A nucleic acid aptamer for lipoprotein-associated phospholipase A2 and its uses

Abstract

This invention relates to nucleic acid aptamers for lipoprotein-associated phospholipase A2 (Lp-PLA2), wherein the nucleic acid aptamer is at least one of B76-2, B76-4, and B76-5, with sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The Lp-PLA2 aptamers provided by this invention fill a gap in the field of Lp-PLA2 aptamers and exhibit high affinity and specificity. Three aptamers with good affinity and specificity for Lp-PLA2 have been screened and verified. These aptamers can be formulated into molecular probes or used as detection reagents in methods or kits for detecting Lp-PLA2, improving accuracy. They can also be used to develop novel Lp-PLA2 biosensing methods for molecular identification, showing great application potential in the early warning of cardiovascular disease events.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the nucleic acid aptamer of lipoprotein-associated phospholipase A2, a marker of atherosclerosis, and its uses. Background Technology

[0002] Lipoprotein-associated phospholipase A2 (LpPLA2) is a 50kD phospholipase produced and secreted by many immune cells, such as macrophages, monocytes, mast cells, and T lymphocytes. Also known as platelet-activating factor acetylhydrolase, LpPLA2 hydrolyzes oxidized phospholipids on the surface of LDL, producing pro-inflammatory byproducts mimicking platelet-activating factors, such as lysophosphatidylcholine and oxidized non-esterified fatty acids. It attracts monocytes to this area, activating leukocytes and stimulating the production of other inflammatory cytokines (such as IL-6 and TNF-α) to mediate the inflammatory response. LpPLA2 further promotes atherosclerosis by attracting smooth muscle cells into the intima, promoting apoptosis and necrosis of macrophages in plaques. In summary, LpPLA2 directly participates in the vascular inflammatory response leading to atherosclerosis, accelerating the rupture of vulnerable plaques, thereby forming thrombi and triggering cardiovascular and cerebrovascular events. It is a novel, independent biomarker for vascular endothelial inflammation.

[0003] LpPLA2, as a highly specific vascular factor, can serve as a dynamic monitoring indicator of vascular inflammation and the degree of atherosclerosis. High levels of LpPLA2 predict a greater susceptibility to plaque rupture, allowing for the assessment and early warning of cardiovascular and cerebrovascular thrombotic risks. Furthermore, LpPLA2 exhibits low biological variability and is less affected by other factors and indicators. Compared to traditional cardiovascular risk prediction indicators, it directly reflects the degree of inflammation in the vascular endothelium; it also demonstrates high specificity and a stronger correlation with plaque instability. LpPLA2 is the only FDA-approved blood test for assessing the risk of atherosclerosis-related coronary artery disease, ischemic stroke, and various thrombotic diseases.

[0004] Currently, methods for detecting enzyme quality include enzyme-linked immunosorbent assay (ELISA), immunoturbidimetric assay, and chemiluminescence assay. Activity detection is achieved through continuous detection using automated biochemical analyzers. However, the ELISA procedure is relatively complex, requiring skilled technicians to minimize operational errors, and the detection time is approximately 2 hours. The LpPLA2 activity detection procedure requires laboratory personnel and large-scale instruments. Furthermore, the preparation of the two monoclonal antibodies and the bioenzyme materials required for the kit is complex, antibody and enzyme activity is easily affected by temperature, and the cost is high. Moreover, kits for both methods are currently lacking in China, far from meeting the actual testing needs of patients with cardiovascular and cerebrovascular diseases. Therefore, developing a novel LpPLA2 detection kit for early warning of cardiovascular events has broad application prospects in home monitoring and clinical testing.

[0005] Nucleic acid aptamers are nucleic acid sequences with targeted functions screened using Systematic Evolution of Ligands (SELEX) technology. They can fold into specific tertiary structures and bind to targets including proteins, small molecules, viruses, bacteria, and cells through hydrogen bonds, electrostatic interactions, hydrophobic interactions, aromatic ring stacking, and van der Waals forces. They play a crucial role in disease diagnosis, targeted drug delivery, environmental monitoring, and food safety analysis. Therefore, finding nucleic acid aptamers with higher affinity and higher specificity for LpPLA2 would facilitate the high-sensitivity and high-specificity detection of LpPLA2. Summary of the Invention

[0006] Given that existing methods can only achieve detection under laboratory conditions, which requires high time, reagent, and labor costs, this invention provides an aptamer for lipoprotein-associated phospholipase A2 and its application. The aptamer has a strong affinity and specificity for lipoprotein-associated phospholipase A2 and can be used for efficient detection of lipoprotein-associated phospholipase A2.

[0007] Accordingly, the present invention also provides a nucleic acid aptamer of lipoprotein-associated phospholipase A2 as a probe for detecting lipoprotein-associated phospholipase A2 and its application in a colorimetric detection method.

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] In one aspect, the present invention provides a method for screening nucleic acid aptamers for the lipoprotein-associated phospholipase A2. Target proteins are immobilized using magnetic beads for LpPLA2 screening, with eight rounds of positive screening followed by magnetic bead-serum protein reverse screening in rounds 2 and 6. Based on the Capture-SELEX screening method, two DNA libraries with the same primers but different lengths of random sequences (40 nt and 50 nt) are used to increase the screening binding probability. In the eight rounds of magnetic bead screening, before the positive screening in rounds 2 and 6, negative screening is performed by adding magnetic beads immobilized with human serum (containing various proteins) to remove non-specifically bound sequences, which are then used as the positive screening libraries for rounds 2 and 6. This further improves the specificity of the nucleic acid aptamers and better meets the practical serum detection requirements for subsequent applications based on nucleic acid aptamers.

[0010] Secondly, this invention screened and obtained a group of nucleic acid aptamers for lipoprotein-associated phospholipase A2. Flow cytometry and surface plasmon resonance experiments verified that three nucleotide sequences, B76-2, B76-4, and B76-5, have excellent affinity and specificity for LpPLA2, as shown in Table 1 (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3), all with orientations of 5'-3'.

[0011] Table 1. A set of aptamer sequences for lipoprotein-associated phospholipase A2 obtained through screening.

[0012]

[0013] The nucleic acid aptamer for lipoprotein-associated phospholipase A2 described in this invention may be at least one of nucleic acid aptamers B76-2, B76-4, and B76-5.

[0014] At least one of the nucleic acid aptamers B76-2, B76-4, and B76-5 has a nucleotide sequence comprising a fixed primer region of 18 nt at both ends and a random sequence region of 40 nt in the middle, wherein the slanted font portion of the primer region is a 10 bp complementary sequence, and the sequences of nucleic acid aptamers B76-2, B76-4, and B76-5 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0015] The aforementioned nucleic acid aptamer, at 25°C, in 100mM Na... + 10uM Mg 2+ Under the given conditions, the spatial structures of the nucleic acid aptamers B76-2, B76-4, and B76-5 are as follows:

[0016]

[0017] The 5' or 3' end of at least one of the nucleic acid aptamers B76-2, B76-4, and B76-5 is chemically modified with a fluorescent group, an amino group, biotin, digoxigenin, or polyethylene glycol.

[0018] Thirdly, the present invention provides the application of the nucleic acid aptamer of the lipoprotein-associated phospholipase A2 in the preparation of reagents, kits or sensors for detecting lipoprotein-associated phospholipase A2.

[0019] The application of the nucleic acid aptamer for lipoprotein-associated phospholipase A2 in the preparation of molecular probes for detecting lipoprotein-associated phospholipase A2. A molecular probe comprising the nucleic acid aptamer described above.

[0020] A kit for specifically recognizing lipoprotein-associated phospholipase A2, comprising the aforementioned nucleic acid aptamer and a complex of gold nanoparticles.

[0021] The application of the nucleic acid aptamer of lipoprotein-associated phospholipase A2 in the detection of lipoprotein-associated phospholipase A2 by the nano-gold colorimetric assay.

[0022] Method principle: The concentration of LpPLA2, a biomarker of atherosclerosis, in serum was assessed by ultraviolet spectrophotometer based on the color-changing properties of gold nanoparticles. Figure 9 The entire detection method was demonstrated, including: First, AuNPs were bound to aptamers B76-2 via electrostatic adsorption. Then, the aptamer probe dissociated from the AuNP surface by inducing a conformational change through the addition of the target LpPLA2. Aggregation occurred at a high salt concentration of 60 mM, and the assay was performed by a rightward shift of the gold nanoparticle peak. The LpPLA2 biomarker was detected at both physiological and pathological levels through colorimetric reactions and changes in absorbance.

[0023] Compared with the prior art, the advantages of the present invention are as follows:

[0024] This invention provides a set of lipoprotein-associated phospholipase A2 aptamers, filling a gap in the field of lipoprotein-associated phospholipase A2 aptamers and exhibiting high affinity and specificity. Three aptamers with good affinity and specificity for lipoprotein-associated phospholipase A2 were screened and validated. These aptamers can be formulated into molecular probes, used as detection reagents, and applied in methods or kits for detecting lipoprotein-associated phospholipase A2, thereby improving accuracy.

[0025] The screening method provided by this invention is simple and reliable. The three lipoprotein-associated phospholipase A2 aptamers provided by this invention have similar affinity and specificity to antibodies, can be artificially synthesized, are low in cost, and are easy to chemically modify.

[0026] The colorimetric detection method based on nano-gold aptamers provided by this invention is simple, visual, and low-cost, and has great application potential in the early warning of malignant cardiovascular diseases. Attached Figure Description

[0027] Figure 1 Schematic diagram of the LpPLA2 aptamer screening process;

[0028] Figure 2 The figure shows the results of the investigation into the binding ability of the secondary library after rounds 3, 6, and 8 screening with MB-LpPLA2;

[0029] Figure 3 This is a diagram showing the homology comparison results of the top 40 candidate aptamer random region sequences obtained after sequencing the libraries in rounds 4 and 8.

[0030] Figure 4 The affinity of four candidate aptamers was determined by flow cytometry and surface plasmon resonance.

[0031] Figure 5 The results show the affinity of LpPLA2 protein with three aptamers at different concentrations and the fitting results of Kd values ​​(the concentrations corresponding to the curves from top to bottom in the figure decrease as indicated by the labels).

[0032] Figure 6 The three aptamers were fitted with secondary structures using Mfold software.

[0033] Figure 7 This is a diagram showing the molecular docking of three aptamers with the target protein LpPLA2.

[0034] Figure 8 Key site analysis for hydrogen bonding between the optimal affinity aptamer B76-2 and LpPLA2.

[0035] Figure 9 This is a schematic diagram of the determination of LpPLA2 by the nano-gold colorimetric method.

[0036] Figure 10 To demonstrate the feasibility of determining LpPLA2 using a nano-gold colorimetric method.

[0037] Figure 11 To determine the absorbance changes of LpPLA2 at physiological and pathological concentrations using a nano-gold colorimetric method.

[0038] Figure 12 The specificity of LpPLA2 determination by nano-gold-aptamer colorimetric method. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0040] Example 1. Screening and Analysis of Nucleic Acid Aptamers

[0041] The screening method for LpPLA2 aptamers is as follows: Figure 1 As shown, it mainly includes the following steps.

[0042] 1. Synthesize a random single-stranded DNA library and primers with the sequences shown below.

[0043] Based on SELEX technology, two single-stranded DNA libraries (Pool B) with different random sequence lengths were designed and synthesized. "N40" and "N50" represent sequences composed of 40 and 50 arbitrary nucleotide bases, respectively. PCR primers designed based on the fixed sequences at both ends were also included. All sequences were synthesized by Shanghai Sangon Biotech Co., Ltd., and the sequence information is shown in Table 2. The primers were prepared into 10 μM stock solutions using ddH2O. The libraries were diluted with binding buffer (PBS containing 4 mM MgCl2) and stored at -20℃ for later use.

[0044] Table 2. DNA library and primer set used for LpPLA2 aptamer screening.

[0045]

[0046]

[0047] 2. Magnetic bead screening method

[0048] Proteins were immobilized using magnetic beads and incubated with corresponding libraries for screening. A total of 8 rounds of positive screening and 2 rounds of negative screening were performed (before the 2nd and 6th rounds of positive screening, respectively).

[0049] (1) Magnetic beads coupled with LpPLA2 (positive sieve magnetic beads)

[0050] Take 50 μL of HisPur Ni-NTA magnetic beads (Thermo Fisher Scientific, catalog number: 88831), wash twice with 200 μL of ddH2O, magnetize the beads, and discard the supernatant. Add 500 μL of PBS (containing 4 mM Mg). 2+ Resuspend the magnetic beads and incubate them with 20 μg of LpPLA2 active protein (Wuhan Yunclone Technology, catalog number: APA867Hu01) at 25°C with horizontal shaking for 2 hours. Then, use a magnet to fish out the magnetic beads, remove the supernatant, and wash twice with PBS to obtain the target immobilized magnetic beads, which are labeled as MB-LpPLA2 for later use.

[0051] (2) Magnetic beads coupled with serum proteins (reverse screening magnetic beads)

[0052] Take 100 μL of carboxyl magnetic beads (Thermo Fisher Scientific, catalog number: 21353), wash four times with 200 μL of ddH2O, and use a magnet to hook the beads, discarding the supernatant. Take 50 μL each of prepared N-hydroxysuccinimide (NHS) at a concentration of 0.1 M and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) at a concentration of 0.4 M, dissolve them at 4 °C, mix them in equal volumes, and quickly add them to the magnetic beads. Incubate at 25 °C for 20 min to activate the carboxyl groups on the surface of the magnetic beads. Use a magnet to hook the magnetic beads, discard the supernatant, wash with ddH2O, and the activated magnetic beads are obtained. Resuspend them in 100 μL of ddH2O. Immediately add 80 μL of healthy volunteer serum to the activated magnetic beads, along with 80 μL of sodium acetate at pH 4.0. Incubate at 25 °C on a shaker for 60 min, allowing the serum protein to couple to the surface of the magnetic beads via amino groups. After conjugation, the magnetic beads were hooked with a magnet, the supernatant was discarded, and 100 μL of 1M ethanolamine (pH 8.5) was added. The mixture was incubated at 25°C on a shaker for 10 min to block unreacted activation sites on the surface of the magnetic beads. The beads were then hooked with a magnet again, the supernatant was discarded, and the beads were washed four times with 200 μL of PBS. These were the magnetic beads conjugated with serum proteins and labeled MB-S.

[0053] (3) Library variation

[0054] Take 1 mL each of 5 μM pool B(40) and pool B(40), anneal at 95 °C, and slowly cool to room temperature for later use.

[0055] (4) Positive sieving and reverse sieving

[0056] Except for rounds 2 and 6, where the secondary libraries were first incubated with carboxyl magnetic beads conjugated to serum proteins and the unbound supernatant was collected as secondary libraries for positive screening, all other rounds used the corresponding secondary libraries directly for positive screening. The sample loading and incubation times for each round are shown in Table 3. Magnetic beads that bound single-stranded DNA were washed three times with 400 μL PBS, then 200 μL ddH2O was added and heated at 95°C for 5 min to separate the single-stranded DNA from the beads. The supernatant was separated while hot, and the ssDNA bound to LpPLA2 was recovered and denoted as E. If it was the first round of positive screening, it was denoted as 1+E, which was used for amplification to prepare the next round of secondary libraries.

[0057] Table 3. Sample dosage and incubation time for LpPLA2 nucleic acid aptamer screening

[0058] Number of filtering rounds serum protein Lp-PLA2 protein Incubation time 1 / 20μg 1h 2 80μL 20μg 1h 3 / 20μg 50min 4 / 20μg 50min 5 / 20μg 40min 6 80μL 20μg 40min 7 / 20μg 30min 8 / 20μg 30min

[0059] (5) Preparation of secondary libraries

[0060] Using target-binding ssDNA as a template, double-stranded DNA was prepared by conventional PCR. The PCR amplification system is shown in Table 4 below. The amplification principle adopted was: if the concentration of template ssDNA (E) was ≥20 ng / μL, 20 rounds of amplification were performed; if it was ≥10 ng / μL, 25 rounds of amplification were performed; and if it was <10 ng / μL, 30 rounds of amplification were performed. Amplification conditions: 95℃, 3 min; 95℃, 30 s, 55℃, 30 s, 72℃, 40 s (the number of cycles was selected according to the initial E concentration); 72℃, 300 s; 4℃, +∞.

[0061] Table 4. Sample loading system for PCR preparation of secondary libraries

[0062] Reagent Name Sample volume E 180μL B-FP 15μL B-RP (Bio) 15μL 5×TaqBuffer 160μL dNTPs (10mM) 10μL <![CDATA[ddH2O]]> 420μL

[0063] (6) Purification of secondary libraries

[0064] Biotin-labeled PCR double-stranded products were obtained by modifying the reverse amplification primers with biotin. After incubating the products with streptavidin magnetic beads (MCE, catalog number: HY-K0208) at room temperature for 2 h, the magnetic bead-PCR complex was recovered. The products were washed twice with PBS, resuspended in 400 μL of ddH2O, and separated from the magnetic beads at 95 °C for 5 min. The supernatant was collected as the corresponding single-stranded secondary library. The nucleic acid concentration was determined by an ultra-micro UV spectrophotometer and prepared for the next round of screening.

[0065] 3. Library Affinity Detection

[0066] During the screening process, the changes in the recognition ability of DNA single-stranded libraries for Lp-PLA2 protein were detected using a CytoFLEX flow cytometer. Secondary libraries from eight rounds of screening were amplified using FAM-modified forward primers and biotin-modified backward primers, respectively. The PCR amplification products were then recovered using streptavidin magnetic beads. The amplified single-stranded libraries with FAM fluorescence were recovered after incubation at 95°C for 5 min and quantified using a micro-volume UV spectrophotometer. 4 μL of the above MB-LpPLA2 was incubated with 200 μg of DNA library for 1 h to investigate the enrichment process of candidate aptamers in each library.

[0067] The results showed that with increasing screening rounds, affinity sequences were gradually enriched in the library, leading to an increase in the binding affinity of ssDNA between the secondary libraries in rounds 1-8 and the magnetic beads, and a gradual increase in the fluorescence shift detected by flow cytometry. Specifically, sequences such as... Figure 2 The figure shown is a graph illustrating the results of the quantitative analysis of the binding force between the positive sieve magnetic beads MB-LpPLA2 and the secondary libraries in rounds 3, 6, and 8.

[0068] 4. Sequencing and analysis of candidate nucleic acid aptamers

[0069] The enriched libraries were sent for sequencing, and the primary structural homology of the DNA was analyzed using ClusterX2 software to select target sequences. For example... Figure 3 The table shows the random region sequence segments and DNA sequence homology comparison results of the top 40 abundant candidate aptamers obtained after sequencing the libraries in rounds 4 and 8. Compared with the G-rich sequences of thrombin-binding aptamers and their analogues, our enriched candidate aptamers also contain G-rich sequences, and the library ends also have a stable 10bp complementary structure, further stabilizing the G-quadruplex conformation. Further analysis of the possible G-quadruplex formation of the nucleotide sequences of the three candidate aptamers using QGRS Mapper software (Quadruplex forming G-RichlSequences, QGRS), website: https: / / bioinformatics.ramapo.edu / QGRS / index.php, yielded the following G-quadruplex prediction results: Table 5 below.

[0070] Table 5. Results of QGRS Mapper software simulation of G-quadruplex candidate aptamers

[0071]

[0072] 5. Verify the affinity of four candidate aptamers for the active protein LpPLA2.

[0073] By coupling 5 μL of magnetic beads with 3 μg of LpPLA2 protein, and then binding them with 5 μL of a candidate aptamer labeled with FAM fluorescence (10 μM), a flow cytometry-detectable signal was provided to record the binding of the aptamer to the target protein. The results are as follows: Figure 4 As shown in A and 4B.

[0074] Flow cytometry results showed that all four candidate sequences had good affinity for the target, with B76-2, B76-4, and B76-5 having similar affinity, while B76-14 had slightly weaker affinity.

[0075] 6. Surface plasmon resonance (SPR) assay for affinity between candidate aptamers and LpPLA2 protein.

[0076] S1 chip surface treatment: First, clean the chip with 50mM NaOH aqueous solution at a flow rate of 30μL / min, with each injection lasting 2min, for a total of 2 times;

[0077] S2 LpPLA2 protein coupling: The chip was activated by injecting the activation mixture at a flow rate of 10 μL / min; channel 4 was activated for 600 s. The LpPLA2 protein was then diluted to 5 μg / mL with 10 mM sodium acetate aqueous solution at pH 5.0 and injected into channel 4 for 1200 s. The LpPLA2 protein coupling amount was 7900 Ru. After the injection, channel 4 was blocked with ethanolamine hydrochloride at pH 8.5 at a flow rate of 10 μL / min for 10 min.

[0078] S3 control channel treatment: The chip was activated by injecting the activation mixed solution at a flow rate of 10 μL / min; the three channels were activated for 600 s. After activation, the three channels were sealed by injecting pH 8.5 ethanolamine hydrochloric acid at a flow rate of 10 μL / min for 10 min.

[0079] S4 detection: The kinetic detection parameters were set using a surface plasmon resonance spectrometer (GE Healthcare, model: Biacore T200). The DNA sequence was diluted to 1 μM with buffer and injected sequentially through channels 3 and 4 at a flow rate of 20 μL / min for 2 min. The dissociation flow rate was 20 μL / min for 2 min. Each sequence sample was regenerated with 1 M NaCl at a flow rate of 20 μL / min for 60 s. After regeneration, the sample was stabilized for 90 s.

[0080] The binding values ​​of LpPLA2 protein to DNA sequences under steady-state conditions are as follows: Figure 4 C, the signal change of the corresponding detection channel minus the control channel is as follows: Figure 4D. The results showed that the target protein was effectively coupled to 7900Ru by dilution with 10mM sodium acetate at pH 5.0. Furthermore, at room temperature, the clone did not bind to the blank channel (which had only undergone activation and blocking treatment). Comparison of the binding between the blank channel and the target protein channel confirmed that at room temperature, the four DNA sequences LpPLA2@76-2, LpPLA2@76-4, LpPLA2@76-5, and LpPLA2@76-14 exhibited affinity for the target protein.

[0081] 7. Surface plasmon resonance (SPR) detection of Kd values ​​of nucleic acid aptamers and LpPLA2 protein

[0082] Channels S1, S2, and S3 follow the same processing steps as described in section 6 above. For the S4 detection channel: use a surface plasmon resonance spectrometer (GE Healthcare, model: Biacore T200) to set the kinetic detection parameters. Take DNA sequences and serially dilute them with buffer as shown in Table 6. Inject the samples sequentially through channels 3 and 4 at a flow rate of 30 μL / min for 3 min. The dissociation flow rate is 30 μL / min for 3 min. Each sequence sample is regenerated with 1M NaCl at a flow rate of 30 μL / min for 60 s. After regeneration, the sample is stabilized for 90 s.

[0083] Table 6. Gradient DNA sequences dissolved and diluted with DPBS to the following concentrations

[0084]

[0085]

[0086] Test results as follows Figure 5 As shown, where, Figure 5 A, 5C, and 5E correspond to the binding of different concentrations of the B76-2, B76-4, and B76-5 sequences to the LpPLA2 target protein. Each curve is the result of channel 4 (response value between the aptamer and the LpPLA2 target protein) minus the result of channel 3 (blank response value). The results show that the three selected sequences, B76-2, B76-4, and B76-5, have high response values ​​to the LpPLA2 protein.

[0087] Provides surface plasmon resonance detection of the dissociation constants of the B76-2, B76-4, and B76-5 sequences and the LpPLA2 protein (as shown below). Figure 5 (As shown in B, 5D, and 5F). Figure 5 As shown in B, 5D, and 5F, the SPR instrument detected strong binding between the three nucleic acid aptamers and the LpPLA2 target protein. The Kd values ​​of each sequence provided by the instrument are shown in Table 7.

[0088] Table 7. Dissociation constants of the three aptamers with LpPLA2

[0089]

[0090] 8. Structural simulation and molecular docking of the three aptamers

[0091] Secondary structure simulation of the three aptamers was performed using Mfold, and the results are as follows: Figure 6 As shown.

[0092] The tertiary structure of single-stranded DNA was fitted using RNA Composer software. Further molecular docking of the target protein LpPLA2 with aptamers B76-2, B76-4, and B76-5 was performed using software from http: / / hdock.phys.hust.edu.cn / . The results are as follows. Figure 7 As shown.

[0093] Pymol software analysis revealed that the optimal aptamer B76-2 exhibits hydrogen-bonded interactions with the target protein LpPLA2. Bases on aptamers with a distance of less than 5 Å between them were identified as potential key active binding sites. The results are as follows... Figure 8 .

[0094] At 25℃, [Na + ] = 100mM, [Mg 2+ Under the condition of 10 μM, simulate the spatial structure of folding 3 aptamers, such as Figure 6 As shown, all three aptamers have a stable stem-ring structure, indicating structural stability.

[0095] Example 2: Detection of lipoprotein-associated phospholipase A2 using the aforementioned nucleic acid aptamer:

[0096] The detection method in this embodiment, using the nucleic acid aptamer described in this invention, can sensitively detect lipoprotein-associated phospholipase A2 (LpPLA2).

[0097] 1. Feasibility verification of LpPLA2 detection by nano-gold colorimetric method

[0098] Figure 9 The diagram illustrates the principle of LpPLA2 detection by colorimetric assay using DNA aptamer B76-2 and AuNPs. The presence of LpPLA2 inhibits the adsorption of aptamers by gold nanoparticles. In the absence of a target, the gold nanoparticle-aptamer complex is in a dispersed state. After the target is added, LpPLA2 competitively adsorbs the aptamer, causing the gold nanoparticles to aggregate.

[0099] The color change resulting from the aggregation of gold nanoparticles due to target competitive recognition of aptamers can be confirmed by absorption spectroscopy. A gold nanoparticle-aptamer complex was prepared by incubating 100 μL of AuNP (0.1 g / L) with 5 μL of B76-2 aptamer (10 μM) at room temperature for 30 min. The feasibility of the colorimetric method for gold nanoparticle aggregation was further verified by referring to the sample addition method shown in Table 8. The results are as follows... Figure 10 As shown, a final concentration of 60 mM NaCl is sufficient to completely aggregate gold nanoparticles, even rendering them colorless. However, the adsorption of the aptamer effectively prevented the aggregation of gold under high salt conditions. The spectral peak of sample 4 shifted to the right from 520 nm to 530 nm, indicating that LpPLA2 bound to aptamer B76-2, restoring the aggregation of gold nanoparticles.

[0100] Table 8. Feasibility Verification Sampling Method

[0101]

[0102]

[0103] 2. Exploration of the detection range of the method

[0104] Lipoprotein-associated phospholipase A2 (LpPLA2) is consistently recommended by guidelines from multiple countries for predicting the risk of cardiovascular and cerebrovascular events. The American Academy of Chemistry (AACE) in the United States clearly states that LpPLA2 levels: <200 ng / mL are normal or low risk; ≥200 and <223 ng / mL are moderate risk; and ≥223 ng / mL are high risk. Based on physiological and pathological concentrations, 0, 100, 150, 200, 250, and 300 ng / mL of the prepared gold nanoparticle-aptamer complex were added, reacted for 30 min, and then 100 μL of NaCl was added to each concentration. The intensity and shift of the UV-Vis spectrum were measured to explore the sensitivity of the detection method. The results are as follows: Figure 11 As shown, the absorbance slowly decreases with increasing target concentration, and the absorption peak red-shifts, indicating that within the physiological and pathological concentration range of 100–300 ng / mL, the aggregation of gold nanoparticles becomes more pronounced with increasing target concentration.

[0105] 3. Specificity verification

[0106] Using myeloperoxidase (MPO), thrombin, and bovine serum albumin (BSA) as non-specific targets, the specificity of the colorimetric method for aptamer competitive recognition of gold nanoparticles was further improved. 0.5 μg of each substance was added to investigate whether it induced gold nanoparticle aggregation. Results are as follows: Figure 12 As shown, only the target substance can cause a significant rightward shift in the spectrum, while the peaks of other substances remain at 520 nm, indicating that the aptamer has specificity in binding to the target.

[0107] The nucleic acid aptamers of this invention are not only applicable to colorimetric analysis detection methods, but combinations of two or more nucleic acid aptamers also have higher sensitivity and are suitable for other detection methods.

[0108] The above description is a general account, specific implementation method and test. However, the present invention is not limited to the above embodiments. Any modifications or improvements based on the present invention, as well as equivalent substitutions or changes to the concept of the present invention, should be covered within the protection scope of the present invention.

Claims

1. A nucleic acid aptamer for lipoprotein-associated phospholipase A2, characterized in that: The nucleic acid aptamer is at least one of B76-2, B76-4, and B76-5, and their sequences are as follows: B76-2:5’- TACCA GTG CG ATGCT CAG GG GGGGG TGGGT GGGGT CACGT CCGGA TGTGTGTCGT GTGC T GAGCA TCG GT AATGA C -3’; B76-4:5’- TACCA GTG CG ATGCT CAG GG GTGGG TGGGT GGGGG AGGAT GGGGG GTGGCCTGAC GTG CT GAGCA TCG GT AATGA C -3’ B76-5:5’- TACCA GTG CG ATGCT CAG GG GTGGG GCGGG TGGGG GAGGG GGCGG AATGGTATGT GTG CT GAGCA TCG GT AATGA C -3’。 2. The nucleic acid aptamer according to claim 1, characterized in that: At 25℃, 100mM Na + 10uM Mg 2+ Under the given conditions, the spatial structures of the nucleic acid aptamers B76-2, B76-4, and B76-5 are as follows: 。 3. The nucleic acid aptamer according to claim 1, characterized in that: The 5' or 3' end of at least one of the nucleic acid aptamers B76-2, B76-4, and B76-5 is chemically modified with a fluorescent group, an amino group, biotin, digoxigenin, or polyethylene glycol.

4. The use of the nucleic acid aptamer of lipoprotein-associated phospholipase A2 as described in any one of claims 1-3 in the preparation of reagents, kits or sensors for detecting lipoprotein-associated phospholipase A2.

5. The use of the nucleic acid aptamer of lipoprotein-associated phospholipase A2 as described in any one of claims 1-3 in the preparation of molecular probes for detecting lipoprotein-associated phospholipase A2.

6. A kit for specifically recognizing lipoprotein-associated phospholipase A2, characterized in that: It comprises the nucleic acid aptamer as described in any one of claims 1-3.

7. A molecular probe, characterized in that: It comprises the nucleic acid aptamer as described in any one of claims 1-3.

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