Design method of DNA aptamer based on directional optimization strategy and application thereof

Abstract

The invention provides a design method and application of a DNA aptamer based on a directional optimization strategy. The method combines methods of OpenMM molecular dynamics simulation, directional point mutation, directional cutting assembly and the like for the first time. Through a directional optimization platform, aptamer sequences of immunosuppressant tacrolimus and mycophenolic acid with higher binding force and specificity are screened out, and an SPR verification technology platform is established. On the basis, a GO load platform is constructed, and simultaneous sensing of tacrolimus and mycophenolic acid is realized. In conclusion, according to the oriented optimization strategy, the selectivity of the aptamer in a complex biological sample can be improved by changing the structure and the function of the aptamer. The proposal and application of the method are expected to provide reference for finding more aptamers for reducing the cost and improving the detection sensitivity and specificity and the like.

Description

Technical Field

[0001] The present invention relates to a therapeutic drug monitoring method and application field, specifically to an aptamer structure optimization design method using a directed optimization strategy and an application of simultaneous monitoring of immunosuppressants mycophenolic acid and tacrolimus. Background Art

[0002] The immunosuppressant tacrolimus (TAC) combined with mycophenolic acid (MMF) is one of the first-choice options for the treatment of lupus nephritis type III / IV+V. The blood concentration of MMF varies greatly from person to person and is affected by factors such as genotype, serum albumin, and creatinine SCr levels. Studies have reported that, especially when patients take mycophenolic acid and tacrolimus at the same time, the AUC of TAC decreases by 19-30%, and the drug metabolism characteristics are more complicated. Therefore, it is necessary to carry out simultaneous detection of tacrolimus and mycophenolic acid concentrations. Currently, the commonly used immunosuppressant detection methods include immunoassay and biochemical technology. Among them, immunoassay has problems such as high cost, complex operation, and susceptibility to interference. Although biochemical technology such as liquid chromatography-mass spectrometry can effectively reflect the blood concentration of immunosuppressants, the application of this technology depends on expensive foreign instruments and equipment and the operation of professional technicians, and it is difficult to apply to medical institutions below the second level and patients at home. Therefore, it is urgent to develop new portable and low-cost immunosuppressant combined monitoring technology at this stage.

[0003] Aptamer sensors are considered to be a promising home monitoring technology and have shown great potential in the field of biosensing. At present, the detection of immunosuppressant aptamer sensors is still in its infancy. Recently, the Seyed team screened a specific tacrolimus aptamer by the systematic evolution ligand index enrichment (SELEX) method, which reached the nanomolar level in serum tacrolimus detection, but its clinical application is still limited and its sensitivity still needs to be improved (J.Pharm.Biomed.Anal.177,2020,112853). Therefore, there is still a lack of innovation in the technical level of aptamer structure optimization.

[0004] To solve the above problems, post-modification aptamer screening technology makes it possible. Different from the SELEX technology that obtains longer aptamer sequences, post-modification aptamer technology focuses more on later structural modification and optimization. Its advantages are that the preferred sequence has a smaller molecular weight and lower synthesis difficulty. It also has the characteristics of low preparation difficulty, improved stability and adjustable structure. Therefore, it has been increasingly studied and applied to the field of sensor structure optimization. For example, Zhang Ge and others created an aptamer PEG modification method to improve the stability of the aptamer. The CN 113129996B patent used a post-modification strategy to screen the preferred aptamer of tetrodotoxin TTX, showing its application prospects in small molecule sensing. However, as of today, there have been no reports on the research of post-modification optimization of aptamers for immunosuppressants, especially in the simultaneous monitoring of tacrolimus and mycophenolic acid. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a design method of DNA aptamers based on a directed optimization strategy, combining molecular dynamics simulation (OpenMM), residue point mutation / tailoring, molecular docking (PLANTS algorithm) and SPR verification to screen high-affinity aptamers, comprising the following steps:

[0006] Step 1: Use OpenMM to perform molecular dynamics simulation on the complex of the original DNA aptamer and the target small molecule to obtain the dynamic structural data of the complex;

[0007] Step 2: Based on the dynamic structure data, the root mean square deviation (RMSD) value of the residues is calculated, and the residues or residue segments whose RMSD value is higher than 1.2 standard deviations from the average value are screened;

[0008] Step 3: Perform point mutations or trim several base pairs of the screened residues to generate optimized DNA aptamer candidate sequences;

[0009] Step 4: Use PLANTS software to perform molecular docking between candidate sequences and target small molecules, and combine ant colony optimization algorithm and AutoDockVina algorithm to screen the top several aptamers with the highest scores;

[0010] Step 5: Prepare the selected aptamers using solid phase synthesis technology, and perform surface plasmon resonance (SPR) verification to select the binding affinity (K D The aptamer with the highest aptamer value was taken as the final optimized product.

[0011] Preferably: in step 1, the conditions for molecular dynamics simulation include:

[0012] Simulation time 150ns;

[0013] Temperature 300K, pressure 1atm;

[0014] The PME method was used for electrostatic interaction, and the Cut-off method was used for van der Waals interaction.

[0015]

[0016] The chemical bond constraints were calculated using the LINCS algorithm with a time step of 2fs.

[0017] Preferably: in step 2, the RMSD value of the residues is calculated by a trajectory analysis tool to determine the residue or residue segment with the largest conformational change; based on the RMSD analysis results, the residues or sequence segments are selected for point mutation or trimming, the selection of mutation sites is based on the residues or residue segments whose RMSD values ​​are greater than 1.2 standard deviations from the average value, the trimming length is determined according to the length of the conformational change region, and 3 to 8 base pairs are trimmed; and the preferred sequence is obtained.

[0018] Preferably: In step 4, molecular docking allows the DNA aptamer to bind to the target small molecule around the binding site. The flexibility was adjusted within the range, and the docking score was calculated using the AutoDock Vina algorithm.

[0019] Preferably: in step 4, the candidate sequence is protonated, and its 3D structure is predicted using Openbabel or other software; docking parameters are set in PLANTS, specifically, the search speed is set to speed1, an ant colony optimization algorithm (ANT System) is used for global search, local search follows a speed priority strategy, and AutoDockVina is selected as the docking algorithm; the docking results of each aptamer and the target small molecule are scored, and the docking score threshold is set to the highest 20% as effective docking, from which the top several aptamers with the highest scores are screened.

[0020] Preferably, in step 5, the above-screened aptamers are prepared using solid phase synthesis technology, and the specific operating steps are as follows: the 5' end of the aptamer is fixed on a solid phase synthesis carrier, and a controlled pore glass can be used as a solid phase support; using an automatic DNA synthesizer, according to a preset DNA sequence, the synthesis is carried out by gradually adding nucleotides one by one, and this process adopts the phosphoamide method; to ensure effective coupling between nucleotides, the coupling time of each step is set to 5-10 minutes; at the same time, phenol phosphoric acid anhydride is added as an activator and N-methylimidazole is used as a catalyst to accelerate the reaction process; during the synthesis process, after each nucleotide is added, an oxidation step is required, and iodine is used to remove the protecting group at the 3' end to ensure that the subsequent nucleotides can be connected smoothly; after the synthesis is completed, a final deprotection and washing step is carried out to remove the protecting group and impurities remaining in the synthesis process; after these steps, a purified single-stranded DNA aptamer is finally obtained.

[0021] Preferably: in step 5, in order to accurately determine the binding strength between the DNA aptamer and the target small molecule, the surface plasmon resonance (SPR) technology is used, and the specific operation process is as follows: the SPR sensor chip is pretreated, the chip is activated with NHS / EDC, and then the DNA aptamer is fixed on the chip surface by covalent bonding; the target small molecule is pumped into the flow cell according to a concentration gradient of 10pM to 10μM, and the flow rate is set in the range of 5-50μL / min; in the process of the target small molecule flowing through the chip surface, the signal change caused by molecular binding is monitored in real time by using SPR technology; the change curve of the SPR signal over time is analyzed, and the binding affinity between the DNA aptamer and the target small molecule is calculated by using a steady-state or kinetic analysis method, and the dissociation constant (K D value) indicates; select binding affinity (K D The aptamer with the highest aptamer value was taken as the final optimized product.

[0022] The present invention also provides a DNA aptamer prepared by the above method, wherein the target small molecule is the immunosuppressant tacrolimus (TAC) or mycophenolic acid (MMF); the nucleotide sequence of the aptamer is SEQ: 1 (for TAC) or SEQ: 2 (for MMF), or a derivative sequence containing a SEQ: 1 / SEQ: 2 binding fragment.

[0023] Preferably: the 5′ end of the aptamer is modified with a fluorescent label; the SEQ: 1 aptamer is labeled with FAM, and the SEQ: 2 aptamer is labeled with VIC.

[0024] The present invention also provides a product for detecting immunosuppressants, comprising:

[0025] The above-mentioned DNA aptamer;

[0026] Graphene oxide (GO) or gold nanoparticles as fluorescence quenching carriers;

[0027] Buffer system, pH range is 5.8~7.4.

[0028] The present invention also provides a method for detecting the concentration of an immunosuppressant in a sample, comprising the following steps:

[0029] Step A: Mixing and incubating the fluorescently labeled aptamer and the sample to be tested to form an aptamer-target complex;

[0030] Step B: adding graphene oxide or nano-gold to form a test system by utilizing its fluorescence quenching properties;

[0031] Step C: detecting the fluorescence intensity of the incubation solution and the test system, and calculating the target concentration according to the linear relationship between the change of fluorescence intensity and concentration;

[0032] Among them, when the target is TAC, the linear relationship is Y=5.895X-0.1307; when the target is MMF, the linear relationship is Y=10.235X+0.0279, X is the concentration, and Y is the fluorescence intensity ratio.

[0033] Preferably, the incubation conditions are light-proof, 20-30° C., 15-45 minutes; and the detection wavelength is set to 665 nm.

[0034] The present invention also provides a detection device, comprising:

[0035] A data acquisition module, used for acquiring fluorescence intensity signals;

[0036] A processing module, calculating the target concentration based on the linear relationship;

[0037] Output module, displays the test results and over-limit warning prompts.

[0038] The present invention also provides a computer-readable storage medium storing a computer program, wherein the program implements the above-mentioned detection method when executed.

[0039] Beneficial Effects

[0040] The present invention innovatively proposes a DNA aptamer post-modification strategy based on a directed optimization strategy. This strategy organically combines OpenMM molecular dynamics simulation, directed point mutation, directed cutting and assembly and other methods for the first time. The present invention builds a directed optimization platform, through which tacrolimus aptamer sequences with higher binding force and specificity are screened. At the same time, an SPR verification technology platform is established, which provides a reliable means for the accurate evaluation of aptamer performance. On this basis, a GO loading platform is constructed to achieve simultaneous sensing detection of tacrolimus and mycophenolic acid. The directed optimization strategy of the present invention can improve the selectivity of the aptamer in complex biological samples by changing its structure and function. This feature enables the aptamer to more accurately identify the target molecule in practical applications and reduce the influence of interference factors.

[0041] The directed optimization technology mentioned here is an aptamer directed optimization method that combines OpenMM molecular dynamics simulation, directed point mutation, and directed cutting and assembly. This method integrates the advantages of multiple technologies and provides a comprehensive and effective way to optimize aptamers.

[0042] The proposal and application of this strategy is expected to provide a reference for the research and development of aptamers. In particular, it has broad application prospects and guiding role in "reducing costs and increasing efficiency" and discovering more aptamers that can reduce costs and improve detection sensitivity and specificity. It will inject new vitality into technological innovation and development in related fields and promote the widespread application of aptamer technology in multiple fields such as biological detection and medical diagnosis.

[0043] At the same time, the detection method of the present invention only requires simple incubation and fluorescence detection steps, and does not require complicated sample pretreatment; the detection method of the present invention only requires the use of nucleic acid aptamers, graphene oxide and fluorescence detectors, and the cost of these materials and equipment is relatively low, thereby reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 :Schematic diagram of the technical route for targeted optimization of aptamer strategy.

[0045] Figure 2 : RMSF plot of the original sequence and TAC under dynamic kinetics simulation.

[0046] Figure 3 : Surface plasmon resonance results of Apt and TAC1~5.

[0047] Figure 4 : 3D composite structures of SEQ1-TAC and SEQ2-MMF.

[0048] Figure 5 : Standard curve of TAC detection by the kit.

[0049] Figure 6 : Standard curve of MMF detected by the kit. DETAILED DESCRIPTION

[0050] The present invention is further explained below in conjunction with the examples. In the following examples, the target small molecule is the immunosuppressant tacrolimus (TAC).

[0051] Example 1

[0052] Reference Figure 1 The optimization strategy shown carries out a directed optimization process, which includes the following steps:

[0053] Step 1: Molecular dynamics simulation

[0054] OpenMM was used to perform molecular dynamics simulation on the complex of the original DNA aptamer and the target small molecule to obtain the dynamic structural data of the complex; specifically, the original DNA aptamer was placed in the center of the water box to ensure that the minimum distance between it and the surface of the water box was The .mol2 structure file of tacrolimus was imported. During the simulation, the electrostatic interaction was calculated using the particle mesh Ewald (PME) method, and the van der Waals interaction was calculated using the Cut-off method. The cut-off distances for both were All chemical bonds were constrained by the LINCS algorithm to keep the bond lengths stable; the integration time step was set to 2 fs; the simulation temperature was maintained at 300 K; the simulation pressure was controlled at 1 atm; and the total simulation time was 150 ns.

[0055] Step 2: Directional Optimization and Structural Analysis

[0056] Based on the dynamic structure data, the root mean square fluctuation (RMSF) values ​​( Figure 2 ), select residues or residue segments with RMSD values ​​greater than 1.2 standard deviations from the average value for point mutation or trimming; the trimming length is determined according to the length of the conformational change region, and the trimming is 3 to 8 base pairs; the specific trimming strategy is: 1. Use the structural trimming method to trim the redundant 35-50 stem-loop region or 3' tail conformation and reorganize it into vApt2-vApt5; 2. Part or all of the bases C46 and U51 are site-directed mutated to T46 and C51 with higher binding affinity to obtain vApt6-vApt16. After obtaining the preferred sequence, the next step of molecular docking is carried out.

[0057] Step 3: Molecular docking

[0058] The optimized DNA aptamer preferred sequences vApt2-vApt16 were molecularly docked with the target small molecule tacrolimus (TAC) using PLANTS software; vApt2-vApt16 were protonated and their 3D structures were predicted using Openbabel or other software; docking parameters were set in PLANTS, the search speed was set to speed1, and the ant colony optimization algorithm (ANTSystem) was used for global search; the speed priority strategy was used for local search, and AutoDockVina was selected as the docking algorithm; vApt2-vApt16 and TAC were allowed to be located around the binding site The docking score threshold was set to the highest 20% as effective docking, and the top 4 aptamers with the highest scores were screened and renamed Apt2~Apt5:SEQ1.

[0059] Step 4: Preparation of aptamers by solid phase synthesis

[0060] The preferred aptamer is prepared by using solid phase synthesis technology, and the specific steps include: fixing the 5' end on a solid phase synthesis carrier; synthesizing according to a preset DNA sequence by an automatic DNA synthesizer, and the synthesis adopts the phosphoamidite method; during the synthesis process, the coupling time of each step is 5-10 minutes, and an activator such as phenol phosphoric acid anhydride chloride and a catalyst N-methylimidazole are added; an oxidation step is performed after each nucleotide is added, and iodine is used to remove the protecting group at the 3' end; after the synthesis is completed, a final deprotection and washing step is performed, and finally 4 purified single-stranded DNA aptamers Apt2~Apt5:SEQ1 are obtained.

[0061] Example 2

[0062] Surface plasmon resonance (SPR) monitoring of the preferred sequence

[0063] The binding strength between DNA aptamers Apt1-Apt5:SEQ1 and target small molecule TAC was detected using surface plasmon resonance (SPR) technology. The specific steps included: fixing the DNA aptamer on the surface of a pretreated SPR sensor chip by covalent bonding, wherein the pretreatment refers to activating the chip with the aid of NHS / EDC reagents to generate active sites on the chip surface that can covalently bind to the DNA aptamer; pumping the target small molecule TAC into the flow cell at a concentration gradient of 10 pM to 10 μM, with the flow rate set at 5-50 μL / min; monitoring the SPR signal changes caused by molecular binding in real time; and calculating the binding affinity (K) between the DNA aptamer and TAC by analyzing the change curve of the SPR signal over time using a steady-state or kinetic analysis method. D value), preferably K D High value fragments ( Figure 3 ). The experimental results show that in the designed Apt2~Apt5:SEQ1 thermodynamic binding K D In the situation analysis, the K of the trimmed Apt5:SEQ1 D The value is 2.6 times higher than that of Apt1, and K D The value is significantly lower than that of other aptamers. The optimization method of mycophenolic acid aptamer is the same as the above steps and will not be repeated here. The 3D composite structures of SEQ2, Apt5: SEQ1-TAC and SEQ2-MMF are shown in Figure 4 .

[0064] Example 3

[0065] Another object of the present invention is to provide a fluorescent sensor and a method of using the same for quantitatively detecting the concentration of tacrolimus and mycophenolic acid in serum samples. The fluorescent sensor uses the binding properties of the targeted optimized oligonucleotides with the immunosuppressants tacrolimus and mycophenolic acid to quantify the concentration of the analyte by the change of the fluorescent signal. The specific steps are as follows:

[0066] Step 1: Labeling of oligonucleotides:

[0067] In one embodiment of the present invention, the DNA aptamer introduces different fluorescent labels at its 5' end to achieve differentiated detection of different drugs. Specifically, the DNA aptamer of SEQ: 1 is labeled with FAM, and the DNA aptamer of SEQ: 2 is labeled with VIC. This design allows multiple drugs to be monitored simultaneously in a single sample, improving the efficiency and accuracy of detection.

[0068] Step 2: Construction of fluorescence sensor:

[0069] A fluorescence sensor based on graphene oxide (GO) was constructed, and the fluorescence quenching property of GO was used to detect tacrolimus. Specifically, the oligonucleotide labeled in the first step was combined with GO to construct a fluorescence sensor. During the construction process, the concentration of GO was controlled in the range of 5-280 μg / mL to achieve the best quenching effect on the fluorescence of the labeled oligonucleotide.

[0070] Step 3: Optimize GO concentration:

[0071] The optimal concentration range of GO was determined to be 5-190 μg / mL by experiments to achieve the best quenching effect on the fluorescence of the labeled oligonucleotide. This step was performed in a phosphate buffer with a pH value of 5.8-7.4 and a temperature controlled at 15-25°C.

[0072] Step 4: Analysis and testing:

[0073] Using the designed and optimized fluorescence sensor, the changes in relative fluorescence intensity are measured to quantitatively detect tacrolimus in different concentration ranges. The specific operation process is as follows:

[0074] Incubation step: First, the labeled DNA aptamer is fully mixed with the serum sample to be tested, and incubated under appropriate conditions to obtain an incubation solution. Subsequently, the graphene oxide is mixed with the incubation solution again and incubated to obtain the system to be tested. The entire incubation process must be strictly controlled, with the incubation temperature maintained at 20-30°C and the incubation time being 15-45 minutes to ensure that the sample fully reacts with each reagent.

[0075] Detection steps: Use professional fluorescence detection equipment to accurately measure the fluorescence intensity of the incubation solution and the test system at 665nm. Based on the linear relationship between the change in fluorescence intensity and the concentration of immunosuppressants, by comparing the fluorescence intensity change values ​​of the incubation solution and the test system, and using scientific and reasonable mathematical models and calculation methods, the concentration of immunosuppressants (such as tacrolimus) in the test sample can be accurately calculated. It is worth noting that this detection step needs to be carried out in a light-proof environment to effectively avoid the degradation of the fluorescence signal due to light and ensure the reliability of the test results. In actual detection, samples containing different concentrations of mycophenolic acid and tacrolimus are fully mixed with labeled oligonucleotides, and then GO is added. According to the above detection process, the quantitative analysis of the tacrolimus concentration is achieved.

[0076] Example 4

[0077] In this example, the detection performance of the fluorescent sensor for tacrolimus and mycophenolic acid was investigated, including the detection limit and the linear response relationship, so as to evaluate the reliability and accuracy of the sensor in actual detection.

[0078] During the experiment, phosphate buffered saline (PBS) and serum samples were selected as the detection environment. The experimental results show that the detection limit of the sensor for tacrolimus in PBS is 1.7nM and in serum samples is 2.8nM. At the same time, the sensor's response to tacrolimus shows a linear relationship with the logarithm of the tacrolimus concentration. When the immunosuppressant is tacrolimus, the linear relationship between the change in fluorescence intensity and the concentration of the immunosuppressant is Y=5.895X-0.1307, where X is the concentration of the immunosuppressant and Y is the ratio of the fluorescence intensity of the test system to the fluorescence intensity of the incubation solution ( Figure 5 ); When the immunosuppressant is mycophenolic acid, the linear relationship between the change in fluorescence intensity and the concentration of the immunosuppressant is Y=10.235X+0.0279, where X is the concentration of the immunosuppressant and Y is the ratio of the fluorescence intensity of the test system to the fluorescence intensity of the incubation solution ( Figure 6 ).

[0079] In summary, the present invention has developed a group of DNA aptamers with high specificity and affinity, whose nucleotide sequences are SEQ: 1 and SEQ: 2. These DNA aptamers are specially designed to efficiently bind to the target immunomodulatory drug and have a lower binding affinity (K D The binding capacity of the aptamer of SEQ:1 is significantly improved compared with the prior art. D The value is 5.3 nM, and the Kd value of the nucleic acid aptamer of SEQ: 2 and mycophenolic acid is 8.3 nM. These data are lower than the reported values ​​in the prior art, thus ensuring high sensitivity and specificity of the detection.

[0080] Group Tacrolimus concentration (n=5, ng / mL) Mycophenolic acid concentration (n=5, ng / mL) HPLC / MS-MS comparison 7.023±3.432 - HPLC / MS-MS comparison - 210.2±65.52 Example 8 7.127±4.821 220.5±60.25

[0081] Sequence Listing

[0082]

[0083] Embodiment 5:

[0084] The detection device in this embodiment is mainly composed of a data acquisition module, a processing module and an output module, and is used to detect the concentration of immunosuppressants in a sample.

[0085] Data acquisition module: It uses a highly sensitive fluorescence detector, which can accurately measure the fluorescence intensity signal at a specific wavelength. When detecting immunosuppressants, the detector is set to detect the fluorescence signal at a wavelength of 665nm. The detector is closely connected to the detection sample pool to ensure that the fluorescence intensity data of the incubation solution and the test system can be stably and accurately obtained, and these analog signals are transmitted to the processing module in real time through the data transmission line.

[0086] Processing module: A high-performance microprocessor is used, which has a pre-stored immunosuppressant concentration calculation program. The program processes the fluorescence intensity data transmitted by the data acquisition module based on the linear relationship (when the target is TAC, Y = 5.895X-0.1307; when the target is MMF, Y = 10.235X + 0.0279, X is the concentration, and Y is the fluorescence intensity ratio). After receiving the fluorescence intensity data, the processing module first calculates the fluorescence intensity ratio, and then substitutes it into the corresponding linear equation to quickly and accurately calculate the concentration of the immunosuppressant in the sample.

[0087] Output module: A liquid crystal display (LCD) is used as an output device to display the test results and over-limit warning prompts. After the processing module calculates the concentration of the immunosuppressant, the data is transmitted to the output module. The output module displays the test results in a clear and easy-to-understand interface. If the detected concentration of the immunosuppressant exceeds the pre-set normal range, the output module will immediately display a striking warning message, such as a flashing red warning icon and a text prompt "Abnormal concentration, please pay attention", to remind relevant personnel to deal with it in time.

[0088] Example 6

[0089] In this embodiment, the computer-readable storage medium stores a computer program for implementing a method for detecting the concentration of an immunosuppressant, and can be run on a device with data processing capabilities, such as a personal computer, a portable detection terminal, and the like.

[0090] Programming: Use Python, C++ and other programming languages ​​to write the program code of the detection method. The code implements each step of the detection method in detail, including data reading, fluorescence intensity ratio calculation, concentration calculation and result output. For example, Python's scientific computing library (such as NumPy, SciPy) is used for data processing and linear equation calculation to ensure the efficiency and accuracy of the program.

[0091] Storage media selection and storage: Choose common computer-readable storage media, such as USB flash drives, solid-state drives (SSDs), or optical disks. Store the written program code in the storage medium through the corresponding writing device, and organize it according to the file system format of the storage medium during storage to ensure that the program file is complete and easy to read.

[0092] Program operation and use: Insert the medium storing the program into the corresponding interface of the target device (such as a personal computer). After the device recognizes the storage medium, the user can find and run the detection program through the file management tool of the operating system. After the program is started, input the fluorescence intensity data of the incubation solution and the test system obtained by the test according to the prompts on the interface. The program automatically executes the internal code, calculates the concentration of the immunosuppressant according to the preset linear relationship, and displays the final test results on the device screen.

Claims

1. A method for designing a DNA aptamer based on a directed optimization strategy, characterized in that: The following steps are involved: Step 1: Use OpenMM to perform molecular dynamics simulation on the complex of the original DNA aptamer and the target small molecule to obtain the dynamic structural data of the complex; Step 2: Based on the dynamic structure data, the root mean square deviation (RMSD) value of the residues is calculated, and the residues or residue segments whose RMSD value is higher than 1.2 standard deviations from the average value are screened; Step 3: Perform point mutations or trim several base pairs of the screened residues to generate optimized DNA aptamer candidate sequences; Step 4: Use PLANTS software to perform molecular docking between candidate sequences and target small molecules, and combine ant colony optimization algorithm and AutoDockVina algorithm to screen the top several aptamers with the highest scores; Step 5: Prepare the selected aptamers using solid phase synthesis technology, and perform surface plasmon resonance (SPR) verification to select the binding affinity (K D The aptamer with the highest aptamer value was taken as the final optimized product.

2. The design method according to claim 1, characterized in that: In step 1, the conditions for molecular dynamics simulation include: Simulation time 150ns; Temperature 300K, pressure 1atm; The PME method was used for electrostatic interaction, and the Cut-off method was used for van der Waals interaction. The chemical bond constraints were calculated using the LINCS algorithm with a time step of 2fs.

3. The design method according to claim 1, characterized in that: In step 4, molecular docking allows the DNA aptamer to bind to the target small molecule around the binding site. The flexibility was adjusted within the range, and the docking score was calculated using the AutoDockVina algorithm.

4. A DNA aptamer prepared by the method according to any one of claims 1 to 3, characterized in that: The target small molecule is the immunosuppressant tacrolimus (TAC) or mycophenolic acid (MMF); the nucleotide sequence of the aptamer is SEQ: 1 (for TAC) or SEQ: 2 (for MMF), or a derivative sequence containing a SEQ: 1 / SEQ: 2 binding fragment.

5. The DNA aptamer according to claim 4, characterized in that: The 5′ end of the aptamer is modified with a fluorescent label; the SEQ: 1 aptamer is labeled with FAM, and the SEQ: 2 aptamer is labeled with VIC.

6. A product for detecting immunosuppressants, characterized in that: include: The DNA aptamer according to claim 4 or 5; Graphene oxide (GO) or gold nanoparticles as fluorescence quenching carriers; Buffer system, pH range is 5.8~7.

4.

7. A method for detecting the concentration of an immunosuppressant in a sample, characterized in that: The following steps are involved: Step A: mixing and incubating the fluorescently labeled aptamer of claim 5 with a sample to be tested to form an aptamer-target complex; Step B: adding graphene oxide or nano-gold to form a test system by utilizing its fluorescence quenching properties; Step C: detecting the fluorescence intensity of the incubation solution and the test system, and calculating the target concentration according to the linear relationship between the change of fluorescence intensity and concentration; Among them, when the target is TAC, the linear relationship is Y=5.895X-0.1307; when the target is MMF, the linear relationship is Y=10.235X+0.0279, X is the concentration, and Y is the fluorescence intensity ratio.

8. The method according to claim 7, characterized in that: The incubation conditions are light-proof, 20-30° C., and 15-45 minutes; and the detection wavelength is set to 665 nm.

9. A detection device, characterized in that: include: A data acquisition module, used for acquiring fluorescence intensity signals; A processing module, calculating the target concentration based on the linear relationship described in claim 7; Output module, displays the test results and over-limit warning prompts.

10. A computer-readable storage medium, characterized in that: A computer program is stored, and when the program is executed, the detection method described in any one of claims 7-8 is implemented.

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