a product for predicting the probability of death of patients with sST2-related diseases; B8) Use in the manufacture of products to assess risk stratification for heart failure.

[0034] In the above uses, the sST2-related disease may be a cardiovascular disease.

[0035] In the above uses, cardiovascular diseases include, but are not limited to, heart failure, atherosclerosis, hypertension, myocardial infarction, coronary heart disease, acute coronary syndrome, aortic aneurysm or aortic dissection.

[0036] The present invention also provides a method for detecting sST2 protein, which comprises labeling any one of the nucleic acid aptamers shown in SEQ ID Nos. 1 to 10 with a reporter group, allowing the nucleic acid aptamer labeled with the reporter group to interact with a test sample, and detecting the signal of the reporter group to detect sST2 protein.

[0037] Additionally, the reporter group may be biotin or a fluorescent group, which may be, but is not limited to, Rhodamine, FAM, FITC, BODIPY, Cy3, Cy5, VIC, HEX, TRT, ROX, JOE, TAMRA.

[0038] The purpose of the above uses and methods may be disease diagnostic, prognostic and / or therapeutic purposes, as well as non-disease diagnostic, prognostic and therapeutic purposes, and their direct purpose may be to obtain information on intermediate results of disease diagnostic, prognostic and / or therapeutic results, as well as non-disease diagnostic, prognostic and therapeutic purposes.

[0039] The nucleic acid aptamers described herein may be single-stranded DNA (ssDNA) aptamers or single-stranded RNA (ssRNA) aptamers.

[0040] The sST2-associated disease described herein may be a disease caused by an abnormality in the IL-33 / ST2 signal pathway.

[0041] The articles of manufacture described herein may be a kit, a test strip, or a biosensor.

[0042] The present invention also provides any of the following uses of any of the nucleic acid aptamers or probes herein: C1) Use in the prevention, improvement or treatment of sST2-related diseases, C2) Use of sST2 protein in in vivo imaging; C3) Use in screening, diagnosis or auxiliary diagnosis of sST2-related diseases; C4) Use in evaluating the prognosis of sST2-related diseases; C5) Use in predicting the probability of death of patients with sST2-related diseases; C6) Use in assessing risk stratification for heart failure; C7) Use in monitoring treatment for sST2-related diseases; C8) Use in purifying sST2 protein.

[0043] In the above uses, the sST2-related disease may be a cardiovascular disease.

[0044] In the above uses, cardiovascular diseases may include, but are not limited to, heart failure, atherosclerosis, hypertension, myocardial infarction, coronary heart disease, acute coronary syndrome, aortic aneurysm, or aortic dissection.

[0045] The present invention also provides a method for screening, diagnosing or auxiliary diagnosing an sST2-related disease, which includes obtaining a sample including serum, blood or plasma from a subject, detecting the content of sST2 protein in the sample using the nucleic acid aptamer described herein, and screening, diagnosing or auxiliary diagnosing an sST2-related disease based on the content of sST2 protein.

[0046] The present invention also provides a method for treating an sST2-related disorder, comprising administering to a subject diagnosed with an sST2-related disorder a nucleic acid aptamer or an sST2 inhibitor bound to a nucleic acid aptamer.

[0047] In the above method, the sST2 inhibitor may be a reagent that inhibits the ST2L / IL-33 signal pathway.

[0048] In the above method, the sST2-related disease may be a cardiovascular disease.

[0049] In the above methods, the cardiovascular disease may include, but is not limited to, heart failure, atherosclerosis, hypertension, myocardial infarction, coronary heart disease, acute coronary syndrome, aortic aneurysm, or aortic dissection.

[0050] The present invention uses MCP-SELEX technology in combination with high-throughput sequencing technology and bioinformatics analysis to reduce the number of screenings and obtain candidate nucleic acid aptamers. Furthermore, by analyzing the affinity and specificity, an ssDNA nucleic acid aptamer that specifically recognizes sST2 protein is obtained. The ssDNA aptamer of the present invention has characteristics such as high specificity, high stability, easy synthesis, and easy labeling of functional groups, and specifically recognizes and binds to sST2 protein, and can be used for detecting sST2 protein and manufacturing biosensors. In addition, the ssDNA aptamer of the present invention is also a potential therapeutic agent for sST2-related diseases, and can be used for manufacturing clinical diagnostic reagents and disease therapeutic agents.

[0051] The present invention provides a highly specific nucleic acid aptamer for detecting sST2, which can be screened in vitro, obtained with high throughput, has a short screening period, is easy to synthesize, has good stability, has high affinity, and is easy to modify and label. Furthermore, the nucleic acid aptamer of the present invention can be used alone or in combination with related drugs, providing new possibilities for the treatment of sST2-related diseases. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1 shows the results of electrophoresis of purified human sST2 protein. [Diagram 2] FIG. 1 shows the percentage of elution rate (first from left), binding rate (second from left), and signal-to-noise ratio (third from left) after the first, second, third, fourth, fifth, sixth, and seventh screening rounds. [Diagram 3] FIG. 1 shows the results of an EMSA experiment in Example 2. [Figure 4] FIG. 13 shows the results of an affinity detection experiment in Example 3 between nucleic acid aptamers Apt4 (SEQ ID No. 4), Apt5 (SEQ ID No. 5) and Apt6 (SEQ ID No. 6) and sST2 protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present invention will be described in detail below by specific embodiments, but the examples shown are only for illustrating the present invention and do not limit the scope of the present invention. The examples provided below can be used as a guide for those skilled in the art to make further improvements, but do not limit the present invention in any way.

[0054] Unless otherwise specified, the experimental methods in the following examples are all conventional methods and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0055] The sST2 protein in the following examples was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (Cat: 10105-H08H). The results of its purification electrophoresis (SDS-PAGE) are shown in FIG.

[0056] Example 1: Screening of nucleic acid aptamers of single-stranded DNA targeting human sST2 protein Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and high-throughput sequencing techniques, bioinformatics analysis was applied to obtain single-stranded DNA (ssDNA) nucleic acid aptamers targeting human sST2 protein.

[0057] The specific steps are as follows: 1. Construction of random ssDNA library The random ssDNA library comprises two primer regions and a 35 base random region, the nucleotide sequence of which is shown in SEQ ID No. 11, in which positions 1-20 of SEQ ID No. 11 are a forward primer region, positions 56-75 of SEQ ID No. 11 are a reverse primer region, and positions 21-55 of SEQ ID No. 11 are the 35 nucleotides N, where N represents A, G, C, or T. Here, the forward primer sequence corresponding to the forward primer region is 5'-GACAGGCAGGACACCGTAAC-3' (SEQ ID No.12), The reverse primer sequence corresponding to the reverse primer region is 5'-GAAGAGGAGGGAGGTAGCAG-3' (SEQ ID No. 13).

[0058] 2. Negative screening using magnetic beads 2-1. 10 μl of 100 mM random ssDNA library was dissolved in 480 ml of binding buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4), heated in a water bath at 95°C for 10 minutes, rapidly cooled in an ice bath for 10 minutes, and allowed to stand at room temperature for 10 minutes to prepare an ssDNA nucleic acid aptamer library that forms a secondary structure. 2-2. The magnetic beads were activated and treated with 50 mg / mL EDC and 50 mg / mL NHS to form carboxylated magnetic beads. These were washed twice with 25 mM MES solution (pH = 5.0), and 10 μL of Binding Buffer was added to disperse the magnetic beads in the Binding Buffer. 2-3. All the activated magnetic beads were added to the ssDNA library solution to make the total volume 500 μL, and after incubation at room temperature for 30 min, the magnetic beads were removed (magnetic separation) and the supernatant was left.

[0059] 3. Screening of ssDNA binding to the target molecule (sST2) using magnetic beads 3-1. 10 μL of 10 μM sST2 protein was added to the supernatant and rotated at room temperature (25° C.) for 1 hour. 3-2, Activate magnetic beads, the procedure is the same as 2-2. 3-3. The activated magnetic beads were added to the ssDNA solution in which sST2 was incubated, and after incubation at room temperature for 30 minutes, the supernatant was discarded to leave the magnetic beads. 3-4. After adding 500 μL of Binding Buffer to the magnetic beads, mix well on a shaker for 5 minutes, leave the beads on a strong magnet for 1 minute, remove the supernatant, and wash the beads 4 times (5 minutes each time), collecting the supernatants from each wash (W1 to W4). 3-5. 1.8 μL of Proteinase K (20 mg / mL) was added to the washed magnetic beads, and the volume was adjusted to 120 μL with Binding Buffer. The mixture was then left to stand in a 52°C incubator for 2 hours. After the reaction was completed, the mixture was heated with shaking at 95°C for 20 minutes to inactivate Proteinase K. 120 μL of the supernatant was removed and used as the eluate (R1) for the first screening.

[0060] 4. Preparation of ssDNA library for next screening 4-1. Prepare PCR small-volume samples using the eluate (R1) as a template, set different amplification times, and determine the cycle with the lowest amplification concentration and the lowest amplified impurity band by gel electrophoresis. Usually, the times set are 5, 9, 12, 15, 18, and 21, and a blank control should be added when performing PCR. The small-volume sample PCR system is shown in Table 1. TIFF2025510610000001.tif6417072°C, samples were removed after completing the specified number of cycles. 4-2. PCR of large amounts of samples After PCR of a small sample, the optimal cycle number is determined by gel electrophoresis experiment, and based on this cycle number, PCR of a large sample is performed, and six to eight 100 μl tubes are required. The large-scale sample PCR system is shown in Table 2. TIFF2025510610000002.tif641704-3, Preparation of single-stranded DNA (secondary library) (1) The PCR products of a large number of samples were sequentially slowly passed through streptomycin-coated agarose beads, and after all the samples had passed through the column, they were washed twice with 200 μL of PBS. (2) 100 μL of NaOH was added, and the liquid that passed through the column was collected as the target single-stranded DNA, which was then neutralized by adding 2 μL of 15 M HCL (hydrochloric acid) and mixed well. (3) The concentration of the collected single-stranded DNA (ssDNA) solution was detected and used as the library for the next screening.

[0061] 5. Detection of enrichment of screening libraries by fluorescent quantitative PCR 5-1. Fluorescent quantitative PCR was performed using the supernatants W1 to W4 collected in step 3-4 and the first eluate R1 as templates. 5-2. The elution rate, binding rate, and signal-to-noise ratio for each screening were obtained from the cycle number and standard curve of the fluorescent quantitative PCR, and the enrichment of the library was evaluated based on these.

[0062] 6. Multiple screening and high-throughput sequencing 6-1. Repeat the screening procedures from 1 to 5, and use the secondary library obtained in the previous screening as the initial library for each screening. When performing screening, increase the screening pressure by reducing the amount of library input and adding negative screening proteins, etc., to increase the enrichment of the ssDNA library. After 6-2 and 7 rounds of screening, the results of fluorescent quantitative PCR showed that the ssDNA library was significantly enriched, much higher than that of the 6th round (the results were shown in Figure 2). Therefore, the 6th and 7th rounds of ssDNA were PCR amplified, the amplified and purified products were subjected to high-throughput sequencing, and the abundances of the sequencing results were compared to determine the top 10 DNA sequences, whose nucleotide sequences are shown in Table 3. TIFF2025510610000003.tif141170

[0063] Example 2. Gel electrophoretic mobility shift assay (EMSA) to detect binding of nucleic acid aptamers to sST2 protein EMSA (Electrophoretic Mobility Shift Assay) is an in vitro technique to detect the binding of proteins to DNA (or RNA) sequences and can be used for qualitative and quantitative analysis. Typically, purified proteins and DNA (or RNA) sequences are incubated in a homogenous environment, and then the protein-DNA complexes and the unbound sequences are separated by non-denaturing polyacrylamide gel electrophoresis (PAGE). The principle of separation is that the protein-DNA complexes migrate slower than the unbound sequences because the macromolecular substances such as proteins are bound to DNA.

[0064] The experimental procedure is as follows. 1. Heat treatment of DNA According to the experimental composition shown in Table 4, the nucleic acid aptamer was added to a buffer solution and nuclease-free water, heated at 95°C for 10 minutes, rapidly cooled on ice for 10 minutes, and allowed to stand at room temperature for 10 minutes. TIFF2025510610000004.tif32170

[0065] 2. Incubation of DNA and target The DNA solution and sST2 protein were mixed gently until homogenous, and the final volume was adjusted to 20 μL. A sample containing only the aptamer without protein was used as a negative control, and incubated at room temperature for 30 min with rotation.

[0066] 3. Gel electrophoresis Non-denaturing PAGE gel electrophoresis was performed on the negative control and the sample, and gel imaging was performed after dye staining and analyzed with imaging software. As shown in Figure 3, the nucleic acid aptamers Apt1 to 10 shown in SEQ ID No. 1 to 10 all had strong binding affinity to sST2, and among them, Apt4 shown in SEQ ID No. 4, Apt5 shown in SEQ ID No. 5, and Apt6 shown in SEQ ID No. 6 had more significant binding affinity.

[0067] Example 3: Measurement of affinity of nucleic acid aptamers Apt4, Apt5, and Apt6 with sST2 protein 1. The sST2 protein to be measured was incubated with the silanized and bifunctionalized optical fiber and left at room temperature for 6 hours. 2. Apt4, Apt5 and Apt6 DNA were labeled with CY5.5 and prepared at different concentrations of 0 nM, 10 nM, 50 nM, 100 nM, 200 nM, 500 nM and 1000 nM. 3. The optical fiber was set in the measurement device, and the nucleic acid aptamers of each concentration were passed through the measurement device in sequence according to the sample injection setting procedure (shown in Table 5). TIFF2025510610000005.tif45170The composition of the buffer in Table 5 is 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, and the regenerant is 2 M NaCl. 4. The fluorescence photometric values ​​of DNA in each concentration gradient entering the measuring device were recorded, and based on the measured fluorescence photometric values, curve fitting was performed using GraphPad Prism software, and the Kd value was calculated using the formula Y=Bmax*X / (Kd+X). The results are shown in Figure 4, the binding value Kd between Apt4 and sST2 protein shown in SEQ ID No. 4 is 127nM, the binding value Kd between Apt5 and sST2 protein shown in SEQ ID No. 5 is 188nM, and the binding value Kd between Apt6 and sST2 protein shown in SEQ ID No. 6 is 173nM.

[0068] The present invention has been described in detail above. Those skilled in the art can carry out the present invention in a broader scope under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention has been described with specific examples, it is understood that the present invention can be further improved. In short, based on the principles of the present invention, the present application is intended to include any changes, uses, or improvements to the present invention that depart from the scope disclosed in the present application and include improvements made using conventional techniques known in the art. Some basic features may be applied within the scope of the following attached claims. [Industrial Applicability]

[0069] The present invention uses MCP-SELEX technology in combination with high-throughput sequencing technology and bioinformatics analysis to reduce the number of screenings and obtain candidate nucleic acid aptamers. Furthermore, by analyzing the affinity and specificity, an ssDNA nucleic acid aptamer that specifically recognizes sST2 protein is obtained. The ssDNA aptamer of the present invention has characteristics such as high specificity, high stability, easy synthesis, and easy labeling of functional groups, and specifically recognizes and binds to sST2 protein, and can be used for detecting sST2 protein and manufacturing biosensors. In addition, the ssDNA aptamer of the present invention is also a potential therapeutic agent for sST2-related diseases, and can be used for manufacturing clinical diagnostic reagents and disease therapeutic agents.

[0070] The present invention provides a highly specific nucleic acid aptamer for detecting sST2, which can be screened in vitro, obtained with high throughput, has a short screening period, is easy to synthesize, has good stability, has high affinity, and is easy to modify and label. This nucleic acid aptamer can be chemically synthesized, is organism-independent, inexpensive, and easy to store. Furthermore, the nucleic acid aptamer of the present invention can be used alone or in combination with related drugs, providing new possibilities for the treatment of sST2-related diseases.

Claims

1. A nucleic acid aptamer characterized in that it is any one of the following: A1) any single stranded DNA having the nucleotide sequence of SEQ ID No. 4, 6, 5, 1, 2, 3, 7, 8, 9 and 10; A2) Any single-stranded DNA having 75% or more identity with the nucleotide sequence defined in A1) and specifically recognizing the sST2 protein; A3) Any single-stranded RNA transcribed from the single-stranded DNA shown in A1) and specifically recognizing the sST2 protein; A4) Any single-stranded DNA or single-stranded RNA which hybridizes under stringent conditions with the nucleotide sequence defined in A1) or A3) and specifically recognizes the sST2 protein.

2. A probe, characterized in that any one of the nucleic acid aptamers according to claim 1 is labeled with a label.

3. A sensor comprising any one of the nucleic acid aptamers according to claim 1 or the probe according to claim 2.

4. A reagent or kit for detecting sST2 protein, comprising any one of the nucleic acid aptamers according to claim 1 or the probe according to claim 2.

5. A pharmaceutical for preventing, ameliorating or treating an sST2-related disease, comprising any one of the nucleic acid aptamers according to claim 1 or the probe according to claim 2.

6. A drug delivery system that specifically targets sST2 protein, comprising any one of the nucleic acid aptamers according to claim 1 or the probe according to claim 2.

7. Use of any of the nucleic acid aptamers according to claim 1 or the probe according to claim 2 in any of the following: B1) Use in the manufacture of medicines or products for preventing, ameliorating or treating sST2-related diseases; B2) Use in detecting sST2 protein or in producing a product for detecting sST2 protein; B3) Use in the manufacture of a product for binding sST2 protein; B4) Use of sST2 protein in the manufacture of products for in vivo imaging; B5) Use in the manufacture of products for screening, diagnosing or auxiliary diagnosing sST2-related diseases; B6) Use in the manufacture of products for the prognosis evaluation of sST2-related diseases; B7) Use in the manufacture of a product for predicting the probability of death of a patient with sST2-related disease; B8) Use in the manufacture of a product for assessing risk stratification for heart failure.

8. The use according to claim 7, characterized in that the sST2-related disease is a cardiovascular disease.

9. 9. The use according to claim 8, characterized in that the cardiovascular disease comprises heart failure, atherosclerosis, hypertension, myocardial infarction, coronary heart disease, acute coronary syndrome, aortic aneurysm or aortic dissection.

10. 1. A method for detecting sST2 protein, comprising: A method characterized by labeling any of the nucleic acid aptamers described in claim 1 with a reporter group, allowing the nucleic acid aptamer labeled with the reporter group to interact with a test sample, and detecting sST2 protein by detecting the signal of the reporter group.

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