A nucleic acid aptamer binding to CD117 protein and its application

Abstract

The present invention discloses a nucleic acid aptamer binding to CD117 protein and its application, wherein the nucleic acid aptamer has a nucleotide sequence that has at least 60% homology with any one of SEQ ID NO.1 to SEQ ID NO.2 and binds to CD117 protein. The nucleic acid aptamer of the present invention has a small molecular weight, stable structure and chemical properties, is easy to store and label, is easy to modify, and can be artificially synthesized in a short period of time. The nucleic acid aptamer obtained by the present invention can be used for CD117 protein detection, purification or imaging, and can also be used to prepare drugs for diagnosis or treatment of abnormal expression of CD117 protein, such as purification to obtain high-sensitivity and high-specificity detection of CD117 protein, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nucleic acid aptamer binding to CD117 protein and application thereof. Background Art

[0002] CD117 protein is a transmembrane protein, belonging to type III tyrosine kinase receptor, a member of the immunoglobulin superfamily, and is divided into three parts: the extracellular region, the transmembrane region, and the cytoplasmic region. The protein is closely related to platelet-derived growth factor in structure. CD117 is the expression product of the c-kit proto-oncogene and is a surface antigen marker of the extracellular region of the stem cell factor receptor (c-kit receptor). Stem cell factor coupling can cause the receptor to form dimers and phosphorylate, induce proliferation and limit apoptosis. CD117 is a type of receptor that plays an important role in cancer progression. It is a cell surface receptor with special structural and biological characteristics that responds to environmental cues by initiating appropriate signal cascades in tumor cells.

[0003] Under normal circumstances, the activation of CD117 requires the participation of the T ligand (stem cell factor, SCF). After CD117 binds to its ligand SCF, it forms a dimer, leading to receptor dimerization, and finally activates transcription factors in the cytoplasm, and initiates a series of downstream signal cascades, regulates gene expression, activates several key effector signaling pathways involved in cell function, and is responsible for maintaining cell functions, such as cell survival, metabolism, cell growth and progression, proliferation, apoptosis, cell migration and cell differentiation. The binding of CD117 to SCF can lead to the activation of multiple signaling pathways, including phosphatidylinositol 3-kinase, phospholipase c-γ, Src kinase, Janus kinase signal transducer and transcription activator, and mitogen-activated protein kinase pathway. Optimal regulation of the CD117 signaling pathway is essential for hematopoiesis, pigmentation, fertility, intestinal motility and nervous system function. Moreover, CD117 is expressed in a variety of normal cells and tissues, such as stem cells, mast cells, germ cells and melanocytes, and plays an important role in the proliferation and differentiation of these cells. The expression of CD117 is not limited to normal tissues, but is also associated with the pathophysiological mechanisms of a variety of human tumors. It is generally believed that CD117 causes tumors mainly in two aspects: one is abnormal gene expression (increased, decreased or missing); the other is that gene deletion or mutation causes changes in the structure and function of CD117. Under normal circumstances, the activation of CD117 requires the participation of SCF, but when it mutates, it can be activated without ligand binding. Mutations in the c-kit gene stimulate the continued proliferation of tumor cells and the loss of control of anti-apoptotic signals, which is conducive to the malignant proliferation of tumors. Oncogenic CD117 receptor tyrosine kinase "driver" mutations have been found in many cancers, such as acute myeloid leukemia (AML), melanoma, mast cell leukemia and gastrointestinal stromal tumors (GIST), and tyrosine kinase inhibitors (TKIs), such as imatinib or sunitinib, have anti-CD117 activity and are commonly used to treat these cancers.

[0004] Aptamers are DNA or RNA molecules separated by systematic evolution of ligands by exponential enrichment (SELEX). They can bind to other targets such as proteins, metal ions, small molecules, peptides, and even whole cells with high affinity and specificity, and therefore show broad prospects in biochemical analysis, environmental monitoring, basic medicine, and new drug synthesis. Compared with antibodies, aptamers have small molecular weight, better stability, easy modification, no immunogenicity, short production cycle, and can be synthesized artificially, eliminating a series of processes such as animal immunization, feeding, protein extraction, and purification. Therefore, if we can find aptamers with higher affinity and high specificity for CD117 protein, it will help to achieve high sensitivity and high specificity detection of CD117 protein, and help to develop drugs targeting CD117 protein.

[0005] Currently, there is no nucleic acid aptamer for CD117 protein. Therefore, it is necessary to find a nucleic acid aptamer with high binding affinity to CD117 protein, good specificity, easy modification and artificial synthesis, good stability, and convenient use. Summary of the invention

[0006] In order to overcome at least one defect of the above-mentioned prior art, the present invention provides a nucleic acid aptamer binding to CD117 protein and its application. A nucleic acid aptamer with small molecular weight, stable chemical properties, easy storage and labeling, which can bind to CD117 protein with high affinity, is obtained by screening, and can be used for detection, diagnosis, imaging and treatment, etc., with broad application prospects.

[0007] The technical solution adopted by the present invention to solve the problem is:

[0008] A nucleic acid aptamer that binds to a CD117 protein, wherein the nucleic acid aptamer has a nucleotide sequence that is at least 60% homologous to any one of SEQ ID NO.1 and SEQ ID NO.2 and binds to the CD117 protein.

[0009] Furthermore, the nucleic acid aptamer has an RNA sequence transcribed from the nucleotide sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.2.

[0010] Furthermore, the nucleic acid aptamer has a nucleotide sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.2.

[0011] Based on the SELEX technology, the inventors designed and synthesized a random single-stranded DNA library and corresponding primers to screen nucleic acid aptamers that have small molecular weight, stable chemical properties, easy storage and labeling and can bind to CD117 protein with high affinity. Thus, some nucleic acid aptamers with high affinity and strong specificity binding to CD117 protein were screened, namely DJ4a (SEQ ID NO.1) and DH09 (SEQ ID NO.2).

[0012] Due to the particularity of the nucleotide sequence, a certain position on the nucleotide sequence of the above-mentioned nucleic acid aptamer can be modified, for example, phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium or isotopization, etc., provided that the nucleic acid aptamer sequence obtained after such modification has desirable properties, for example, it can have an affinity for binding to CD117 protein equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or although the affinity is not significantly improved, it has higher stability. It is understandable that the nucleotide sequence having at least 30%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% homology with the nucleic acid aptamer provided by the present invention and binding to the CD117 protein, for example, the nucleotide sequence shown in any one of the above-mentioned nucleic acid aptamers can be deleted or a partial sequence can be added, and still has a high affinity with the CD117 protein, and is still within the scope of protection of the present invention.

[0013] In other words, the above aptamers, whether partially substituted or modified, have substantially the same or similar molecular structures, physicochemical properties and functions as the original aptamers, and can be used to bind to the CD117 protein.

[0014] Furthermore, the nucleotide sequence of the nucleic acid aptamer is modified, and the modification method includes one or more of phosphorylation, methylation, amination, sulfhydrylation, replacement of oxygen with sulfur, replacement of oxygen with selenium, and isotopization.

[0015] Furthermore, the nucleic acid aptamer has a sequence that is at least 60% homologous to any one of SEQ ID NO.1 and SEQ ID NO.2 and is obtained by modifying the nucleotide sequence that binds to the CD117 protein.

[0016] Furthermore, the transformation includes at least one of the following transformation methods:

[0017] (1) Connecting a fluorescent marker to the aptamer;

[0018] (2) Connecting radioactive substances to the aptamer;

[0019] (3) Connecting therapeutic substances to the aptamer;

[0020] (4) connecting biotin to the nucleic acid aptamer;

[0021] (5) connecting digoxigenin to the aptamer;

[0022] (6) connecting a nanoluminescent material to the nucleic acid aptamer;

[0023] (7) Connecting a small peptide to the nucleic acid aptamer;

[0024] (8) Connecting siRNA to the nucleic acid aptamer.

[0025] The present invention provides a conjugate or derivative of a nucleic acid aptamer, wherein the nucleic acid aptamer has a nucleotide sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.2; the conjugate of the nucleic acid aptamer comprises a fluorescent marker; and the derivative of the nucleic acid aptamer comprises a phosphorothioate skeleton or a peptide nucleic acid that binds to a CD117 protein and is formed by modifying the nucleotide sequence skeleton of the nucleic acid aptamer or the conjugate of the nucleic acid aptamer.

[0026] The aptamer conjugate of the present invention refers to connecting other groups to the aptamer, such as fluorescent markers with marking functions, such as FAM, radioactive substances, therapeutic substances, biotin, digoxin, nanoluminescent materials, small peptides, siRNA or enzyme markers, so that the modified aptamer sequence has desirable properties, for example, it can have an affinity for binding to CD117 protein equal to or higher than that of the parent aptamer sequence before modification, or has higher stability although the affinity is not significantly improved.

[0027] The nucleic acid aptamer derivatives of the present invention are obtained by transforming the nucleotide sequence of the nucleic acid aptamer into a phosphorothioate backbone that binds to the CD117 protein, or are peptide nucleic acids that bind to the CD117 protein that are transformed from the nucleic acid aptamer or the conjugate of the nucleic acid aptamer in any of the above technical solutions. The condition is that the derivatives have a molecular structure, physicochemical properties and functions that are substantially the same or similar to the original nucleic acid aptamer, and all bind to the CD117 protein.

[0028] The term "phosphorothioate backbone" as used in the present invention has the meaning generally understood by those of ordinary skill in the art, and refers to that the non-bridging oxygen atoms of the phosphodiester backbone of RNA and DNA aptamers can be replaced by one or two sulfur atoms, resulting in a phosphorothioate backbone with phosphorothioate or phosphorodithioate bonds, respectively. Such phosphorothioate backbones are known to have increased binding affinity to their targets, as well as enhanced resistance to nuclease degradation.

[0029] The term "peptide nucleic acid" used in the present invention has the meaning generally understood by those of ordinary skill in the art, and refers to an artificially synthesized DNA molecule analog, first reported by Nielsen et al. in 1991. Oligonucleotide mimics linked by peptide bonds were synthesized using N-2-(aminoethyl)-glycine units instead of sugar-phosphate backbones as repeating structural units, and are called peptide nucleic acids. Since peptide nucleic acids (PNA) do not have phosphate groups like those on DNA or RNA, there is a lack of electrical repulsion between PNA and DNA, making the binding strength between the two greater than the binding strength between DNA.

[0030] The present invention also provides a derivative of a nucleic acid aptamer, wherein the derivative of the nucleic acid aptamer comprises a phosphorothioate skeleton or a peptide nucleic acid that binds to a CD117 protein and is formed by modifying the nucleotide sequence skeleton of the nucleic acid aptamer or a conjugate of the nucleic acid aptamer.

[0031] The present invention also provides a use of a nucleic acid aptamer and a nucleic acid aptamer derivative, specifically using the above-mentioned nucleic acid aptamer and nucleic acid aptamer derivative, and the use thereof includes at least one of the following:

[0032] (1) Quantitative or qualitative detection of CD117 protein;

[0033] (2) purifying CD117 protein;

[0034] (3) Imaging of CD117 protein;

[0035] (4) preparing drugs targeting CD117 protein;

[0036] (5) Preparing reagents or drugs for diagnosing and treating abnormal CD117 expression.

[0037] The present invention also provides a CD117 protein detection kit prepared by using the nucleic acid aptamer.

[0038] Furthermore, the kit includes the nucleic acid aptamer, or a conjugate or derivative of the nucleic acid aptamer.

[0039] In summary, the present invention provides a method having the following technical effects:

[0040] 1. SEQ ID NO.1 and SEQ ID NO.2 are designed to have stable secondary structures, which can maintain their conformation under various physiological conditions, ensuring high affinity and specific binding with CD117 protein. This stability enables nucleic acid aptamers to maintain their functions in complex biological environments.

[0041] 2. SEQ ID NO.1 and SEQ ID NO.2 are relatively short and simple in structure, which facilitates rapid and efficient preparation by chemical synthesis, which not only reduces production costs but also improves the feasibility of mass production.

[0042] 3. SEQ ID NO.1 and SEQ ID NO.2 can be modified by various chemical modification methods, such as 2'-O-methyl ribonucleotides, locked nucleic acids (LNA), etc., to enhance their stability and anti-degradation ability. In addition, these modifications will not affect the binding properties of nucleic acid aptamers to CD117 protein.

[0043] 4. The optimized nucleic acid aptamers show good chemical stability during storage and transportation and can be stored for a long time at room temperature, reducing the need for special storage conditions.

[0044] 5. SEQ ID NO.1 and SEQ ID NO.2 can be easily achieved by fluorescent labeling, radioactive labeling or other labeling methods, which are helpful to provide visual signals in detection and imaging applications. ;

[0045] 6. SEQ ID NO.1 and SEQ ID NO.2 can be used to develop highly sensitive and specific detection methods, such as ELISA, immunoprecipitation and flow cytometry, etc. These methods can accurately detect CD117 protein in samples and are suitable for clinical diagnosis and scientific research analysis.

[0046] 7. Due to their high specific binding ability, SEQ ID NO.1 and SEQ ID NO.2 can be used for affinity chromatography to efficiently purify CD117 protein from complex mixtures, which is of great significance for protein research and drug development.

[0047] 8. SEQ ID NO.1 and SEQ ID NO.2 can be used as targeting ligands and coupled with drug molecules to prepare drugs targeting CD117 protein. Such drugs can specifically act on cells expressing CD117 protein, improve therapeutic effects and reduce side effects.

[0048] 9. SEQ ID NO.1 and SEQ ID NO.2 can be used to develop diagnostic kits to detect the expression level of CD117 protein and help diagnose related diseases at an early stage. In addition, they can also be used to prepare drugs for treating abnormal CD117 expression, such as targeted therapy in cancer treatment.

[0049] 10. Through fluorescent labeling or radioactive labeling, SEQ ID NO.1 and SEQ ID NO.2 can be used for in vivo imaging to monitor the distribution and dynamic changes of CD117 protein in vivo, providing important information for disease progression and treatment effect evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The molecular retention rate of the positive and negative screening elutions obtained by qPCR (real-time fluorescence quantitative PCR) screening for 7 rounds in Example 1;

[0051] Figure 2 This is a schematic diagram of the binding ability of the single-chain library obtained by the 1st, 4th and 7th rounds of screening, the initial Lib13 random single-chain library, the control His small peptide and the target protein CD117 detected by flow cytometry in Example 1;

[0052] Figure 3 This is a data diagram of affinity detection of nucleic acid aptamer DJ4a and CD117 protein using SPR (surface plasmon resonance) in Example 2;

[0053] Figure 4 This is a data diagram of the affinity detection of nucleic acid aptamer DH09 and CD117 protein using SPR (surface plasmon resonance) in Example 2;

[0054] Figure 5 Figure 3 is a graph showing the affinity detection data of the nucleic acid aptamer DJ4a and five other proteins in Example 3;

[0055] Figure 6 Figure 3 is a graph showing the affinity detection data of nucleic acid aptamer DH09 and five other proteins in Example 3;

[0056] Figure 7 It is a schematic diagram of the results of the dot hybridization experiment based on the nucleic acid aptamer DJ4a and different concentrations of CD117 protein in Example 5;

[0057] Figure 8 This is a schematic diagram of the results of the dot hybridization assay based on nucleic acid aptamer DJ4a and the CD117 protein and control protein in Example 4;

[0058] Fig. 9 Schematic diagram of the results of the dot hybridization experiment based on nucleic acid aptamer DH09 and different concentrations of CD117 protein in Example 5.

[0059] Fig.10 This is a schematic diagram of the results of the dot hybridization assay based on nucleic acid aptamer DH09 and the detection of CD117 protein and control protein in Example 4. DETAILED DESCRIPTION

[0060] For better understanding and implementation, the technical solutions in the embodiments of the present invention will be clearly and completely described and discussed below in conjunction with the accompanying drawings of the present invention. Obviously, what is described here is only a part of the examples of the present invention, not all the examples. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0061] In order to facilitate the understanding of the embodiments of the present invention, the following will be further explained by taking specific embodiments as examples in conjunction with the drawings, and each embodiment does not constitute a limitation on the embodiments of the present invention.

[0062] In the description of the present invention, it should be noted that the terms "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, they cannot be understood as limiting the present invention.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0064] Example 1: Screening of ssDNA nucleic acid aptamers binding to CD117 protein

[0065] The method for screening a ssDNA nucleic acid aptamer that binds to the CD117 protein in this embodiment comprises the following steps:

[0066] 1. Synthesize the random single-stranded DNA library and primers shown in the following sequence:

[0067] Random single-stranded DNA library:

[0068] 5'-TTCAGCACTCCACGCATAGC(36N)CCTATGCGTGCTACCGTGAA-3'(SEQ ID NO.3);

[0069] Among them, "36N" represents a sequence formed by connecting 36 arbitrary nucleotide bases. The library was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0070] Primer information is shown in Table 1 and was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0071] Table 1. Primers and their sequences

[0072]

[0073] Among them, S in the primer name represents the forward primer, and A in the primer name represents the reverse primer.

[0074] The primers were prepared into 100 μM stock solution with ddH2O and stored at -20°C for future use.

[0075] 2. Magnetic bead screening

[0076] The magnetic bead method was used for screening, with a total of 7 rounds of screening. The screening conditions for each round are shown in Table 2.

[0077] Table 2. Screening conditions for each round of CD117 protein

[0078]

[0079]

[0080] DPBS buffer: potassium chloride 0.2 g / L, potassium dihydrogen phosphate 0.2 g / L, sodium chloride 8 g / L, disodium hydrogen phosphate dodecahydrate 2.8975 g / L; pH 7.4, stored at 25°C.

[0081] DPBS-T buffer: DPBS buffer contains 5 mM Mg2+ and 0.02% Tween20.

[0082] The specific screening process is as follows:

[0083] 1) Carboxyl magnetic beads coupled to CD117 protein (positive screening magnetic beads)

[0084] Take 45 μL of CD117 protein (purchased from ABclonal, Uniport ID: P10721, concentration of 1 mg / mL), add 555 μL of 10 mM sodium acetate at pH 4.5 and mix well to obtain the diluted CD117 protein, and place on ice for later use.

[0085] Take 300 μL of carboxyl magnetic beads (Jiangsu Zecheng Biotechnology Co., Ltd., catalog number: FM2221), wash once with 300 μL of 20mM NaOH, then wash twice with 300 μL of ultrapure water, fish the magnetic beads with a magnet, and remove the supernatant. Take 300 μL of the prepared NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution), mix them in equal volumes, add them to the magnetic beads, and incubate at 25°C for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads. After the incubation, remove the supernatant and immediately wash once quickly with 300 μL of ultrapure water to reduce protein self-coupling. Add the above-mentioned sodium acetate diluted protein and incubate it on a vertical mixer at 25°C for 60 minutes. The CD117 protein is coupled to the surface of the magnetic beads through the amino groups on the surface of the protein.

[0086] After the coupling is completed, place the coupling tube on the magnetic rack, discard the supernatant, take 300μL 1M ethanolamine pH8.5 and add it to the magnetic beads, incubate on a 25℃ vertical mixer for 10min to block the unreacted activation sites on the surface of the magnetic beads. Place it on the magnetic rack and discard the blocking solution. Wash the magnetic beads 4 times with 300μL of DPBS-T buffer and resuspend it, which is the magnetic beads coupled with CD117 protein, marked as MB-CD117, and stored at 4℃.

[0087] 2) Carboxyl magnetic beads coupled to His peptide (counter-screening magnetic beads)

[0088] Same as step 1), CD117 protein was replaced with 3 μL His peptide (concentration 20 mg / mL), the volume of sodium acetate buffer was changed to 597 μL, the volume of coupled carboxyl magnetic beads was changed to 500 μL, and the rest of the steps were the same. The magnetic beads coupled with His peptide were labeled MB-His. His peptide is 10 consecutive histidines and was synthesized by GenScript Biotech Co., Ltd.

[0089] 3) Library dissolution and denaturation treatment

[0090] Take the Lib13 random single-stranded nucleotide library (1OD) synthesized by Bio-Industry, centrifuge at 12000rpm for 10min, centrifuge the library to the bottom of the tube, dissolve it to 7μM with DPBS buffer, mix it and dispense it into PCR tubes for denaturation treatment. The treatment process is as follows: set the PCR instrument program to 95℃ for 10min. The purpose of this step is to unwind the folded chain, then keep it at 4℃ for 5min, and then balance it to room temperature. Then add Mg2+ (final concentration of 5mM), BSA (final concentration of 0.5mg / mL), and HSDNA (final concentration of 0.1mg / mL) to the library and mix it to provide the ionic environment required for single-stranded DNA, reduce nonspecific binding and increase competitiveness. This is the library after denaturation treatment.

[0091] 4) Positive screening

[0092] Take 50 μL MB-CD117, magnetically remove the supernatant, wash twice with 200 μL DPBS-T buffer and remove the supernatant, add 200 μL of Lib13 library treated in step 3) and mix with it, and incubate on a vertical mixer at 25°C for 40 minutes. Place on a magnetic stand, remove the supernatant, retain the magnetic beads, and wash the magnetic beads 4 times with 200 μL DPBS buffer. Finally, add 100 μL ultrapure water to the washed magnetic beads, boil in a boiling water bath for 10 minutes, collect the supernatant, and mark it as elution-CD117.

[0093] 5) Reverse screening

[0094] The second round of screening added a counter-screening step. Take 50μL MB-His in an EP tube, remove the supernatant by magnetic suction, wash twice with 200μL DPBS-T buffer to remove the supernatant, add 100μL of the secondary ssDNA library obtained in the first round of screening, mix well and incubate on a vertical mixer at room temperature for 40 minutes. At the end of incubation, collect the supernatant as the ssDNA library used for the second round of positive screening. Use this method to adjust the screening pressure for 2 to 7 rounds. After each round of counter-screening, wash the remaining magnetic beads with 200μL DPBS buffer 4 times, and then add 100μL ultrapure water, boil in a boiling water bath for 10 minutes, collect the supernatant (i.e., the counter-screening supernatant), and mark it as elution-His.

[0095] 6) Preparation of secondary library

[0096] The nucleic acid molecule in step 4) elution-CD117 was used as a template and amplified by ordinary PCR. The method is as follows: 90 μL of template elution-CD117 was added to 900 μL PCR mix and mixed, 10 μL of ultrapure water was added, and the template and PCR mix mixture was divided into 100 μL / tube and added to the PCR tube. The amplification conditions are as follows: 95℃ pre-denaturation for 2 minutes, 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 30 seconds, a total of 25 cycles, and stored at 4℃. The PCR raw materials in the PCR mix are prepared by dNTPs (P031-02) purchased from Novozymes and rtaq enzyme (R500Z) purchased from Takara Biotech.

[0097] The amplified product was purified using commercially available Tiandirenhe SA magnetic beads (SM017010) to prepare a secondary library for the next round of screening. Prepare 80 μL SA magnetic beads, wash twice with 500 μL DPBS-T buffer, discard the supernatant by magnetic suction, add 1000 μL of the PCR product in step 5), and then add 1 / 5 volume of 4M sodium chloride aqueous solution of the PCR product, incubate on a shaker at room temperature for 30 minutes, and discard the supernatant by magnetic suction. Wash the magnetic beads three times with DPBS buffer-T, remove the supernatant, add 100 μL of 40mM sodium hydroxide aqueous solution, incubate at room temperature for 3 minutes, and then remove the magnetic beads by magnetic suction. Then add 4 μL of 1M hydrochloric acid to the supernatant to neutralize the single strand, and then add 104 μL of 2×DPBS buffer to dilute and neutralize the salt concentration, and finally obtain 208 μL of the secondary library dissolved in 1×DPBS buffer, which can be used as the library for the next round of screening.

[0098] The magnetic bead method was repeated for 7 rounds of screening, each operation using the secondary library obtained in the previous operation as the starting nucleic acid library, and the operation was performed according to steps 3), 4), 5) and 6).

[0099] 7) Library affinity detection

[0100] The library enrichment was determined based on the qPCR Cq value of elution during each round of positive and negative screening. If the amount of template eluted from each round of positive screening increased (Cq advanced) with the increase in screening rounds and both the amplification curve and the melting curve reached a stable state, the screening was stopped. Figure 1 The retention rate of each round of molecules was calculated based on the elution Cq values ​​of the positive and negative screens. The retention rate of the positive screen gradually increased, indicating that the library was effectively enriched. After the screening, the recognition ability of the DNA single-stranded library with MB-CD117 and MB-His was detected by flow cytometry. The specific operation is as follows:

[0101] The single-stranded libraries obtained in the 1st, 4th, and 7th rounds of screening and the initial Lib13 random single-stranded nucleotide library were selected for denaturation treatment: 40 μL of 400 nM single-stranded library was taken, Mg2+ (final concentration of 5 mM) and BSA (final concentration of 0.5 mg / mL) were added and mixed, and 2 μL of MB-CD117 was added respectively, and incubated in the dark at 25°C on a vertical mixer for 1 hour. Place on a magnetic stand, discard the supernatant, retain the magnetic beads, wash 4 times with 500 μL DPBS-T buffer, and resuspend with it, and mark them as CD117+Pool1, CD117+Pool4, CD117+Pool7, and CD117+Random.

[0102] Under the same conditions, step 1) MB-CD117 was replaced with step 2) MB-His as a control, and the products were labeled CD117-Pool1, CD117-Pool4, CD117-Pool7, and CD117-Random.

[0103] Prepare 2 μL of carboxyl magnetic beads, discard the supernatant by magnetic aspiration, add 600 μL of DPBS-T buffer and resuspend as a blank control, marked as NC.

[0104] The above 9 samples were tested by flow cytometry. The flow cytometry images are shown in Figure 2 shown.

[0105] Figure 2 The flow fluorescence displacement of the single-stranded library and the positive screening target in each round also showed an increasing trend and had no binding with the initial random library, indicating that the nucleic acid molecules in the library that can bind to the positive screening target are effectively enriched. With the increase of screening rounds, the library's recognition ability for the target gradually increases, that is, the affinity gradually increases. CD117+Pool7 is much higher than CD117+Pool1 and CD117+Pool4, meeting the sequencing requirements, and the resulting library is analyzed by high-throughput sequencing. At the same time, the flow fluorescence displacement of the library and the control reverse screening target did not change, indicating that the library has no affinity for the reverse screening target, thereby confirming the effectiveness of the reverse screening step and ensuring that the screened nucleic acid aptamer has good specificity.

[0106] 3. Analysis and identification of nucleic acid aptamers obtained after screening: After high-throughput sequencing analysis of the obtained enriched library products, several sequences were selected and synthesized by Genewise Biotechnology (Jiangsu) Technology Co., Ltd., and the affinity was tested.

[0107] In subsequent tests, two sequences with the strongest binding ability were identified from the sequences obtained in the final 7th round. They were nucleic acid aptamers with nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, and they were named DJ4a and DH09, respectively.

[0108] Example 2: Surface Plasmon Resonance (SPR) Detection of the Affinity of CD117 Protein Aptamer and CD117 Protein

[0109] Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize nucleic acid aptamers DJ4a (SEQ ID NO.1) and DH09 (SEQ ID NO.2), which were diluted with DPBS buffer (containing 5 mM Mg2+) to 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, and 15.625 nM, respectively.

[0110] 1. Couple the CD117 protein to the second channel on the surface of the CM5 chip. The specific method is as follows: first clean the chip with 50mM NaOH, inject 20μL, flow rate 10μL / min, then mix the two reagents of equal volume of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and inject 105μL to activate the chip, flow rate 10μL / min. Dilute the CD117 protein with 10mM sodium acetate at pH 4.5 to a final concentration of 50μg / mL and inject it, the injection volume is 190μL, the flow rate is 10μL / min. After the injection is completed, inject ethanolamine to block the chip, flow rate 10μL / min, and inject 129μL. The first channel is treated as above, the steps of coupling His small peptide, activation and blocking steps are exactly the same, as the control channel.

[0111] 2. Detection: A surface plasmon resonance instrument (GE Healthcare, model: Biacore 8K) was used to set the detection parameters. The diluted 2 nucleic acid aptamer samples flowed through channels 1 and 2 in turn. The program for each nucleic acid aptamer was as follows: injection 30 μL / min, time 3 min, dissociation 30 μL / min, time 3 min, regeneration 2 M NaCl 30 μL / min, time 45 s.

[0112] The affinity test data of nucleic acid aptamers DJ4a, DH09 and CD117 protein are shown in Figure 3 , Figure 4 ,in Figure 3 This is the affinity test diagram of DJ4a and CD117 protein; Figure 4 Figure 3 is the affinity test diagram of DH09 and CD117 protein. The affinity KD values ​​of CD117 protein aptamer DJ4a, DH09 and CD117 protein are shown in Table 3. Figure 3 and Figure 4 These data indicate that both nucleic acid aptamers were detected to have strong binding with CD117 protein using SPR instrument.

[0113] Table 3. Affinity of nucleic acid aptamers to CD117 protein

[0114]

[0115]

[0116] Example 3: Surface Plasmon Resonance (SPR) Detection of the Specificity of CD117 Protein Aptamer and CD117 Protein

[0117] TF protein (Uniport ID: P13726, purchased from Nearshore Biotechnology Co., Ltd.); TROP2 protein (Uniport ID: P09758, purchased from Nearshore Biotechnology Co., Ltd.); FOLR1 protein (Uniport ID: P15328, purchased from Nearshore Biotechnology Co., Ltd.); Nectin-4 protein (Uniport ID: Q96NY8, purchased from Nearshore Biotechnology Co., Ltd.); FAP protein (Uniport ID: Q12884, purchased from Nearshore Biotechnology Co., Ltd.).

[0118] In this embodiment, TF protein, TROP2 protein, FOLR1 protein, Nectin-4 protein, and FAP protein are used to replace CD117 protein, respectively. The method of fixing CD117 protein to the SPR chip for testing is the same as that in Example 2. TF protein, TROP2 protein, FOLR1 protein, Nectin-4 protein, and FAP protein are coupled to the second channel of the five channels on the surface of the CM5 chip, and His protein is coupled to the first channel. DJ4a and DH09 nucleic acid aptamers diluted to 500 nM are injected in sequence.

[0119] The affinity test data of nucleic acid aptamer DJ4a and the above five proteins are shown in Figure 5 ; The affinity test data of nucleic acid aptamer DH09 and the above five proteins are shown in Figure 6 .Depend on Figures 5-6 It can be seen that the nucleic acid aptamers DJ4a and DH09 cannot bind to the five proteins TF, TROP2, FOLR1, Nectin-4, and FAP. This shows that the CD117 nucleic acid aptamer obtained through screening has very high specificity in binding to the target.

[0120] Example 4: Detection of CD117 protein by dot blot hybridization based on nucleic acid aptamers

[0121] Suzhou Genewise Biotech Co., Ltd. was commissioned to synthesize biotin-modified nucleic acid aptamers DJ4a and DH09.

[0122] Dot hybridization experiments were performed on biotin-modified aptamers DJ4a and DH09, respectively, and the steps were as follows:

[0123] 1. Dissolve CD117 protein in DPBS buffer to 0.500mg / mL, 0.250mg / mL, 0.125mg / mL, 0.063mg / mL, 0.032mg / mL, and 0.016mg / mL as test samples; dissolve the control proteins His small peptide, TROP2, FOLR1, Nectin-4, and FAP in DPBS buffer to 0.500mg / mL as control samples. Take 1 8cm×2cm test strip (nitrocellulose membrane, 0.2μm, GE Amersham, catalog number 10600001), spot 2μL of the test sample and control sample on the nitrocellulose membrane, and air dry for 40 minutes.

[0124] 2. Then put the test strip into a 5mL centrifuge tube, add 3mL of 10% BSA to soak it, and block it on a shaker at room temperature for 1 hour. After blocking, wash it with DPBS-T for 5 minutes, repeat the washing 3 times, and aspirate it clean.

[0125] 3. Transfer the test strip to a new 5 mL centrifuge tube, add 3 mL of DPBS buffer (containing 5 mM Mg2+) dissolved in biotin-modified nucleic acid aptamer DJ4a (500 nM) to soak it, incubate it on a shaker at room temperature for 30 min, wash it with DPBS-T for 2 min after the incubation, repeat the washing 3 times, and aspirate it clean.

[0126] 4. Transfer the test strip to a new 5 mL centrifuge tube, dilute HRP-Streptavidin (purchased from Beyotime Biotech 1 mg / ml): DPBS buffer at a ratio of 1:2000, take 3 mL and add it to soak it, incubate it on a shaker at room temperature for 30 minutes. After the incubation, wash it with DPBS-T for 2 minutes, repeat the washing 3 times, and aspirate it clean.

[0127] 5. Transfer the test strip to a clean PE glove, take the ECL color development kit (purchased from Beyotime Biotech), mix 100 μL of solution A and 100 μL of solution B, soak the surface of the test strip, and incubate in the dark for 5 minutes.

[0128] 6. Imaging system observation and photography: The instrument used was the ImageQuantTM LAS 4000 digital imaging system of GE Healthcare Life Sciences. The results are shown in Figure 7 , 8 .

[0129] 7. Under the same conditions, 500nM biotin-modified aptamer DJ4a was replaced with 500nM biotin-modified aptamer DH09. The results are shown in Fig. 9 , 10 .

[0130] The results of dot hybridization experiments of biotin-modified aptamer DJ4a and different concentrations of CD117 protein are shown in Figure 7 As shown; the results of the dot hybridization experiment of biotin-modified nucleic acid aptamer DJ4a with CD117 protein and control protein are shown Figure 8 As shown; the results of the dot hybridization experiment of the biotin-modified nucleic acid aptamer DH09 and different concentrations of CD117 protein are shown Fig. 9 As shown; the results of the spot hybridization experiment of biotin-modified nucleic acid aptamer DH09 with CD117 protein and control protein are shown in Fig.10 As shown;

[0131] Depend on Figure 7 and Fig. 9 It can be proved that both nucleic acid aptamers can be used to accurately detect 0.125mg / mL CD117 protein. Figure 8 and Fig.10 It can be seen that compared with the spots of the control proteins His small peptide, TROP2, FOLR1, Nectin-4, and FAP, the CD117 protein is obvious. This shows that the biotin-modified nucleic acid aptamers DJ4a and DH09 can be used for the detection of membrane hybridization CD117 protein, and the control proteins His small peptide, TROP2, FOLR1, Nectin-4, and FAP are almost colorless, which further proves that DJ4a and DH09 can specifically bind to the CD117 protein.

[0132] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above-mentioned technical features. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A nucleic acid aptamer that binds to CD117 protein, characterized in that: The nucleic acid aptamer has a nucleotide sequence that is at least 60% homologous to any one of SEQ ID NO. 1 and SEQ ID NO. 2 and binds to the CD117 protein.

2. A nucleic acid aptamer binding to CD117 protein according to claim 1, characterized in that: The nucleic acid aptamer has an RNA sequence transcribed from the nucleotide sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.

2.

3. A nucleic acid aptamer binding to CD117 protein according to claim 2, characterized in that: The nucleic acid aptamer has a nucleotide sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.

2.

4. A nucleic acid aptamer binding to CD117 protein according to claim 3, characterized in that: The nucleotide sequence of the nucleic acid aptamer is modified, and the modification method includes one or more of phosphorylation, methylation, amination, sulfhydrylation, replacement of oxygen with sulfur, replacement of oxygen with selenium, and isotopization.

5. The nucleic acid aptamer binding to CD117 protein according to claim 4, characterized in that: The nucleic acid aptamer has a sequence obtained by modifying a nucleotide sequence that has at least 60% homology with any one of SEQ ID NO.1 and SEQ ID NO.2 and binds to the CD117 protein.

6. The nucleic acid aptamer binding to CD117 protein according to claim 2, characterized in that: The transformation includes at least one of the following transformation methods: (1) Connecting a fluorescent marker to the aptamer; (2) Connecting radioactive substances to the aptamer; (3) Connecting therapeutic substances to the aptamer; (4) connecting biotin to the nucleic acid aptamer; (5) connecting digoxigenin to the aptamer; (6) connecting a nanoluminescent material to the nucleic acid aptamer; (7) Connecting a small peptide to the nucleic acid aptamer; (8) Connecting siRNA to the nucleic acid aptamer.

7. A derivative of a nucleic acid aptamer, characterized in that: The derivatives of the nucleic acid aptamer include a phosphorothioate backbone or a peptide nucleic acid that binds to the CD117 protein and is formed by modifying the nucleotide sequence backbone of the nucleic acid aptamer or the conjugate of the nucleic acid aptamer.

8. Use of the nucleic acid aptamer according to any one of claims 1 to 6 and the nucleic acid aptamer derivative according to claim 7, characterized in that: Include at least one of the following uses: (1) Quantitative or qualitative detection of CD117 protein; (2) Purification of CD117 protein; (3) Imaging of CD117 protein; (4) preparing drugs targeting CD117 protein; (5) Preparing reagents or drugs for diagnosing and treating abnormal CD117 expression.

9. A CD117 protein detection kit prepared using the nucleic acid aptamer according to any one of claims 1 to 6.

10. A CD117 protein detection kit according to claim 9, characterized in that: The kit includes the nucleic acid aptamer, or a conjugate or derivative of the nucleic acid aptamer.

Images