Nucleic acid aptamer for specifically recognizing monocyte chemotactic protein-1 as well as modification and application of nucleic acid aptamer

Abstract

The invention belongs to the technical field of biological medicines, and discloses a nucleic acid aptamer capable of specifically recognizing monocyte chemotactic protein-1 and application of the nucleic acid aptamer. The nucleotide sequence of the nucleic acid aptamer comprises any one of the sequences shown as SEQ ID No.3 and / or SEQ ID No.4. The SELEX technology is adopted for screening to obtain the high-affinity nucleic acid aptamer capable of specifically recognizing MCP-1, and the performance of the nucleic acid aptamer is further improved through combined application of optimization strategies. The aptamer disclosed by the invention has the characteristics of high affinity, strong specificity, good stability, low immunogenicity, low preparation cost, easiness in modification and marking and the like, and can be used for capturing MCP-1 in a system, detecting MCP-1 in vivo and in vitro, developing MCP-1 therapeutic drugs in related diseases, constructing a targeted drug delivery system and the like. The nucleic acid aptamer disclosed by the invention has huge potential in practical application.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a nucleic acid aptamer that specifically recognizes monocyte chemoattractant protein-1, and the modification and application thereof. Background Art

[0002] Monocyte Chemoattractant Protein-1 (MCP-1), also known as Monocyte Chemotactic and Activating Factor (MCAF), is a secreted protein encoded by the human gene CCL2 (C-CMotif Chemokine Ligand 2) and belongs to the CC chemokine subfamily (β-chemokines). MCP-1 regulates the migration and activation of monocytes, macrophages, and memory T cells by specifically binding to the CCR2 receptor, acting as the "start switch" of the inflammatory cascade. In ophthalmology, abnormal expression of MCP-1 has been shown to be directly associated with a variety of blinding eye diseases, including age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, uveitis, and cataracts in high myopia. For example, in dry AMD, MCP-1 drives macrophage infiltration into the sub-Bruch's membrane deposition area, promoting complement activation and geographic atrophy; while in wet AMD, it accelerates the formation of choroidal neovascularization (CNV) by synergizing with VEGF. In addition, in the intraocular fluid of patients with AMD, DR, glaucoma, uveitis, and high myopia cataract, the concentration level of MCP-1 has a significant dose-dependent relationship with the occurrence and development of eye diseases. Therefore, the development of ligand molecules that specifically recognize MCP-1 not only lays an important foundation for the preparation of MCP-1 diagnostic reagents, the development of neutralizing drugs, and the construction of targeted drug delivery systems in clinical diseases, but also provides efficient molecular tools for the capture, enrichment, and purification of MCP-1 in basic research.

[0003] Aptamers, as a new type of biorecognition molecule, can specifically recognize and bind to their targets with high affinity. Aptamers are typically obtained from synthetic random nucleic acid libraries using the in vitro screening method Systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have broad application prospects in multiple fields of analytical diagnosis and medical research due to their chemical synthesis, ease of labeling and modification, broad target recognition, freedom from immunogenicity and immune constraints, high binding affinity, and strong targeting specificity. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the present invention aims to provide a high-affinity nucleic acid aptamer that specifically binds to MCP-1. The present invention first screens the initial sequences of the nucleic acid aptamer, Seq2 and Seq39, for specific binding to MCP-1 using magnetic bead SELEX technology. Then, through truncation optimization, the core sequences, Seq2T and Seq39T, of the nucleic acid aptamer that specifically recognize MCP-1 are obtained. Then, through directed coupling, a dimer of the nucleic acid aptamer is obtained that has extremely high binding affinity for MCP-1.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides, in a first aspect, a nucleic acid aptamer that specifically recognizes MCP-1, wherein the nucleic acid aptamer comprises at least one of 1) to 5):

[0006] 1) A single-stranded DNA having a nucleotide sequence including the sequence shown in SEQ ID No. 3 and / or SEQ ID No. 7;

[0007] 2) a single-stranded DNA having at least 75% identity with the nucleotide sequence defined in 1) and specifically recognizing monocyte chemoattractant protein-1;

[0008] 3) deleting or adding one or more nucleotides from the nucleotide sequence defined in 1) and specifically recognizing single-stranded DNA of monocyte chemoattractant protein-1;

[0009] 4) a single-stranded RNA transcribed from the nucleotide sequence defined in any one of 1) to 3) and specifically recognizing monocyte chemoattractant protein-1;

[0010] 5) Any single-stranded DNA or single-stranded RNA that hybridizes with the nucleotide sequence specified in 1) or 2) or 3) under stringent conditions and specifically recognizes monocyte chemoattractant protein-1.

[0011] The second aspect of the present invention provides a use of a nucleic acid aptamer that specifically recognizes monocyte chemoattractant protein-1 in at least one of the following B1) to B6):

[0012] B1) Use in the preparation of a product for binding monocyte chemoattractant protein-1;

[0013] B2) Use in the preparation of a product for detecting monocyte chemoattractant protein-1;

[0014] B3) Use in the preparation of a product targeting monocyte chemoattractant protein-1;

[0015] B4) Use in the preparation of inhibitors of monocyte chemoattractant protein-1;

[0016] B5) Use in the preparation of screening, diagnosis or auxiliary diagnosis of monocyte chemoattractant protein-1 related diseases;

[0017] B6) Use in the preparation of a drug or product for preventing, improving or treating monocyte chemoattractant protein-1 related diseases.

[0018] The third aspect of the present invention provides a product for binding or detecting monocyte chemoattractant protein-1, wherein the product comprises the above-mentioned nucleic acid aptamer.

[0019] Preferably, the product is selected from the group consisting of a reagent, a kit, a membrane strip, a sensor and a chip.

[0020] The fourth aspect of the present invention provides a drug for preventing, improving or treating monocyte chemoattractant protein-1 related diseases, wherein the drug contains the above-mentioned nucleic acid aptamer.

[0021] A fifth aspect of the present invention provides a drug delivery system specifically targeting monocyte chemoattractant protein-1, wherein the drug delivery system comprises the above-mentioned nucleic acid aptamer.

[0022] A sixth aspect of the present invention provides a nucleic acid aptamer derivative, wherein the nucleic acid aptamer derivative is obtained by labeling the nucleic acid aptamer with a labeling substance.

[0023] A seventh aspect of the present invention provides a method for detecting monocyte chemoattractant protein-1, which comprises labeling a reporter group on the nucleic acid aptamer as described above, allowing the nucleic acid aptamer labeled with the reporter group to interact with monocyte chemoattractant protein-1, and detecting monocyte chemoattractant protein-1 by detecting the signal of the reporter group.

[0024] The present invention provides a nucleic acid aptamer that specifically recognizes monocyte chemoattractant protein-1, and its modification and application. Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention immobilizes the MCP-1 protein on the surface of magnetic beads and uses the magnetic bead SELEX technology to screen nucleic acid aptamers, obtaining a group of high-affinity nucleic acid aptamers that specifically bind to MCP-1. The performance of the nucleic acid aptamers is further improved by the combined application of optimization strategies such as truncation and dimerization.

[0026] 2. As molecular tools for targeting MCP-1, the nucleic acid aptamers of the present invention offer advantages such as high affinity, strong specificity, good stability, low immunogenicity, low preparation cost, and ease of modification and labeling. They can be used for capturing MCP-1 in systems, detecting MCP-1 in vivo and in vitro, developing therapeutic drugs for MCP-1 in ocular diseases, and constructing targeted drug delivery systems, thus possessing broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown is a schematic diagram of SELEX technology screening.

[0028] Figure 2 Displayed is a monitoring graph of ssDNA recovery.

[0029] Figure 3 Shown are the binding and dissociation curves of nucleic acid aptamers Seq2 and Seq39 with MCP-1.

[0030] Figure 4 Shown are the predicted secondary structures of nucleic acid aptamers Seq2 and Seq39.

[0031] Figure 5 Shown are the binding and dissociation curves of nucleic acid aptamers Seq2T and Seq39T with MCP-1.

[0032] Figure 6 Shown are the binding and dissociation curves of dimeric aptamers DA2TN2T, DA39TN39T, and DA2TN39T with MCP-1.

[0033] Figure 7 The figure shows the Transwell assay to evaluate the aptamer-targeted inhibition of MCP-1-mediated RF / 6A cell migration. DETAILED DESCRIPTION

[0034] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0035] In the present invention, magnetic bead SELEX technology is first used for screening to obtain initial sequences Seq2 and Seq39 of nucleic acid aptamers that specifically bind to MCP-1. Truncation optimization is then performed to obtain core sequences Seq2T and Seq39T of nucleic acid aptamers that target and recognize MCP-1. Directed coupling is then performed to obtain a dimer of nucleic acid aptamers with extremely high binding affinity to MCP-1. The nucleic acid aptamers of the present invention have the characteristics of high affinity, strong specificity, good stability, low immunogenicity, low preparation cost, and easy modification and labeling. They can specifically bind to MCP-1 protein and can be used for detecting MCP-1 protein and preparing products targeting MCP-1 protein, such as sensors, to screen, diagnose or assist in the diagnosis of MCP-1-related diseases. Furthermore, the nucleic acid aptamers of the present invention are also potential drugs for MCP-1 protein-related diseases and can be used to prepare reagents or drugs for clinical prevention, improvement or treatment.

[0036] The present invention provides a nucleic acid aptamer that specifically recognizes MCP-1, wherein the nucleic acid aptamer comprises at least one of 1) to 5):

[0037] 1) A single-stranded DNA having a nucleotide sequence including the sequence shown in SEQ ID No. 3 and / or SEQ ID No. 4;

[0038] 2) a single-stranded DNA having at least 75% identity with the nucleotide sequence defined in 1) and specifically recognizing monocyte chemoattractant protein-1;

[0039] 3) deleting or adding one or more nucleotides from the nucleotide sequence defined in 1) and specifically recognizing single-stranded DNA of monocyte chemoattractant protein-1;

[0040] 4) a single-stranded RNA transcribed from the nucleotide sequence defined in any one of 1) to 3) and specifically recognizing monocyte chemoattractant protein-1;

[0041] 5) Any single-stranded DNA or single-stranded RNA that hybridizes with the nucleotide sequence specified in 1) or 2) or 3) under stringent conditions and specifically recognizes monocyte chemoattractant protein-1.

[0042] In a specific embodiment, the nucleic acid aptamer is in a single copy or multi-copy form, and the multi-copy SEQ ID No. 3 or SEQ ID No. 4 are connected by a linker sequence. In the nucleic acid aptamer containing both SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 3 and SEQ ID No. 4 are also connected by a linker sequence. Further, the linker sequence is a flexible linker sequence. Preferably, the flexible linker sequence is a T repeat sequence. More preferably, the sequence of the nucleic acid aptamer is selected from any one of SEQ ID Nos. 1 to 7.

[0043] In a preferred embodiment, the nucleotide sequence in 2) is specifically obtained by substituting one or more bases (specifically, 1-10, 1-5, or 1-3) in the nucleotide sequence of SEQ ID No. 3 and / or SEQ ID No. 4. The nucleotide sequence in 2) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or greater homology with either SEQ ID No. 3 and / or SEQ ID No. 4.

[0044] In a preferred embodiment, the nucleotide sequence of the nucleic acid aptamer is modified, and the modified nucleic acid aptamer specifically recognizes monocyte chemoattractant protein-1, and the modification is selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, replacement of oxygen with sulfur, replacement of oxygen with selenium, and isotopization.

[0045] In a preferred embodiment, a signal molecule and / or an active molecule and / or a functional group and / or a radionuclide is linked to the nucleotide sequence of the nucleic acid aptamer, and the linked nucleic acid aptamer specifically recognizes monocyte chemoattractant protein-1.

[0046] To achieve the above-mentioned purpose, another aspect of the present invention provides use of a nucleic acid aptamer that specifically recognizes monocyte chemoattractant protein-1 in at least one of the following B1) to B6):

[0047] B1) Use in the preparation of a product for binding monocyte chemoattractant protein-1;

[0048] B2) Use in the preparation of a product for detecting monocyte chemoattractant protein-1;

[0049] B3) Use in the preparation of a product targeting monocyte chemoattractant protein-1;

[0050] B4) Use in the preparation of inhibitors of monocyte chemoattractant protein-1;

[0051] B5) Use in the preparation of screening, diagnosis or auxiliary diagnosis of monocyte chemoattractant protein-1 related diseases;

[0052] B6) Use in the preparation of a drug or product for preventing, improving or treating monocyte chemoattractant protein-1 related diseases.

[0053] According to the technical solution of the present invention, the monocyte chemoattractant protein-1-related disease is selected from eye-related diseases, and the eye-related diseases include age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, uveitis and high myopia cataract.

[0054] In order to achieve the above object, another aspect of the present invention provides a nucleic acid aptamer derivative, wherein the nucleic acid aptamer derivative is a substance obtained by labeling the nucleic acid aptamer with a labeling substance.

[0055] As a preferred embodiment, the label is selected from a signal molecule and / or a functional group. The label refers to any atom or molecule that is used to provide a detectable (preferably quantifiable) effect and can be attached to a nucleic acid. Labels include, but are not limited to, dyes; radiolabels, such as 32P; conjugated coupling groups, such as biotin; haptens, such as digoxigenin (DIG); chemiluminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight measurement, quantum dots, electrochemistry, X-ray diffraction or absorption, magnetism, immunoenzyme labeling reaction, filter paper-based immunoassay, affinity precipitation, affinity chromatography, enzyme activity, microscopic projection or scanning imaging, super-resolution imaging, cell tracking, in vivo nanoparticle tracking imaging in animals or humans, 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, microbiome, RNA (mRNA, lnRNA, snRNA), miRNA, etc. The label can be a charged moiety (positive or negative charge) or can be selected as needed and can be charge neutral. The label can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., direct sequence reading). The label can also be used for targeted drug delivery.

[0056] In some embodiments, the label is a fluorophore, a colorimetric label, a quantum dot, biotin, and other label molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cyclic olefins for click reactions, and initiator groups for polymer labeling), and can also be selected from polypeptides / protein molecules, LNA / PNA, non-natural amino acids and their analogs (such as peptoids), non-natural nucleic acids and their analogs (nucleotide mimics) and nanostructures (including inorganic nanoparticles, NV-centers, aggregation / assembly-induced emission molecules, rare earth ion ligand molecules, polymetallic oxygen clusters, etc.).

[0057] In some embodiments, the fluorophore is selected from fluorescein dyes, rhodamine dyes, and cyanine dyes.

[0058] Preferably, the fluorescein dye includes standard fluorescent groups and their derivatives, such as fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), Cy5, Cy3, Quasar 670, Alexa Fluor 488 / 647, etc.

[0059] Preferably, the rhodamine dyes include R101, tetraethyl rhodamine (RB200) and carboxytetramethylrhodamine (TAMRA).

[0060] Preferably, the cyanine dyes are mainly selected from two categories, one is thiazole orange (TO), oxazole orange (YO) series and dimer dyes thereof, and the other is polymethine series cyanine dyes.

[0061] In some embodiments, the fluorophore can also be selected from the following dyes: diphenylethylene, naphthalimide, coumarins, acridines, pyrenes, and the like.

[0062] The label is usually placed at the 5' end of the probe sequence, but can also be placed at the 3' end by changing the modification bond (such as -OH or -NH bond).

[0063] In one embodiment, the 5' end of the nucleic acid aptamer described in SEQ ID No. 1-7 is labeled with biotin. The nucleic acid aptamer derivative is then injected into the blood or incubated with relevant tissues and cells in vitro. The expression of monocyte chemoattractant protein-1 can then be recognized and imaged by a fluorescence recognition instrument.

[0064] Another aspect of the present invention provides a product for binding or detecting monocyte chemoattractant protein-1, wherein the product comprises the above-mentioned nucleic acid aptamer.

[0065] According to the technical solution of the present invention, the product is selected from the group consisting of reagents, test kits, membrane strips, sensors and chips.

[0066] Preferably, the kit further comprises one or more of Taq DNA polymerase, dNTP, PCR buffer, and Mg2+ required for PCR amplification.

[0067] Preferably, the various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0068] Another aspect of the present invention provides a drug for preventing, improving or treating monocyte chemoattractant protein-1 related diseases, wherein the drug contains the above-mentioned nucleic acid aptamer.

[0069] As a preferred embodiment, the drug further contains one or more pharmaceutically acceptable carriers.

[0070] Preferably, the pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant.

[0071] The present invention provides a drug-carrying system for specifically targeting monocyte chemoattractant protein-1 on the other hand, and the drug-carrying system includes the above-mentioned nucleic acid aptamer. By related technology (such as emulsification diffusion technology, etc.), drugs (such as MTX, etc.) are loaded into drug-carrying microparticles (such as PLGA, etc.), and nucleic acid aptamers are linked to drug-carrying microparticles by methods such as electrostatic coupling, a drug-carrying system with targeted drug delivery to cells (such as retinal pigment epithelial cells, endothelial cells, smooth muscle cells, monocytes / macrophages, fibroblasts, epithelial cells, glial cells, tumor cells, etc.) expressing monocyte chemoattractant protein-1 can be constructed. This targeted drug delivery system is based on the strong specific recognition effect of the above-mentioned nucleic acid aptamer on monocyte chemoattractant protein-1, realizing low toxicity and efficient targeted drug delivery.

[0072] As a preferred embodiment, the drug delivery system can be a liposome drug delivery system, a polymer micelle drug delivery system, a polymer disc drug delivery system or a nanoparticle drug delivery system.

[0073] As a preferred embodiment, the drug delivery system is used for targeted transport and / or point-specific release of drugs.

[0074] The present invention also provides a method for detecting monocyte chemoattractant protein-1, which comprises labeling a reporter group on the nucleic acid aptamer as described above, allowing the nucleic acid aptamer labeled with the reporter group to interact with monocyte chemoattractant protein-1, and detecting monocyte chemoattractant protein-1 by detecting the signal of the reporter group.

[0075] As a preferred embodiment, the reporter group may be biotin or a fluorescent group, and the fluorescent group is selected from Rhodamine, FAM, FITC, BODIPY, Cy3, Cy5, VIC, HEX, TRT, ROX, JOE, TAMRA but is not limited thereto.

[0076] As a more preferred embodiment, the reporter group is biotin.

[0077] In the present invention, the purpose of the above-mentioned uses or methods may be disease diagnosis, disease prognosis and / or disease treatment, and their purpose may also be non-disease diagnosis, non-disease prognosis and non-disease treatment; their direct purpose may be to obtain information on intermediate results of disease diagnosis results, disease prognosis results and / or disease treatment results, and their direct purpose may be non-disease diagnosis, non-disease prognosis and / or non-disease treatment.

[0078] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0079] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0080] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0081] In the following examples, experimental procedures, where specific conditions are not specified, generally followed conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0082] Example 1: Construction of an aptamer screening library and its primers

[0083] 1. Construction of an 80-nucleotide ssDNA library

[0084] Contains the initial oligonucleotide library sequence: 5′-AGCAGCACAGAGGTCAGATG-N 40-CCTATGCGTGCTACCGTGAA-3′; wherein N represents any one of the bases A, T, C, or G. 40 represents a random sequence of 40 nucleotides in length.

[0085] 2. Primer Construction

[0086] Upstream primer: 5′-AGCAGCACAGAGGTCAGATG-3′ (SEQ ID No. 8);

[0087] Downstream primer 1: 5′-TTCACGGTAGCACGCATAGG-3′ (SEQ ID No. 9);

[0088] Downstream primer 2: 5′-poly(dA20)-Spacer18-TTCACGGTAGCACGCATAGG-3′ (SEQ ID No. 10).

[0089] Example 2: Screening of MCP-1 nucleic acid aptamers

[0090] like Figure 1 As shown in the figure, to obtain high-affinity nucleic acid aptamers that specifically bind to MCP-1, MCP-1 (i.e., CCL2) protein was immobilized on the surface of magnetic beads (Thermo Fisher Scientific, Waltham, MA) using EDC / NHS chemical coupling, and a total of 12 rounds of screening were performed. To improve screening efficiency, blank magnetic beads were introduced for reverse screening starting from the fifth round, and free competitive targets, including BSA, HAS, GTX, ATP, TNF-α, VEGF165, PDGF-BB, GDF15, LCN 1, MMP-9, AZGP1, ANGPTL4, CTGF, CNTF, and BDNF, were gradually added to the forward system for co-incubation to further improve screening specificity.

[0091] The specific screening process is as follows: (1) Take 50 μL of the MCP-1 magnetic beads prepared above, rinse several times with screening buffer (PBS containing 1 mM MgCl2 and 1 mM CaCl2, pH 7.2), add blocking buffer (screening buffer containing 0.1 mg / ml yeast tRNA and 1 mg / mL BSA) and incubate for 30 minutes. (2) Dissolve the ssDNA library in screening buffer, incubate in a 95°C water bath for 10 minutes, quench in an ice bath for 5 minutes, and place at room temperature for 10 minutes. Then, add it to the blocked MCP-1 magnetic beads and incubate at room temperature with low speed rotation. (3) After the incubation is completed, rinse several times with screening buffer to remove unbound ssDNA, then add 100 μL of enzyme-free water, heat at 95°C for 10 minutes, and recover the ssDNA specifically bound to MCP-1. (4) The eluted ssDNA was used as a template for PCR amplification. The reaction system included: 10 μL of Hot start premix (5x); 2.5 μL of upstream primer and downstream primer 2 (10 μM); 5 μL of template; finally, sterile water was added to make up the system to 50 μL, for a total of 40 tubes. The amplification conditions were: 94°C, pre-denaturation for 1 min; 95°C, denaturation for 30 s; 60°C, annealing for 30 s; 72°C, extension for 30 s; and finally, 72°C, extension for another 2 min; for a total of 20 cycles. (5) Urea-denatured loading buffer was added to the amplified PCR library. The sample was incubated at 95°C for 10 min, cooled in an ice bath for 5 min, and placed at room temperature for 5 min. The sample was then loaded onto a 12% urea-denatured polyacrylamide gel well and electrophoresed at a constant voltage of 300 V. (6) After electrophoresis, add 20 mL of ddH2O and 5 μL of nucleic acid fluorescent dye to a clean dish, mix thoroughly, place the gel in it, and shake gently on a horizontal shaker. (7) After staining for 5-10 minutes, place the gel on a fluorescent imaging system, cut and recover the lower end of the ssDNA library into a 2 mL test tube, add 1.5 mL of ddH2O, boil the gel in boiling water for 30 minutes, and then centrifuge to recover the supernatant. (8) The ssDNA in the purified supernatant was recovered using a Qiagen® kit (Qiagen, Hilden, Germany) and redissolved in screening buffer for the next round of screening.

[0092] The above screening process was repeated until the 12th round, and the ssDNA recovery rate increased significantly ( Figure 2 ). Therefore, the screening was stopped, and the enriched library was subjected to high-throughput sequencing and multiple sequence alignment analysis, and finally the initial sequences of the nucleic acid aptamers Seq2 and Seq39 were obtained, and the sequences are shown in Table 1 SEQ ID No. 1-2.

[0093] Table 1 Aptamer sequences and their affinities

[0094]

[0095] Example 3: Determination of the intermolecular interaction between nucleic acid aptamers and MCP-1 using biomembrane interferometry

[0096] Biomembrane interferometry is a real-time molecular interaction analysis method. Its principle is that the instrument transmits white light onto the sensor surface. After passing through the biofilm layer of the sensor, the light is reflected. The frequency of the reflected light is affected by the thickness of the biofilm. Some frequencies of the reflected light constructively interfere with the incident light, while others interfere destructively. These interfering light waves are detected by a spectrometer, forming an interference spectrum, which is displayed as the relative shift intensity of the interference spectrum. Therefore, as the number of molecules bound to the sensor surface increases or decreases, the spectrometer detects a shift in the interference spectrum in real time. This shift directly reflects changes in the biofilm thickness on the sensor surface. Specifically, when the aptamer immobilized on the sensor biofilm interacts with MCP-1 in solution, the biofilm thickness changes, resulting in a relative shift. This relative shift increases and decreases with the amount of MCP-1 bound, eventually reaching an equilibrium state, and the corresponding binding and dissociation curves and affinity constants are generated in real time.

[0097] The specific implementation process is as follows: (a) After dissolving the biotin-labeled aptamer in the screening buffer, place it in a 95°C water bath for 10 minutes, quench it in an ice bath for 5 minutes, and leave it at room temperature for 10 minutes to promote the formation of a stable spatial structure; (b) 200 μL of screening buffer, aptamer, MCP-1 (PeproTech Inc., Rocky Hill, USA or Sino Biological Inc., Beijing, China) and screening buffer are added to a 96-well plate in sequence; (c) Streptavidin-coated sensors (Sartorius Trading (Shanghai) Co., Ltd.) are immersed in each reaction well in sequence according to the program set by the instrument, and the process goes through five steps: sensor equilibration, aptamer solidification, rinsing, MCP-1 binding and dissociation. The results are shown in Figure 2. Figure 3 A. Figure 3 As shown in Figure B, the binding affinity constants of the aptamers Seq2 and Seq39 for MCP-1 were 82.6 and 7.16 nM, respectively, demonstrating the high sensitivity of Seq2 and Seq39 in binding to the target protein. The specificity of the binding of Seq2 and Seq39 to MCP-1 was also examined. Compared to the target protein, Seq2 and Seq39 showed little binding to other proteins (HSA), demonstrating the specificity of the binding of Seq2 and Seq39 to the target protein.

[0098] Example 4: Optimization and modification of nucleic acid aptamers

[0099] In order to further improve the performance of nucleic acid aptamers, the present invention introduces two optimization strategies: sequence truncation and dimer construction, and applies them in combination. Figure 4 A. Figure 4 As shown in Figure B, based on QGRS and Mfold predictions, it was found that the nucleic acid aptamer Seq2 folded into a G-tetramer structure, while Seq39 had a double stem-loop structure. By truncating the redundant sequences at both ends of the nucleic acid aptamer, the core structure sequences Seq2T and Seq39T were generated, as shown in Table 1 SEQ ID No. 3-4, and their binding strength to MCP-1 was increased, respectively 11.7nM ( Figure 5 A) and 3.98 nM ( Figure 5 B), which may be due to the reduction of steric hindrance when the nucleic acid aptamer binds to MCP-1, indicating that Seq2T and Seq39T have high sensitivity in binding to the target protein. In addition, the specificity of Seq2T and Seq39T binding to MCP-1 was detected. Compared with the target protein, Seq2T and Seq39T had almost no binding to other proteins (HSA), indicating that Seq2T and Seq39T have specific binding to the target protein ( Figure 5 In order to obtain a nucleic acid aptamer with extremely high affinity, the present invention connected Seq2T and Seq39T in series through a T30 linker to obtain dimers of three nucleic acid aptamers, DA2TN2T, DA39TN39T and DA2TN39T, respectively. The sequences are shown in Table 1 SEQ ID No. 5 to 7. Biomembrane interferometry detection found that the binding affinities of DA2TN2T and DA39TN39T to MCP-1 were similar to those of their monomers, which were 34.9 nM ( Figure 6 A) and 8.05 nM ( Figure 6 B), indicating that Seq2T and Seq39T only bind to one epitope of MCP-1. It is worth noting that the binding affinity of DA2TN39T to MCP-1 is several dozen times that of its monomer (K D 0.274nM, Figure 6 C), indicating that Seq2T and Seq39T act on different epitopes of MCP-1, respectively, and that DA2TN39T can significantly increase the binding strength to MCP-1 by virtue of its bivalent binding mode, indicating that DA2TN2T, DA39TN39T, and DA2TN39T bind to the target protein with high sensitivity. Simultaneously, the binding specificity of DA2TN2T, DA39TN39T, and DA2TN39T to MCP-1 was detected. Compared with the target protein, DA2TN2T, DA39TN39T, and DA2TN39T showed almost no binding to other proteins (HSA), indicating that DA2TN2T, DA39TN39T, and DA2TN39T bind to the target protein specifically.

[0100] Example 5: Determination of the biological efficacy of nucleic acid aptamers in targeting and inhibiting MCP-1

[0101] In order to further clarify the biological efficacy of nucleic acid aptamer targeted inhibition of MCP-1, a cell migration experiment was performed using a 24-well Transwell culture plate with a pore size of 8.0 μm. Pretreated or unpretreated RF / 6A cells were seeded in the upper chamber. 600 μL of DMEM culture medium containing 10% fetal bovine serum was added to the lower chamber for all groups. Except for the control group (ctrl), 40 ng / m of MCP-1 protein (PeproTech Inc., Rocky Hill, USA), and nucleic acid aptamers Seq2T, Seq39T and DA2TN39T were added to the lower chamber of the other groups. After incubation for 12 hours, the non-migrated cells on the surface of the upper chamber were removed, and the remaining cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The cells were then observed and counted at 40 times magnification using an inverted microscope. Figure 7 As shown, Seq2T, Seq39T, and DA2TN39T all significantly inhibited MCP-1-mediated RF / 6A cell migration, with the dimeric aptamer DA2TN39T exhibiting the strongest inhibitory effect. Therefore, the present invention provides a set of high-affinity aptamers that specifically bind to MCP-1, demonstrating significant potential for practical applications.

[0102] The present invention uses magnetic bead SELEX technology to screen and obtain initial sequences Seq2 and Seq39 of nucleic acid aptamers that specifically bind to MCP-1. Through truncation optimization, core sequences Seq2T and Seq39T of nucleic acid aptamers that target and recognize MCP-1 are obtained. Through directional coupling, a dimer of nucleic acid aptamers with extremely high binding affinity to MCP-1 is obtained. Analysis of the affinity of the nucleic acid aptamers of the present invention to the MCP-1 protein and their effect on inhibiting cell migration reveals that the nucleic acid aptamers of the present invention have the advantages of high affinity, strong specificity, and good stability. Furthermore, they have the advantages of low immunogenicity, low preparation cost, and easy modification and labeling. They can specifically recognize the MCP-1 protein and be used for detecting the MCP-1 protein and preparing biosensors. Furthermore, the nucleic acid aptamers of the present invention can also participate in the development of potential therapeutic drugs for MCP-1 protein-related diseases and can be used to prepare reagents for clinical diagnosis or drugs for treating diseases.

[0103] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods and compositions in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A nucleic acid aptamer that specifically recognizes monocyte chemoattractant protein-1, the nucleic acid aptamer comprising at least one of 1) to 5): 1) A single-stranded DNA having a nucleotide sequence including the sequence shown in SEQ ID No. 3 and / or SEQ ID No. 4; 2) a single-stranded DNA having at least 75% identity with the nucleotide sequence defined in 1) and specifically recognizing monocyte chemoattractant protein-1; 3) deleting or adding one or more nucleotides from the nucleotide sequence defined in 1) and specifically recognizing single-stranded DNA of monocyte chemoattractant protein-1; 4) a single-stranded RNA transcribed from the nucleotide sequence defined in any one of 1) to 3) and specifically recognizing monocyte chemoattractant protein-1; 5) Any single-stranded DNA or single-stranded RNA that hybridizes with the nucleotide sequence specified in 1) or 2) or 3) under stringent conditions and specifically recognizes monocyte chemoattractant protein-1.

2. The nucleic acid aptamer according to claim 1, wherein The nucleic acid aptamer is in a single copy or multiple copy form. Preferably, the multiple copies of SEQ ID No. 3 or SEQ ID No. 4 are connected by a linker sequence. In a nucleic acid aptamer containing both SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 3 and SEQ ID No. 4 are also connected by a linker sequence. And / or, the nucleotide sequence of the nucleic acid aptamer is modified, and the modified nucleic acid aptamer specifically recognizes monocyte chemoattractant protein-1, and the modification is selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopization; And / or, a signal molecule and / or an active molecule and / or a functional group and / or a radionuclide is connected to the nucleotide sequence of the nucleic acid aptamer, and the connected nucleic acid aptamer specifically recognizes monocyte chemoattractant protein-1.

3. The nucleic acid aptamer according to claim 2, wherein The linker sequence is a flexible linker sequence; preferably, the linker sequence flexible linker sequence is a T repeat sequence.

4. The nucleic acid aptamer according to claim 3, wherein The sequence of the nucleic acid aptamer is selected from any one of SEQ ID No. 1 to 7.

5. Use of the nucleic acid aptamer according to any one of claims 1 to 4 in at least one of B1) to B6): B1) Use in the preparation of a product for binding monocyte chemoattractant protein-1; B2) Use in the preparation of a product for detecting monocyte chemoattractant protein-1; B3) Use in the preparation of a product targeting monocyte chemoattractant protein-1; B4) Use in the preparation of inhibitors of monocyte chemoattractant protein-1; B5) Use in the preparation of screening, diagnosis or auxiliary diagnosis of monocyte chemoattractant protein-1 related diseases; B6) Use in the preparation of a drug or product for preventing, improving or treating monocyte chemoattractant protein-1 related diseases.

6. The use according to claim 5, characterized in that The monocyte chemoattractant protein-1 related diseases include eye-related diseases. Preferably, the eye-related diseases include age-related macular degeneration, diabetic retinopathy, glaucoma, uveitis and high myopia cataract.

7. A nucleic acid aptamer derivative, wherein the nucleic acid aptamer derivative is a substance obtained by labeling the nucleic acid aptamer according to any one of claims 1 to 4 with a label, preferably, the label is selected from a signal molecule and / or a functional group.

8. A product for binding or detecting monocyte chemoattractant protein-1, characterized in that The product comprises the nucleic acid aptamer according to any one of claims 1 to 4.

9. The product according to claim 8, characterized in that The product is selected from the group consisting of reagents, kits, membrane strips, sensors and chips.

10. A drug for preventing, improving or treating a disease related to monocyte chemoattractant protein-1, comprising the nucleic acid aptamer according to any one of claims 1 to 4. 11 . A drug delivery system specifically targeting monocyte chemoattractant protein-1, comprising the nucleic acid aptamer according to any one of claims 1 to 4 .

12. A method for detecting monocyte chemoattractant protein-1, comprising labeling a reporter group on the nucleic acid aptamer according to any one of claims 1 to 4, allowing the nucleic acid aptamer labeled with the reporter group to interact with monocyte chemoattractant protein-1, and detecting monocyte chemoattractant protein-1 by detecting a signal from the reporter group.

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