Nucleic acid aptamer specifically binding to matrix metalloproteinase-9 and application of nucleic acid aptamer

Abstract

The invention relates to a nucleic acid aptamer specifically bound with matrix metalloproteinase-9 and application of the nucleic acid aptamer, and belongs to the field of biological medicines.The high-affinity nucleic acid aptamer Apt6 specifically bound with the matrix metalloproteinase-9 is obtained through screening of a magnetic bead SELEX technology, and the nucleic acid aptamer Apt6 specifically bound with the matrix metalloproteinase-9 is obtained through optimization strategies such as mutation and tailoring. According to the present invention, the high affinity nucleic acid aptamers Apt6M3 and Apt6M3T, which are specifically bound with the matrix metalloproteinase-9, are further obtained, the CD result shows that the Apt6M3 is folded into the stable parallel G-tetramer structure, the Apt6M3T is folded into the high-stability anti-parallel G-tetramer structure, and the binding affinity is increased to 1.07 nM; the aptamer disclosed by the invention can be used for preparing an MMP-9 detection reagent, a kit or a sensor; preparing an MMP-9 capturing, separating and purifying preparation; preparing a diabetic retinopathy diagnostic reagent or kit; preparing an age-related macular degeneration diagnostic reagent or kit; an MMP-9 targeted drug delivery system is constructed, and drugs are neutralized or blocked.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and in particular, relates to a nucleic acid aptamer specifically binding to matrix metalloproteinase-9 and an application thereof. Background Art

[0002] Matrix metalloproteinase-9 (MMP-9) is a member of the matrix metalloproteinase family and is mainly involved in the metabolic balance of the extracellular matrix. MMP-9 plays an important role in the remodeling of the extracellular matrix of tissues such as the cornea, sclera, and retina. In the pathogenesis of age-related macular degeneration (AMD), especially wet AMD, MMP-9 can promote the growth of choroidal neovascularization induced by vascular endothelial growth factor. Therefore, the detection of MMP-9 levels in blood and intraocular fluid is of great significance for the early diagnosis and evaluation of treatment effects of AMD. In addition, under diabetic retinopathy (DR), oxidative stress and inflammatory response caused by hyperglycemia can lead to dysfunction of retinal vascular endothelial cells and smooth muscle cells. MMP-9 is involved in retinal neovascularization and the destruction of the blood-retinal barrier. Therefore, by detecting the level of MMP-9 in vitreous humor, it can not only assist in the diagnosis of DR, but also assess the severity and prognosis of the disease according to its concentration changes. At present, the methods for detecting MMP-9 mainly include enzyme-linked immunosorbent assay and immunohistochemistry. However, for eye diseases such as AMD and DR, the amount of intraocular fluid samples available is extremely small. The above method may have errors in small sample testing and is often difficult to achieve accurate measurement. Therefore, more precise and sensitive detection technology is needed to adapt to the characteristics of eye samples.

[0003] As a new type of biorecognition molecule, nucleic acid aptamers can specifically recognize and bind to corresponding targets with high affinity. They are ssDNA or RNA molecules obtained from random nucleic acid libraries using the systematic evolution of ligands by exponential enrichment (SELEX) technology. Nucleic acid aptamers can be used for the capture, detection and imaging of specific targets, as well as the development of corresponding therapeutic drugs and the construction of targeted drug delivery systems, due to their advantages such as chemical synthesis, strong affinity and high specificity, simple and flexible chemical modification, good stability, low immunogenicity and toxicity. Therefore, nucleic acid aptamers have great application potential and development value in both diagnosis and treatment. Summary of the invention

[0004] In view of the problems raised in the background technology, the purpose of the present invention is to provide a nucleic acid aptamer that specifically binds to matrix metalloproteinase-9. Another purpose of the present invention is to provide the application of the nucleic acid aptamer in preparing MMP-9 detection reagents, kits or sensors; preparing MMP-9 capture, separation and purification preparations; preparing diabetic retinopathy diagnostic reagents or kits; preparing age-related macular degeneration diagnostic reagents or kits; constructing MMP-9 targeted drug delivery systems, neutralizing or blocking drugs, etc.

[0005] In a first aspect of the present invention, a nucleic acid aptamer that specifically binds to matrix metalloproteinase-9 is provided, wherein the nucleotide sequence of the nucleic acid aptamer is as follows: Table 1 Nucleic acid aptamers that specifically bind to and recognize matrix metalloproteinase-9

[0006] In a second aspect, the present invention provides use of the nucleic acid aptamer described above in the preparation of a MMP-9 detection reagent, a kit or a sensor.

[0007] The third aspect of the present invention provides the use of the nucleic acid aptamer described above in the preparation of MMP-9 capture, separation and purification preparations.

[0008] In a fourth aspect, the present invention provides use of the nucleic acid aptamer described above in the preparation of a diagnostic reagent or kit for diabetic retinopathy.

[0009] In a fifth aspect, the present invention provides use of the nucleic acid aptamer described above in the preparation of a diagnostic reagent or kit for age-related macular degeneration.

[0010] In a sixth aspect, the present invention provides the use of the above-mentioned nucleic acid aptamer in constructing a MMP-9 targeted drug delivery system, a neutralizing or blocking drug.

[0011] Beneficial effects of the present invention The nucleic acid aptamer Apt6 of the present invention was first screened by magnetic bead SELEX technology, and the nucleic acid aptamers Apt6M3 and Apt6M3T were further obtained by optimization schemes such as mutation and tailoring. They have the performance of specific recognition and high affinity binding to MMP-9, which provides a highly specific tool for the detection of disease markers.

[0012] Stable secondary structure: CD results showed that Apt6M3 formed a stable parallel G-tetramer structure, while Apt6M3T formed a highly stable antiparallel G-tetramer structure. These stable secondary structures contribute to the stability and functionality of nucleic acid aptamers in biological environments.

[0013] High binding affinity: The binding affinity of nucleic acid aptamers reaches the nanomolar level, which means that they can bind to MMP-9 at extremely low concentrations, which is crucial for improving detection sensitivity and accuracy.

[0014] Multifunctional application: The nucleic acid aptamer of the present invention can be used to prepare MMP-9 detection reagents, kits or sensors, which are of great value in the diagnosis of diseases such as diabetic retinopathy and age-related macular degeneration.

[0015] Drug delivery system: Nucleic acid aptamers can be used to construct MMP-9 targeted drug delivery systems, which may include the release of specific therapeutic drugs after specific recognition and binding with MMP-9, providing a new approach for disease treatment.

[0016] Capture and purification: In the field of biotechnology, nucleic acid aptamers can be used to capture, separate and purify MMP-9, which is crucial for studying the function and regulatory mechanism of MMP-9. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the magnetic bead SELEX technology screening of the present invention; Figure 2 is a circular dichroism spectrum diagram of the nucleic acid aptamer Apt6 of the present invention; Figure 3 is a circular dichroism spectrum diagram of the nucleic acid aptamer Apt6M3 of the present invention; Figure 4 This is the circular dichroism spectrum of the nucleic acid aptamer Apt6M3T of the present invention Figure 5 is the binding and dissociation curve of the nucleic acid aptamer Apt6 of the present invention; Figure 6 is the binding and dissociation curve of the nucleic acid aptamer Apt6M3 of the present invention; Figure 7 This is the binding and dissociation curve of the nucleic acid aptamer Apt6M3T of the present invention. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0019] In order to more clearly illustrate the present invention, it is described in detail through the following examples.

[0020] The experimental methods in the following examples where specific conditions are not specified are generally carried out according to 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 conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention.

[0021] Example 1 Screening library and primer components

[0022] 1. Construct a ssDNA library with a length of 80 nucleotides, the sequence is as follows: 5′-AGCAGCACAGAGGTCAGATG-N 40 -CCTATGCGTGCTACCGTGAA-3′; wherein N represents any one of the bases A, T, C, G, N 40 Represents a random sequence of 40 nucleotides in length.

[0023] 2. Primer Construction Upstream primer: 5′-AGCAGCACAGAGGTCAGATG-3′ (SEQ ID NO. 4) Downstream primer 1: 5′-TTCACGGTAGCACGCATAGG-3′ (SEQ ID NO.5) Downstream primer 2: 5′-poly (dA20)-Spacer18-TTCACGGTAGCACGCATAGG-3′ (SEQ ID NO. 6).

[0024] Example 2 Nucleic Acid Aptamer Screening

[0025] like Figure 1 As shown in the figure, in order to obtain high-affinity nucleic acid aptamers that specifically bind to MMP-9, MMP-9 was immobilized on the surface of magnetic beads by EDC / NHS chemical coupling, and 10 rounds of screening were performed. In order to improve the screening efficiency, blank magnetic beads were introduced for reverse screening from the 5th round, and competitive reverse targets were added to the forward system for co-incubation in each round to further improve the screening specificity.

[0026] The specific screening process is: (1) Take 50 μL of MMP-9 magnetic beads and use screening buffer (containing 2 mM MgCl 2After rinsing with PBS, pH 7.2 (unless otherwise specified below, the screening buffer composition is the same as herein), a blocking buffer (a screening buffer containing 0.1 mg / mL tRNA and 1 mg / mL BSA) was added and incubated for 30 minutes to obtain blocked MMP-9 magnetic beads.

[0027] (2) The ssDNA library of Example 1 was dissolved in screening buffer, incubated at 95°C in a water bath for 10 min, quenched in an ice bath for 5 min, and allowed to stand at room temperature for 10 min. The ssDNA library was then added to the blocked MMP-9 magnetic beads and incubated at room temperature with low speed rotation.

[0028] (3) After incubation, rinse several times with screening buffer to remove unbound ssDNA, then add 100 μL of enzyme-free water and incubate in a 95°C water bath for 10 min to recover ssDNA that specifically binds to MMP-9.

[0029] (4) The eluted ssDNA was used as a template for PCR amplification. The reaction system included: 10 μL of Hotstart premix (5x); 2.5 μL of upstream and downstream primers (10 μM); 5 μL of template; finally, sterile water was added to replenish 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.

[0030] (5) Add urea-denatured loading buffer to the amplified PCR library, incubate in a 95°C water bath for 10 min, quench in an ice bath for 5 min, and place at room temperature for 5 min before loading onto a 12% urea-denatured polyacrylamide gel well and performing electrophoresis at a constant voltage of 300 V.

[0031] (6) After electrophoresis, add 20 ml ddH 2 O and 5 μL nucleic acid fluorescent dye, mix thoroughly and place the gel in it, and shake gently on a horizontal shaker. (7) After staining, place the gel on a fluorescent imaging system, cut and recover the ssDNA at the bottom into a 2 mL test tube, and add 1.5 mL of ddH 2 O, boil the gelatin in boiling water for 30 min, and then recover the supernatant by centrifugation.

[0032] (8) Through QIAEX ® II kit to recover ssDNA in the purified supernatant and re-dissolve it in screening buffer for the next round of screening.

[0033] The above screening process was repeated, and by the 10th round, the ssDNA recovery rate increased significantly. The screening was stopped, and the enriched library was subjected to high-throughput sequencing, and finally the nucleic acid aptamer Apt6 (nucleotide sequence as SEQ ID NO.1) was obtained. Using BLI detection, it was found that Apt6 could bind to MMP-9 with high affinity and strong specificity ( Figure 5 ).

[0034] Example 3 Optimization and identification of nucleic acid aptamers

[0035] In order to further improve the performance of nucleic acid aptamers, optimization strategies such as mutation and tailoring were introduced. First, circular dichroism (CD) spectroscopy was used to detect the Figure 2 ), it was found that the nucleic acid aptamer Apt6 has characteristic positive and negative absorption bands at 280 nm and 245 nm, respectively, which indicates that Apt6 has formed a B-DNA structure to a large extent. Based on the prediction of QRGS online software (https: / / bioinformatics.ramapo.edu / QGRS / index.php), it was found that Apt6 can also fold into a G-tetramer structure containing 2 G-tetrad planes. In order to further improve the structural stability, 13C in the Apt6 sequence was mutated to 13G and 25T was mutated to 25G, thereby forming a G-tetramer structure containing 3 G-tetrad planes, named Apt6M3 (nucleotide sequence as shown in SEQ ID NO.2). Using BLI detection, it was found that Apt6M3 not only maintained the same affinity and specificity as Apt6 ( Figure 5 , Figure 6 ), and CD results showed that Apt6M3 folded into a stable parallel G-tetramer structure ( Figure 3 ), these stable secondary structures contribute to the stability and functionality of nucleic acid aptamers in biological environments. Furthermore, by truncating the redundant sequences at both ends of the G-tetramer, the core structure sequence of the G-tetramer, Apt6M3T (nucleotide sequence as SEQ ID NO.3), was generated. Apt6M3T not only maintains high recognition specificity for MMP-9, but also increases the binding affinity to 1.07 nM ( Figure 7 In addition, CD results show that Apt6M3T has characteristic positive and negative absorption bands at 296 nm and 265 nm, respectively ( Figure 4 ), which indicates that Apt6M3T folds into a highly stable antiparallel G-tetramer structure, which in turn leads to full exposure of the binding site, thereby improving the affinity of targeted binding. The nucleic acid aptamer of the present invention has important application prospects in the field of biomedicine due to its high specificity, high affinity and stable secondary structure, especially in disease diagnosis and treatment.

[0036] Example 4 Analysis of molecular interactions using biomembrane interferometry (BLI)

[0037] BLI is a real-time analysis method for molecular interactions. The principle is that the instrument emits white light to the sensor surface, and the light will be reflected after passing through the biofilm layer of the sensor, and the frequency of the reflected light will be affected by the thickness of the biofilm layer. Some frequencies of reflected light will produce constructive interference with the incident light, while others will be destructively interfered. These interference light waves are detected by the spectrometer to form an interference spectrum, which is displayed as the relative displacement intensity of the interference spectrum. Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer will detect the displacement of the interference spectrum in real time, and this displacement directly reflects the change in the thickness of the biofilm on the sensor surface. That is, when the nucleic acid aptamer fixed on the sensor biofilm interacts with the molecules in the solution (such as MMP-9), the thickness of the biofilm layer changes, resulting in a relative displacement. This relative displacement increases or decreases with the increase or decrease in the amount of the molecule bound, and finally reaches an equilibrium state and gives the corresponding binding curve, dissociation curve and affinity constant in real time.

[0038] The specific implementation process is as follows: (a) After the biotin-labeled aptamer is dissolved in the screening buffer, it is placed in a 95°C water bath for 10 min, ice-cooled for 5 min, and left at room temperature for 10 min to form a stable spatial structure; (b) 200 μL of screening buffer, aptamer, MMP-9 protein and screening buffer are added to the 96-well plate in sequence; (c) The streptavidin-coated sensor is immersed in each reaction well in sequence according to the program set by the instrument, and goes through five steps: sensor balancing, aptamer solidification, rinsing, MMP-9 (or BSA) binding and dissociation. The results are shown in Figure 2. Figure 5 , Figure 6 and Figure 7 As shown in the figure, nucleic acid aptamers Apt6, Apt6M3 and Apt6M3T can bind to MMP-9 with high affinity and strong specificity, and the binding affinity constants are 26.3, 24.0 and 1.07 nM, respectively. The binding affinity of nucleic acid aptamers reaches the nanomolar level, which means that they can bind to extremely low concentrations of MMP-9, which is crucial for improving detection sensitivity and accuracy.

[0039] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

[0040] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A nucleic acid aptamer that specifically binds to matrix metalloproteinase-9, characterized in that: The nucleic acid aptamer is selected from one or more of the following sequences: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) the nucleotide sequence shown in SEQ ID NO.2; (3) The nucleotide sequence shown in SEQ ID NO.

3.

2. Use of the nucleic acid aptamer that specifically binds to matrix metalloproteinase-9 as claimed in claim 1 in the preparation of a MMP-9 detection reagent, a kit or a sensor.

3. Use of the nucleic acid aptamer that specifically binds to matrix metalloproteinase-9 as claimed in claim 1 in the preparation of MMP-9 capture, separation and purification preparations.

4. Use of the nucleic acid aptamer that specifically binds to matrix metalloproteinase-9 as claimed in claim 1 in the preparation of a diagnostic reagent or kit for diabetic retinopathy.

5. Use of the nucleic acid aptamer that specifically binds to matrix metalloproteinase-9 as claimed in claim 1 in the preparation of a diagnostic reagent or kit for age-related macular degeneration.

6. Use of the nucleic acid aptamer that specifically binds to matrix metalloproteinase-9 as claimed in claim 1 in constructing a MMP-9 targeted drug delivery system, neutralizing or blocking drug.

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