CN112210587B

CN112210587B - Nucleic acid aptamer design method based on single nucleotide molecule docking

Info

Publication number: CN112210587B
Application number: CN202010924315.0A
Authority: CN
Inventors: 黄强; 宋梦华; 刘建平; 颜志超; 李园园
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2020-09-04
Filing date: 2020-09-04
Publication date: 2021-04-30
Anticipated expiration: 2040-09-04
Also published as: CN112210587A

Abstract

The invention belongs to the technical field of nucleic acid aptamer design, and particularly relates to a nucleic acid aptamer design method based on single nucleotide molecule docking. The invention starts from the experimental structure of the target small molecule, and obtains the nucleotide position combined around the target small molecule through hydration docking; subsequently assembling the discrete nucleotides into a complete aptamer; and finally, detecting the binding capacity of the designed aptamer and the target small molecule by adopting an MST (minimum shift keying) experiment. The invention has been used in aptamer design of toxin small molecule Okadaic Acid (OA); the toxin is a widely distributed marine organism toxin, and can cause diarrhea type shellfish poisoning, so that the detection of OA in marine products has important significance for food safety. An aptamer OA-D1 of the target OA is designed by a calculation method, and the MST experiment verifies that the equilibrium dissociation constant of the aptamer and OA is 75.6 nM; the calculation design method is reasonable and effective.

Description

Nucleic acid aptamer design method based on single nucleotide molecule docking

Technical Field

The invention belongs to the technical field of nucleic acid aptamer design, and particularly relates to a nucleic acid aptamer design method.

Background

Aptamers are ribonucleic acids (RNA) or single-stranded deoxyribonucleic acids (ssDNA) that generate high affinity for specific targets and can fold to form stable secondary or tertiary structures through hydrogen bonding between bases in the strand (2, 3). Aptamers generally consist of dozens of nucleotides, have small relative molecular mass, stable properties, and are easy to prepare and modify, and can be combined with targets such as ions, small molecules, proteins, cells, microorganisms, and the like. The aptamer combined with the small molecule target can specifically recognize and distinguish tiny differences among small molecules, is usually used as a recognition molecule of a chemical and microbial sensor, and is widely applied to the fields of food, environmental monitoring and the like (4).

Aptamers are mainly composed of SELEX (Systematic Evolution of L)The basic principle of the method is that the method is obtained by the screening of the technology of (2,3) the method comprises the following steps: first, a large amount (about 10) was synthesized^14-17Strip) random DNA or RNA oligonucleotide strands, followed by multiple cycles of screening and enrichment to select oligonucleotide strands that bind to the target molecule with high specificity and high affinity. Along with the circulation, the specificity and the affinity of the selected oligonucleotide chains for combining with the target molecules are gradually improved, and finally, one or more oligonucleotide chains which are specifically combined with the target molecules and have the highest affinity are obtained, namely, the oligonucleotide chains are the aptamers of the target molecules. However, the conventional SELEX technology tends to have problems of low efficiency and low hit rate (5). This is because the pool of oligonucleotides does not synthesize all possible oligonucleotide strands, and there are differences in the synthesis rates of different types of oligonucleotide strands, which affect the screening of aptamers.

Therefore, it is urgently needed to provide a computational design method to specifically design the aptamer targeting the target small molecule. The aptamer can be divided into two parts. The first part directly interacts with a target small molecule and contributes most of the binding free energy to the aptamer-target small molecule compound; the second part is a mini-hairpin structure and provides connection for forming a complete nucleic acid aptamer.

Disclosure of Invention

It is an object of the present invention to provide a method for designing aptamers using computational techniques to obtain aptamers specifically targeting small molecules of a target.

It is another object of the present invention to provide aptamers specifically targeting small target molecules obtained by the above design methods.

The method for designing the aptamer by adopting the computing technology adopts molecular docking and molecular dynamics simulation to obtain the position of the nucleotide combined around the target small molecule, thereby pertinently designing the aptamer of the target small molecule and simultaneously obtaining the stable conformation of the aptamer-target small molecule compound; and finally, detecting the binding capacity of the designed aptamer and the target small molecule by adopting an MST (minimum shift keying) experiment. The method of the present invention is called a nucleic acid aptamer design method based on Single-nucleotide molecular Docking, and is denoted as SDTA (Single-nucleotide Docking the Assembly) design method.

The method for designing the aptamer based on single nucleotide molecule docking provided by the invention comprises the following specific steps.

The first step is as follows: carrying out hydration docking of mononucleotide to obtain the position of the mononucleotide combined on the surface of the target small molecule;

the first step is aimed at obtaining the position of the single nucleotide bound on the surface of the target small molecule. Molecular docking enables rapid search for the proper orientation and conformation of the ligand molecule, optimizing the match of shape and interaction of the ligand and receptor molecules (6). The software used for the hydration docking is an AutoDock molecular docking software package (6), the receptor molecules are small target molecules, and the ligand molecules are nucleotides (A, C, G and T); in the first docking process, the receptor molecules are target small molecules, and the ligand molecules are nucleotides (A, C, G, T). In the subsequent docking process, the receptor molecules are the target small molecules and the nucleotides retained after docking, and the ligand molecules are still the respective nucleotides. During the docking process, the hydration docking phase of a single nucleotide is considered to be over when the nucleotide that is the docking ligand is no longer in contact with the target small molecule. The molecular docking adopts an Algorithm of Lamark Genetic Algorithm (LGA), the Algorithm combines the Genetic Algorithm and local search, the Genetic Algorithm is used for global search, and the local search is used for energy optimization; the docking method is semi-flexible docking, namely in the docking process, the conformation of a receptor molecule is not changed, and the conformation of each nucleotide is changed within a certain range. In order to increase the accuracy of molecular docking, the effect of water needs to be considered in the docking process, so the force field used is a hydrated docking force field (7) with discrete displaceable water and desolvation entropy. Meanwhile, in order to enable the nucleotide to be capable of sufficiently searching conformation, the length, the width and the height of a butt joint lattice are both set to be 100A; the spacing between lattice points is 0.375A, and the center of the abutting lattice is the geometric center of the receptor molecule.

The second step is that: assembling discrete nucleotides to obtain a nucleic acid short chain combined with a target small molecule;

through hydration docking in the first step, nucleotides combined with the target small molecules are obtained and are distributed around the small molecules in a discrete shape; discrete nucleotides are assembled into nucleic acid short chains through the distance relationship among the nucleotides, and a foundation is laid for obtaining a complete nucleic acid aptamer; the specific method comprises the following steps:

since in a standard DNA strand the distance between the atoms C3 '/C4' of a nucleotide and the atoms C4 '/C3' of an adjacent nucleotide is around 5 a. Using this distance as a standard, adjacent nucleotides are determined by measuring the distance between the C3 '/C4' atoms between nucleotides; while maximizing the assembly of discrete nucleotides together and ultimately into multiple isolated nucleic acid short strands.

To rationalize the structure of the nucleic acid short strand, the energy of the nucleic acid short strand is minimized. In this process, in order not to affect the interaction with the target small molecule, the side chain atoms are fixed, and only the main chain atoms are moved. The energy minimization adopts a GROMACS-5.1.4 molecular dynamics software package (1), an AMBER99bsc1 force field (8) and an SPC water model to model a nucleic acid short chain system; in the simulated system, the nucleic acid short chain was placed in the center of the water box and the minimum distance from the surface of the water box was 10 a; and inserting metal cations and corresponding anions to keep the solution environment neutral and achieve the required ion concentration; finally, the energy minimization is carried out (for example, 5,000 steps) by using the steepest descent method, and the nucleic acid short chain with reasonable structure is obtained.

The third step: performing molecular dynamics simulation to obtain stable conformation of the nucleic acid short chain-target aptamer complex;

after the nucleic acid short chain with reasonable structure is obtained in the last step, the nucleic acid short chain and the target small molecule are taken as a whole to carry out molecular dynamics simulation so as to obtain stable complex conformation. Wherein, the universal AMBER force field parameter of the target micromolecule is generated by adopting an Antechalber software package carried by AmberTools; meanwhile, a GROMACS-5.1.4 molecular dynamics software package, an AMBER99bsc1 force field and an SPC water model are adopted to model a simulation system; in a simulated system, a plurality of nucleic acid short chains as a whole are placed in the center of a water box, and the minimum distance from the surface of the water box is 10A; in order to keep consistent with the solution environment, metal cations and corresponding anions are inserted to keep the solution environment neutral and achieve the required ion concentration; the simulation process adopts periodic boundary conditions, static and Van der Waals interaction are respectively calculated by adopting PME and Cut-off methods, and the truncation distance is 14A; all chemical bonds are constrained by the LINCS algorithm; wherein the step length of the integration time is 1-3 fs; the specific process is as follows: firstly, minimizing the energy of a simulation system by using a steepest descent algorithm; then a v-throttle thermostat is adopted to control the system to balance the temperature, and the berendsen pressure is coupled to balance the pressure; finally, the molecular dynamics simulation is carried out on the system in an md integrator (9) for a time length of not less than 50 ns.

The fourth step: assembling nucleic acid short chains into a complete nucleic acid aptamer by adopting a mini-hairpin structure;

after the nucleic acid short chain conformation stably combined with the target small molecule is obtained, a mini-hairpin structure is adopted as a connecting component, and the isolated nucleic acid short chain is assembled into a complete nucleic acid aptamer. The Mini-hairpin structure has good thermal stability, and the nucleic acid sequence is short, so that the stable connection effect can be achieved (10). The stem structure of the Mini-hairpin comprises 3-4 pairs of complementary base pairs, and the ring structure comprises 3-4 isolated base pairs. The 3D structure of Mini-hairpin can be obtained by means of the RNA 3D structure modeling module (11) in the Rosetta program. To obtain a structurally sound aptamer, energy minimization is performed (see step two for energy minimization of short nucleic acid strands), during which the atoms on the short nucleic acid strands are immobilized and only the atoms on the mini-hairpin are moved.

The fifth step: performing molecular dynamics simulation to obtain stable conformation of the aptamer-target small molecule compound;

after obtaining the aptamer with reasonable structure, performing molecular dynamics simulation by taking the aptamer and the target small molecule as a whole to obtain the stable conformation of the aptamer-target small molecule compound. Wherein, the universal AMBER force field parameter of the target micromolecule is generated by adopting an Antechalber software package carried by AmberTools; the simulation system adopts a GROMACS-5.1.4 molecular dynamics software package, an AMBER99bsc1 force field and an SPC water model for modeling; in the simulated system, the nucleic acid aptamer was placed in the center of the water box with a minimum distance of 15 a from the surface of the water box; simultaneously inserting cations and corresponding anions to keep the solution environment neutral and achieve the required ion concentration; the simulation process adopts periodic boundary conditions, electrostatic interaction and van der Waals interaction are respectively calculated by adopting a PME (12) and Cut-off method, and the truncation distance is 14A; all chemical bonds are constrained by the LINCS algorithm; wherein the step length of the integration time is 1-3 fs; the temperature and the pressure of the system are maintained through v-restraint and berendsen coupling (13), and finally molecular dynamics simulation with the time length not less than 100 ns is carried out on the system.

And a sixth step: measuring the binding affinity of the aptamer and the target small molecule by adopting an MST (Messaging kit) experiment;

the microcalorie Thermophoresis (MST) (14) utilizes the Thermophoresis phenomenon of molecules, and analyzes the interaction between molecules by measuring Thermophoresis changes caused by tiny changes such as hydration layers, molecular sizes and charges when the molecules are coupled under a temperature gradient. For each set of MST experiments, the concentration of 16 target small molecules is designed to be used for 16 capillaries, and the target small molecules are diluted by a gradient dilution method 1:1, wherein the concentration of the nucleic acid aptamer in each capillary is fixed; the fluorescent label is marked on the circular structure of the aptamer mini-hairpin. The coupling of the aptamer and the target small molecule can influence the process of the thermophoresis, so that the change of a fluorescence signal value after the thermophoresis is caused. By measuring the fluorescence change signal of the MST process of the sample solution under different binding degrees, the dissociation equilibrium constant K of the binding reaction can be obtained by fitting_dThe value is obtained.

In one embodiment of the invention, the target small molecule is Okadaic Acid (OA) (15).

The invention also relates to the aptamer obtained by the design method. Comprising a high affinity aptamer OA-D1 that targets OA (15).

The SDTA design method provided by the invention obtains the binding position of mononucleotide by a molecular docking method, assembles discrete nucleotide into complete aptamer by molecular dynamics simulation, and detects the binding capacity of the aptamer and a target small molecule by adopting an MST (minimum shift keying) experiment. The aptamer is usually obtained by screening by a SELEX technology, but the SELEX experiment period is long, the cost consumption is high, and the screening success rate cannot be ensured. Molecular docking allows specific recognition of the binding site of the target small molecule, and molecular dynamics simulation allows for stable complex conformation. Thus, using this computational design approach, aptamers that target small molecules can be specifically designed.

Drawings

FIG. 1 is a flow chart of a method for computational design of aptamers.

FIG. 2 shows the chemical structure of OA.

FIG. 3 is a schematic representation of the OA complex of discrete nucleotides.

FIG. 4 is a schematic diagram of the nucleic acid short chain: OA complex.

FIG. 5 is a schematic diagram showing binding energy and stable conformation of the OA system, which is a nucleic acid short chain, with time.

FIG. 6 shows the sequence of aptamer OA-D1 and its secondary structure.

FIG. 7 is a graph showing the binding energy and stable conformation of the OA-D1: OA system over time.

FIG. 8 shows the MST assay of aptamer OA-D1.

Detailed Description

The specific process of the method of the present invention is further illustrated below by taking the calculation design method of the aptamer OA-D1 targeting OA (Okadaic acid) as an example.

The first step is as follows: carrying out hydration docking of mononucleotide to obtain the position of mononucleotide combined on the surface of small toxin molecule;

the first step is aimed at obtaining the position of the single nucleotide bound to the OA surface. The molecular docking method enables rapid search for the major binding sites of OA and the binding sites of the individual nucleotides (A, C, G, T) on their surface. In the first docking, the receptor molecule is a toxin small molecule OA, the structure of the receptor molecule is from a PDB database (ID:2I4E), and the ligand molecule is each nucleotide; in the next molecular docking, the receptor molecules are toxin small molecules OA and the nucleotides retained after docking, and the ligand molecules are still the respective nucleotides. The software used for docking is an AutoDock molecular docking software package; the docking algorithm is the Lamark Genetic Algorithm (LGA); the docking method is semi-flexible docking, namely in the docking process, the conformation of a receptor molecule is not changed, and the conformation of each nucleotide is changed within a certain range. In order to increase the accuracy of the docking, the effect of water needs to be considered in the docking process, so the force field used for docking is a hydrated docking force field with discrete replaceable water and desolvation entropy. To ensure that the nucleotides are able to search for the conformation sufficiently, the length, width and height of the abutting lattice are all set to 100 a. The spacing between lattice points was 0.375 a, with the lattice center being the geometric center of each acceptor molecule.

The second step is that: assembling discrete nucleotides to obtain a short nucleic acid chain bound to a small toxin molecule;

by the first step of hydration docking, positions of discrete nucleotides bound to the OA surface can be obtained, for a total of 10 nucleotides, as shown in fig. 3. Because the distance between the atoms C3 '/C4' of a nucleotide and the atoms C4 '/C3' of an adjacent nucleotide in a standard DNA strand is around 5 a. By measuring the distance between the C3 '/C4' atoms between nucleotides (<10 a) and maximally connects all discrete nucleotides, which are assembled into two short nucleic acid chains, named SC1 (short chain 1) and SC2, respectively. Each nucleotide is numbered in the order of the docking, and the two short strands of nucleic acid are 5 '-4T-2C-5A-7G-3' and 5 '-6C-3G-8T-1A-10G-3', respectively. In order to rationalize the structure of the nucleic acid short strand, it is energy minimized. In this process, the side chain atoms of the nucleic acid short chain are fixed and only the main chain atoms can move. The energy minimization adopts a GROMACS-5.1.4 molecular dynamics software package, an AMBER99bsc1 force field and an SPC water model to model a nucleic acid short chain system; in the simulated system, the nucleic acid short chain was placed in the center of the water box and the minimum distance from the surface of the water box was 10 a; and insert Na⁺And Cl^-Keeping the solution environment neutral and enabling the ion concentration to reach 0.15 nmol/L; then, energy minimization was performed by 5,000 steps using the steepest descent method to obtain a nucleic acid short chain with a rationalized structure, as shown in FIG. 4.

The third step: performing molecular dynamics simulation to obtain stable conformation of the nucleic acid short chain-toxin small molecule compound;

by energy minimization, structurally rationalized nucleic acid short strands can be obtained, followed by molecular dynamics modeling of 50 ns to obtain a stable conformation of the nucleic acid short strand-OA complex. The conformation of the complex is altered by molecular dynamics simulations, indicating that after assembly of discrete nucleotides into nucleic acid short strands, the interaction of the nucleotides with OA is subsequently adjusted in order to rationalize the structure, and finally a stable complex conformation is obtained, as shown in fig. 5. Meanwhile, the free binding energy of each frame in the simulated track is calculated by adopting a semi-empirical force field formula in the AutoDock. As can be seen from FIG. 5A, the free binding energy of the nucleic acid short chain: OA complex system was about-12 kcal/mol, indicating that the nucleic acid short chain has a strong interaction with OA.

The fourth step: assembling the nucleic acid short chain into a complete nucleic acid aptamer by adopting a mini-hairpin structure;

after the stable conformation of the nucleic acid short chain-OA compound is obtained, the nucleic acid short chain needs to be assembled into a complete nucleic acid aptamer, and a mini-hairpin structure can be used as a connecting component. The Mini-hairpin structure has good thermal stability and a short nucleic acid sequence, so that the Mini-hairpin structure can play a role in stable connection. To ensure the isolation of the two short strands of nucleic acid, RNAStructure will be used to predict the secondary structure comprising different mini-hairpin sequences, as shown in FIG. 6, the final mini-hairpin sequence obtained being 5 '-CGCGTAGCG-3'. The 3D structure of the mini-hairpin was then 3D modeled by the RNA 3D structure modeling module in the Rosetta program (11). The end bases of the two short strands of nucleic acids SC1 and SC2 were 4T and 10G, respectively, and the distance between P atoms in these two nucleotides was 19 a, which was similar to the distance between P atoms in complementary paired nucleotides. Therefore, the mini-hairpin is adopted to connect the two nucleotides, and the nucleic acid short chain is assembled into the complete aptamer. And energy minimization of this aptamer to give a structurally sound aptamer OA-D1. In this process, the atoms on the short nucleic acid chain will be immobilized, and only the atoms on the mini-hairpin will be moved.

The fifth step: performing molecular dynamics simulation to obtain stable conformation of the aptamer-toxin small molecule complex;

after obtaining the intact aptamer OA-D1, molecular dynamics simulation was performed to obtain a stable conformation of OA-D1: OA complex due to the addition of the mini-hairpin structure (FIG. 7B). As can be seen, OA stably binds between the two short strands of nucleic acid of aptamer OA-D1. At the same time, the free binding energy of the complex will be calculated for each frame of the simulated trajectory. As shown in FIG. 7A, the interaction between OA and OA-D1 is very stable, the average free binding energy of the complex system is still-12 kcal/mol, and the free binding energy of the complex system is similar to that of the nucleic acid short chain: OA complex system, which indicates that the addition of mini-hairpin does not affect the interaction between the aptamer and OA.

the binding capacity of the aptamer OA-D1 and OA is determined by a micro-calorimetry (MST) experiment. The MST experiment utilizes the thermophoretic phenomenon of molecules, and analyzes the interaction between molecules by measuring thermophoretic changes caused by small changes such as hydration layer, molecular size and surface charge when the molecules are coupled under a temperature gradient. This method has been widely used to analyze the interaction between proteins and proteins, proteins and nucleic acids, proteins and small molecules, nucleic acids and nucleic acids, nucleic acids and small molecules. The target small molecule OA used in the experiment is purchased from Taiwan algae research Co., Ltd, and the aptamer and the rest of the chemical reagents are purchased from Shanghai biological engineering Co., Ltd. The apparatus used for the experiments was a Monolith NT.115 intermolecular interactor from Nano tester Technologies, Germany.

1. Sample preparation: the buffer solution used for the MST experiment of the invention is 100 mmol/L Tris, 30 mmol/L NaCl and 4 mmol/L MgCl₂The pH of the system was maintained at 7.4. After the buffer preparation, the solution was filtered through a 0.2 μm aqueous filtration membrane to remove particles from the solution. The aptamer used in the experiment is diluted to 280 nmol/L by using a buffer solution, 200 mu L of nucleic acid sample is placed in a heater to be heated at 95 ℃ for 10 minutes, then ice-bath is carried out for 5 minutes, and finally standing is carried out for 5 minutes at room temperature.

2. MST experiment detection: MST experiments for measuring K_dIn the value, the OA solution is sequentially diluted into 16 groups by adopting a gradient dilution method. The specific operation is as follows: prepared were 16 PCR tubes, numbered 1 to 16. To the first tube was added 20. mu.L of a 100. mu. mol/L OA solution. ② 10. mu.L of buffer solution was added to each of the 2 nd to 16 th tubes. Thirdly, transferring 10 mu L of the solution in the previous tube to the next tube in sequence, mixing the solution fully and uniformly, and finally sucking 10 mu L of the solution from the 16 th tube 20 mu L. Finally, 16 tubes of 10. mu.L OA solution with successively halved concentration were obtained. And fourthly, sequentially adding 10 mu L of 0.28 mu mol/L fluorescence labeled aptamer solution into each tube. The binding site of OA is in the middle of the nucleic acid short chain, so that the fluorescent label (6-FAM) is linked to the loop T base of the mini-hairpin structure of the aptamer. In this case, the initial concentration of OA diluted in the system was 50. mu. mol/L, and the concentration of the aptamer was 0.14. mu. mol/L. Incubated for MST experiments at room temperature in the dark for 2 h.

After the concentrations of aptamers OA-D1 and OA were determined, the samples were placed in the capillary and in turn placed in the grooves of the capillary holder, which was loaded into the MST instrument and the instrument door closed. And (3) sequentially filling the final concentration of OA in 16 capillaries on the operation interface, then setting MST Power to 40%, and clicking Start Cap Scan of the operation interface to check whether the initial fluorescence values of the scanned 16 capillaries are consistent (ensuring that the concentrations of the fluorescence labeling molecules are consistent). If the inconsistency will affect the collection and fitting of data, the deviation of fluorescence values is typically controlled to within 10%. Whether the molecules are gathered on the surface of the capillary can be judged by scanning the sample in the capillary, and when the scanning result is a smooth peak-shaped graph, the fact that the molecules of the sample are not gathered on the tube wall is indicated. When the result display is normal, the Start MST Measurement button may be clicked to perform the MST experiment. And after the experiment is finished, importing the data into MO. The results of the measurements are shown in FIG. 8, K for OA-D1_dThe value was 75.6 nM, indicating a high affinity of this aptamer for OA. The aptamer design method based on the toxin small molecule OA is proved to be reasonable and effective.

Reference character selection

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Claims

1. A method for designing a nucleic acid aptamer based on single nucleotide molecule docking is characterized by comprising the following specific steps:

the first step is as follows: carrying out single nucleotide hydration docking to obtain a single nucleotide position combined on the surface of the target small molecule;

wherein, the software used for the hydration docking is an AutoDock molecular docking software package, the receptor molecule is a target micromolecule, and the ligand molecule is each nucleotide (A, C, G, T); the algorithm adopted by molecular docking is a Lamark Genetic Algorithm (LGA), the algorithm combines a genetic algorithm and local search, the genetic algorithm is used for global search, and the local search is used for energy optimization; the docking method adopts semi-flexible docking, namely in the docking process, the conformation of a target small molecule is not changed, and the conformation of each nucleotide is changed in a certain range; in order to increase the accuracy of molecular docking, the effect of water is considered in the docking process, so the force field used is a hydrated docking force field with discrete replaceable water and desolvation entropy; to ensure that the nucleotides are able to search for a sufficient conformation, the length, width and height of the butt lattice are both set to 100 a; the spacing between lattice points is 0.375A, and the center of the butt lattice is the geometric center of the target small molecule;

obtaining nucleotides combined with the target small molecule by hydration docking in the first step, wherein the nucleotides are distributed around the small molecule in a discrete shape; discrete nucleotides are assembled into nucleic acid short chains through the distance relationship among the nucleotides, and a foundation is laid for obtaining a complete nucleic acid aptamer; the specific method comprises the following steps:

determining an adjacent nucleotide by measuring the distance between the C-3 'and C-4' atoms between nucleotides in a standard DNA chain at a distance of 5A between the atom C-3 'of the nucleotide and the atom C-4' of the adjacent nucleotide or at a distance of 5A between the atom C-4 'of the nucleotide and the atom C-3' of the adjacent nucleotide using this distance as a standard; simultaneously, the discrete nucleotides are assembled together to the maximum extent, and finally, a plurality of isolated nucleic acid short chains are assembled;

energy minimization of the nucleic acid short strand in order to rationalize the structure of the nucleic acid short strand; in this process, the side chain atoms are fixed, and only the main chain atoms are moved; the energy minimization adopts a GROMACS-5.1.4 molecular dynamics software package, an AMBER99bsc1 force field and an SPC water model to model a nucleic acid short chain system; in the simulated system, the nucleic acid short chain was placed in the center of the water box and the minimum distance from the surface of the water box was 10 a; and inserting metal cations and corresponding anions to keep the solution environment neutral and achieve the required ion concentration; finally, performing energy minimization by using a steepest descent method to obtain a nucleic acid short chain with a reasonable structure;

the third step: performing molecular dynamics simulation to obtain the stable conformation of the nucleic acid short chain-target small molecule compound;

after the nucleic acid short chain with reasonable structure is obtained in the last step, the nucleic acid short chain and the target small molecule are taken as a whole to carry out molecular dynamics simulation so as to obtain stable complex conformation; wherein, the universal AMBER force field parameter of the target micromolecule is generated by adopting an Antechalber software package carried by AmberTools; meanwhile, a GROMACS-5.1.4 molecular dynamics software package, an AMBER99bsc1 force field and an SPC water model are adopted to model a simulation system; in a simulated system, a plurality of nucleic acid short chains as a whole are placed in the center of a water box, and the minimum distance from the surface of the water box is 10A; in order to keep consistent with the solution environment, metal cations and corresponding anions are inserted to keep the solution environment neutral and achieve the required ion concentration; the simulation process adopts periodic boundary conditions, static and Van der Waals interaction are respectively calculated by adopting PME and Cut-off methods, and the truncation distance is 14A; all chemical bonds are constrained by the LINCS algorithm; wherein the step length of the integration time is 1-3 fs; the specific process is as follows: firstly, minimizing the energy of a simulation system by using a steepest descent algorithm; then a v-throttle thermostat is adopted to control the system to balance the temperature, and the berendsen pressure is coupled to balance the pressure; finally, performing molecular dynamics simulation on the system in an md integrator, wherein the time length of the molecular dynamics simulation is not less than 50 ns;

after a nucleic acid short chain conformation stably combined with a target small molecule is obtained, a mini-hairpin structure is adopted as a connecting component, and an isolated nucleic acid short chain is assembled into a complete nucleic acid aptamer; the stem structure of the Mini-hairpin comprises 3-4 pairs of complementary base pairs, and the ring structure comprises 3-4 isolated base pairs; the 3D structure of the Mini-hairpin is obtained by an RNA 3D structure modeling module in the Rosetta program; in order to obtain a nucleic acid aptamer with reasonable structure, energy minimization is carried out, in the process, atoms on a short chain of nucleic acid are fixed, and only atoms on mini-hairpin are moved;

after obtaining the complete aptamer, performing molecular dynamics simulation by taking the aptamer and the target small molecule as a whole to obtain stable complex conformation; wherein, the universal AMBER force field parameter of the target micromolecule is generated by adopting an Antechalber software package carried by AmberTools; the simulation system adopts a GROMACS-5.1.4 molecular dynamics software package, an AMBER99bsc1 force field and an SPC water model for modeling; in the simulated system, the nucleic acid aptamer was placed in the center of the water box with a minimum distance of 15 a from the surface of the water box; in order to keep consistent with the solution environment, metal cations and corresponding anions are inserted to keep the solution environment neutral and achieve the required ion concentration; the simulation process adopts periodic boundary conditions; the electrostatic interaction and the Van der Waals interaction are respectively calculated by adopting a PME method and a Cut-off method, and the truncation distance is 14A; all chemical bonds are constrained by the LINCS algorithm; wherein the step length of the integration time is 1-3 fs; maintaining the temperature and the pressure of the system through v-restraint and berendsen coupling, and finally performing molecular dynamics simulation on the system for a time length of not less than 100 ns;

and a sixth step: detecting the bonding strength of the aptamer and the target micromolecule by adopting a micro-calorimetric electrophoresis method MST experiment;

for each set of MST experiments, the concentration of 16 target small molecules is designed to be used for 16 capillaries, and the target small molecules are diluted by a gradient dilution method 1:1, wherein the concentration of the nucleic acid aptamer in each capillary is fixed; the fluorescent label is marked on the circular structure of the aptamer mini-hairpin; the coupling of the aptamer to the target small molecule affects the process of thermophoresis, thereby resulting in a change in the fluorescence signal value after thermophoresis; by measuring the change signal of the MST process fluorescence of the sample solution under different binding degrees, the dissociation equilibrium constant K of the binding reaction can be obtained by fitting_dA value;

wherein the targetThe small target molecule is Okadaic Acid (OA); the aptamer was designed and has the sequence OA-D1

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