Structure-based optimized design method for aptamer
阅读说明：本技术 一种基于结构的核酸适配体优化设计方法 (Structure-based optimized design method for aptamer ) 是由 黄强 宋梦华 刘建平 章琪 李干 于 2019-09-18 设计创作，主要内容包括：本发明属于核酸适配体设计技术领域,具体为一种基于结构的核酸适配体优化设计方法。本发明从SELEX技术筛选所得核酸适配体的3D结构出发,通过自发结合模拟获得小分子结合核酸适配体所形成复合物的3D结构；并分析复合物的结构和关键结合位点,利用所得信息指导高亲和力核酸适配体的优化设计；进而采用核酸适配体表面结合能的能貌图评估设计方案。本发明已用于特异结合毒素GTX1/4的核酸适配体GO18-T-d的优化设计中；该核酸适配体通过SELEX技术筛选所得,亲和力为75.63 nM。通过优化得到GO18-T-d的一个截短型核酸适配体(GO18-T-d_S),采用MST实验验证该核酸适配体的亲和力提高了20倍；说明本优化方法是合理且有效的。(The invention belongs to the technical field of nucleic acid aptamer design, and particularly relates to a structure-based nucleic acid aptamer optimal design method. The invention starts from the 3D structure of the aptamer obtained by SELEX technology screening, and obtains the 3D structure of a compound formed by small molecule combined aptamer through spontaneous combination simulation; analyzing the structure and key binding sites of the complex, and guiding the optimal design of the high-affinity aptamer by using the obtained information; further, the design scheme was evaluated using energy profiles of the surface binding energy of the aptamer. The invention has been used in the optimized design of the aptamer GO18-T-d which specifically binds to the toxin GTX 1/4; the aptamer was screened by SELEX technology with an affinity of 75.63 nM. A truncated aptamer (GO18-T-d _ S) of GO18-T-d is obtained through optimization, and the affinity of the aptamer is improved by 20 times through MST experiment verification; the optimization method is reasonable and effective.)
1. A nucleic acid aptamer optimal design method based on a structure is characterized by comprising the following specific steps:
the first step is as follows: performing ligand spontaneous binding unbiased molecular dynamics simulation to obtain a stable 3D structure of a complex formed by the target small molecule and the aptamer;
the method comprises the steps of generating general AMBER force field parameters of target small molecules by adopting an Antechamp software package carried by AmberTools, modeling a nucleic acid aptamer system by adopting a Gromacs-5.1.4 molecular dynamics software package, an AMBER99bsc1 force field and an SPC water model in order to avoid the influence of long-range interaction after the force field parameters of the small molecules are obtained, wherein in the simulation system, the nucleic acid aptamer is arranged in the center of a water cube, the minimum distance between the surface of the water cube and the nucleic acid aptamer is 15 Å, 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, in the simulation process, periodic boundary conditions are adopted, Van der Waals interaction between static electricity and Van der Waals is respectively calculated by adopting a PME method, the truncation distance is 14 Å, all chemical bonds are constrained by LINCS algorithm, wherein the integration time is 1-3 pressure, the temperature and the end-circle coupling temperature are maintained, and the final length of the molecular dynamics system is simulated by a linear algorithm, and the final simulation time of the molecular dynamics system is not less than 1 mu.
The second step is that: analysis of Complex Structure and Key binding sites
Obtaining molecular dynamics simulation tracks of the aptamer and the target small molecule from the first step, wherein the tracks show the whole process of binding the small molecule to the aptamer and a stable binding state; analyzing the interaction between the aptamer and the target micromolecule during the stable structure period according to the molecular dynamics simulation track of the target micromolecule combined to the aptamer to obtain the key combination site of the aptamer;
since the binding between the target small molecule and the aptamer is mainly through hydrogen bonds and van der waals interactions, in order to determine the key binding site, the number of hydrogen bonds formed between each nucleotide and the small molecule and the probability of being nearest to the small molecule are adopted to characterize the two interaction forces within the structural stability time; in the analysis of van der waals interactions, the distance between each atom of the nucleic acid aptamer and each atom of the target small molecule in each frame is calculated, and the nucleotide to which the atom of the smallest distance belongs is identified;
the third step: designing high affinity aptamers
Optimizing and designing the aptamer by analyzing the combination process of the target small molecule and the aptamer, the structure of the compound and the key combination site; wherein, the binding process of the target small molecule and the aptamer provides the orientation and the way of the small molecule entering the binding site; the structure of the complex provides a binding mode for the aptamer to the small molecule; ranking the importance of the nucleotides according to the information of the key binding sites; based on this information, nucleotides at specific sites are specifically mutated, aptamers are extended or truncated, and the affinity of the aptamers is increased;
the fourth step: mapping the surface binding energy of aptamer
The surface binding energy field plays a leading role in the probability of ligand micromolecules binding on the surface of the nucleic acid aptamer, for a given simulation system, the binding free energy of each frame of ligand micromolecules binding the nucleic acid aptamer in the MD simulation is calculated by using a semi-empirical energy function in the AutoDock-4.2, and then the energy appearance graph of the surface binding energy of the nucleic acid aptamer is drawn by using the discrete energy value data sets; in order to locate all target small molecules in the same coordinate system, the aptamer is set as a sphere, and then the geographic coordinate system is used for defining the MD position of the small molecules; then, unfolding the sphere into a two-dimensional plane graph by adopting an equiangular projection method, and converting the longitude and the latitude of the micromolecule into a horizontal coordinate and a vertical coordinate respectively; smoothing the discrete binding free energy of the aptamer and the small molecule by adopting a thin plate spline method, and projecting the smooth discrete binding free energy onto a two-dimensional plane; the affinity of different aptamers was evaluated based on a profile of their surface binding energy.
2. The method for optimally designing the nucleic acid aptamer based on the structure as claimed in claim 1, wherein the nucleic acid aptamer is G-tetrad type GO18-T-d, and the target small molecule is targeting GTX 1/4; and optimizing to obtain an aptamer which is marked as GO18-T-d _ S.
3. An aptamer obtained by the optimal design method according to claim 1.
4. An aptamer obtained by the optimal design method according to claim 2.
Technical Field
The invention belongs to the technical field of nucleic acid aptamer design, and particularly relates to a nucleic acid aptamer optimal 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 within the strand (1, 2). 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 (3).
The aptamer is mainly obtained by SELEX (Systematic Evolution of Ligands by exponential enrichment ligand Evolution) (1,2) technical screening, and the basic principle is as follows: first, a large amount (about 10) was synthesized14-17Strip) 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 often has the problems of low efficiency and low hit rate. 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 propose a post-SELEX (post-SELEX) optimization design method to improve the affinity of the aptamer to the target small molecule, but rational optimization design requires structural information of the binding of the aptamer to the target small molecule. Theoretically, the 3D structure of the complex formed by the small molecule binding aptamer can be obtained by X-ray crystallography (4) and Nuclear Magnetic Resonance (NMR) experimental methods (5). However, aptamers have great flexibility, and these methods are difficult to apply in large quantities.
Disclosure of Invention
An object of the present invention is to provide a method for optimizing aptamers based on the structure of the complex and key binding sites, using molecular dynamics simulation, denoted as post-SELEX optimization.
Another object of the present invention is to provide a truncated high affinity variant of aptamer GO18-T-d (GO18-T-d _ S) targeting
The method for optimizing the aptamer by adopting molecular dynamics simulation provided by the invention obtains the stable structure and the key binding site of the complex formed by the target small molecule binding aptamer by adopting spontaneous binding simulation, and avoids the problem that the aptamer is difficult to obtain the 3D structure through an experimental method due to strong flexibility. Affinity enhanced aptamers are then rationally designed by analyzing the structure and key binding sites of the complex and the optimization protocol is evaluated using a profile of the aptamer surface binding energy. Provides a reasonable and effective post-SELEX optimization method for the aptamer obtained by SELEX screening.
Specifically, the structure-based aptamer optimization method provided by the invention is marked as a post-SELEX optimization method, and comprises the following specific steps:
the first step is as follows: unbiased molecular dynamics simulation of ligand spontaneous binding
The first step is aimed at obtaining a stable 3D structure of complexes formed by target small molecules binding to aptamers.a simulation of unbiased Molecular Dynamics (MD) of ligand spontaneous binding not only enables reliable determination of binding paths and binding sites of ligands, but also enables stable conformation of complexes (6) where the Generic AMBER Force Field (GAFF) parameters of target small molecules will be generated (7) using AMBER tools self-contained Antechamber software package after obtaining force field parameters of small molecules, distance of small molecules to aptamer surface should be above 15 Å to avoid influence of long-range interaction, while using gromac-5.1.4 molecular dynamics software package, AMBER99bsc1 force field and SPC water model to model the aptamer system (7,8) in a simulation system where aptamer is placed in the center of water cube, minimum distance of water surface to aptamer 15, minimum distance of aptamer to Particle aptamer 15, insert ion into corresponding ionic systems (cs 32) to keep the ionic interaction time of complex between
The second step is that: analysis of Complex Structure and Key binding sites
After the last step is finished, molecular dynamics simulation tracks of the aptamer and the target small molecule are obtained, and the tracks show the whole process of binding the small molecule to the aptamer and a stable binding state. The 3D atomic structure of the aptamer complex contributes to rational optimization of design. According to the invention, the interaction between the aptamer and the target micromolecule during the stable structure is analyzed according to the molecular dynamics simulation track of the target micromolecule combined to the aptamer, so as to obtain the key combination site of the aptamer. Because the binding between the target small molecule and the aptamer is mainly through hydrogen bonds and van der waals interactions, in order to determine the key binding site, the number of hydrogen bonds formed between each nucleotide and the small molecule and the probability of being nearest to the target small molecule are adopted to characterize the two interaction forces in the structural stability time; in the analysis of van der waals interactions, the distance between each atom of the nucleic acid aptamer and each atom of the target small molecule in each frame is calculated, and the nucleotide to which the atom of the smallest distance belongs is identified.
The third step: designing high affinity aptamers
By analyzing the binding process of the target small molecule and the aptamer, the structure of the complex and the key binding site, the aptamer is rationally and optimally designed. Specifically, the binding process of the target small molecule and the aptamer provides the orientation and the way of the small molecule to enter the binding site; the structure of the complex provides a binding mode for the aptamer to the small molecule; information on key binding sites allows ranking the importance of nucleotides. Based on this information, therefore, the nucleic acid aptamers are specifically mutated at a specific site, extended or truncated, so that the affinity of the aptamers is increased. The lack of such information can only be used for blind experiments such as batch mutation, which results in waste of time and money.
The fourth step: mapping the surface binding energy of aptamer
The surface binding energy field dominates the probability of ligand small molecules binding to the surface of the aptamer (11). For a given simulation system, the binding free energy of the small molecules of the ligand binding aptamer in each frame in the MD simulation is calculated by using a semi-empirical energy function in the AutoDock-4.2(12), and then an energy profile of the surface binding energy of the aptamer is drawn by using the discrete energy value data sets. To locate all target small molecules in the same coordinate system, the aptamer is set as a sphere, and then the geographic coordinate system is used to define the MD position of the small molecule. Then, the sphere is expanded into a two-dimensional plane graph by an isometric projection method, and the longitude and the latitude of the small molecule are respectively converted into an abscissa and an ordinate. The discrete binding free energy of the aptamer and the small molecule is smoothed by a Thin-plate spline (Thin-plate Splines) method and projected onto a two-dimensional plane. The affinity of different aptamers was evaluated based on a profile of their surface binding energy.
In one embodiment of the invention, the aptamer is G-tetrad type GO18-T-d, and the target small molecule is targeting
The post-SELEX optimization method provided by the invention obtains a stable structure of a complex through spontaneous binding simulation, and adopts an energy profile graph of the surface binding energy of the aptamer to evaluate the binding capacity and probability of the aptamer and a small molecule. Rational optimization design requires 3D atomic structural information of the complex, and since the aptamer has strong flexibility, it is difficult to obtain the 3D configuration by conventional experimental methods, and spontaneous binding molecular dynamics simulation can well replace this process. The energy profile of the surface binding energy may guide the increase in affinity both in binding capacity and in binding probability. Thus, using this optimization method, aptamers can be rationally optimized to increase their affinity.
Drawings
FIG. 1 is a flow chart of a post-SELEX optimization method.
FIG. 2 shows the GO18-T-d sequence and the GTX1/4 structural formula.
FIG. 3 is a graph of the binding energy of the GO18-T-d: GTX1/4 system as a function of time.
FIG. 4 is a diagram showing the structural change of GO18-T-d: GTX1/4 complex.
FIG. 5 is an overlay of the ten lowest energy GO18-T-d: GTX1/4 complexes.
FIG. 6 shows the probability and number of hydrogen bonds formed that each nucleotide in GO18-T-d is closest to GTX 1/4.
FIG. 7 is a graph of the energy profile of the surface binding energy of aptamers.
FIG. 8 shows MST experiments with aptamers.
Detailed Description
The specific process of the method is further illustrated by taking the optimized design method of G-quadruplet aptamer GO18-T-d of targeted GTX1/4 (gonyautoxin 1/4).
The first step is as follows: performing spontaneous binding molecular dynamics simulation
The first step is a spontaneous binding simulation without preference, with the aim of obtaining a stable and reasonable GO18-T-D: GTX1/4 complex 3D structure. The sequence of aptamer GO18-T-d and the structure of toxin small molecule GTX1/4 are shown in FIG. 2. The structure of GO18-T-D consists of 3D-Nus (13) and Aut in VMD (14)The GAFF parameter of the toxin molecule GTX1/4 is generated by adopting an Antechamber software package carried by AmberTools, after the force field parameter of the small molecule is obtained, the distance between the small molecule GTX1/4 and the GO18-T-d surface is more than 15 Å in order to avoid the influence of interaction, and simultaneously, the Gromacs-5.1.4 molecular dynamics software package, the AMBER99bsc1 force field and an SPC water model are adopted to model a nucleic acid aptamer simulation system, in the simulation system, the nucleic acid aptamer is arranged at the center of a water cube, the minimum distance between the surface of the water cube and the nucleic acid aptamer is 15 Å, because the nucleic acid aptamer GO18-T-d is a G-tetrad, a metal cation Mg-is required to be arranged at the centers of two planes of the G-tetrad2+On the one hand, the structure of the G-quadruplex is stabilized, and on the other hand, the small molecule GTX1/4(15) is attracted. In order to ensure the consistency of the experiment and the simulation, the metal cation and the anion added into the solution are respectively Mg2+And Cl-In order to ensure that the central ion of the G-tetrad is Mg2+The simulation process adopts periodic boundary conditions, electrostatic and van der Waals interactions are calculated by PME and Cut-off methods respectively, the truncation distance is 14 Å, all chemical bonds are constrained by LINCS algorithm, and the integration time step is 2 fs.. the simulation system is firstly subjected to 5000-step energy minimization by a steepest descent algorithm, then is subjected to 50ps of balance at the temperature of 300K by a v-rescale thermostat control system, is subjected to 50 ps. of balance at the pressure of 1atm by berendsen pressure coupling, and finally is subjected to molecular dynamics simulation of the length of the system in an md integrator of 1 mu s.
The second step is that: analysis of Complex Structure and Key binding sites
Through spontaneous binding simulation, molecular dynamics simulation tracks of GO18-T-d and GTX1/4 can be obtained, the tracks show the whole process of combining GTX1/4 to GO18-T-d, as shown in FIG. 3 and FIG. 4, GTX1/4 is free in solution before 11.0 ns, then enters a binding region from the right side of A16, is combined with a main chain part of 5 ' -end of GO18-T-d, after 168 ns, the interaction of the main chain of 5 ' -end and GTX1/4 is weakened, GTX1/4 is greatly adjusted in self conformation, finally crosses over G-tetrad, in 100 ns simulation, 5 ' -end is structurally adjusted under the action of GTX1/4, finally forms a stable complex conformation at 360.7ns, as shown in FIG. 5, the stable complex conformations are formed by stacking of G3-5-7 ns, the stable complex conformations are formed by stacking of G27-T-5-T27-T-d, G-T-d, and G-T-d, and G-3-G-5-G-5-C are combined together at a stable planar structure, and a stable planar structure with a stable spacing of a stable planar structure formed by a narrow spacing between G27-G-7-G27-G-7-G19, a central transition, a planar structure, a stable planar structure, a linear transition, a.
According to the invention, the interaction between GTX1/4 and GO18-T-d during the stable structure is analyzed according to the molecular dynamics simulation track of the small molecule GTX1/4 combined to GO18-T-d, so as to obtain the key binding site of the aptamer. Unlike the contributing forces that lead to GO18-T-d forming a stable conformation by itself, there is no pi-pi stacking interaction between GO18-T-d and GTX1/4, mainly van der Waals and hydrogen bonding interactions. The probability that a nucleotide is closest to the small molecule, GTX1/4, is therefore used to characterize van der Waals interactions, and the number of hydrogen bonds formed by a nucleotide with a small molecule characterizes hydrogen bonding interactions. In the analysis of van der Waals interactions, the distance between each atom of the nucleic acid aptamer and each atom of GTX1/4 in each frame was calculated, and the nucleotide to which the atom of the smallest distance belongs was identified. The specific process is as follows: taking a total of 2000 frames of 700-900 ns as a structural stability stage, and calculating to obtain the nearest nucleotide to a small moleculeThe average probability. As shown in FIG. 6, GTX1/4 tended to bind to four major nucleotides: a2, C3, T7, and G12 with probabilities of 0.06, 0.27, and 0.59, respectively. Calculation of Hydrogen bonds all possible hydrogen bonds were analyzed using the gmxhbond module from Gromacs-5.1.4, the criterion for hydrogen bond definition being: r is less than or equal to rHB=0.35nm,α≤αHB=30 °. Hydrogen bonds the average number of hydrogen bonds formed by GTX1/4 with each nucleotide was still calculated using an average 2000 frame method. Similar to the probability of being closest to GTX1/4, most of the hydrogen bonds are formed with GTX1/4 and a2, C3, T7 and G12, the number of hydrogen bonds being 0.93, 0.84, 1.97 and 3.37, respectively. In addition, GTX1/4 also formed 0.31 hydrogen bonds with G13. The results, ordered by binding contribution, are: g12>T7>A2 is more than or equal to C3. The order of the strength of binding helps rationally design high affinity aptamers.
The third step: designing affinity-enhanced aptamers
The molecular dynamics simulation traces show that the 5' -end has strong flexibility before GO18-T-d forms a stable conformation. Therefore, the formation of stable pi-pi stacking of 5' -end with A16 is more difficult, and it will block the entry of GTX1/4 into the binding site. Therefore, the 5' -end truncated partial nucleotide is considered to be capable of exposing the binding site of GO18-T-d and improving the binding efficiency of GTX1/4 and GO 18-T-d. Also, as seen in the second step, T7 has strong hydrogen bonding and van der Waals interactions with GTX1/4, in order to prevent the direct exposure of T7 to the solution from causing increased volatility, therefore, the first 5 nucleotides were truncated by the 5' -end, leaving T6 and T7. By comprehensively analyzing the structure and key binding sites of the complex, a novel aptamer is designed and is marked as GO18-T-d _ S.
The fourth step: mapping the surface binding energy of aptamer
To evaluate the optimized aptamers, an energy profile of the surface binding energy of two aptamers, GO18-T-d and GO18-T-d _ S, was plotted. For a given aptamer GTX1/4 system, the binding free energy of GTX1/4 binding aptamers per frame in MD simulations was calculated using the semi-empirical energy function in AutoDock-4.2. These discrete energy value data sets are then used for plottingProfiling of surface binding energy of aptamer. To locate all small molecules in the same coordinate system, we set the aptamer as a sphere and then use the geographic coordinate system to define the MD position of
To elucidate the binding ability of aptamers to GTX1/4, the binding energies were classified into four classes based on local energy minima: deep potential well (binding energy less than or equal to-8.0 kcal. mol)-1) Shallow potential well (-8.0 < binding energy ≦ -4.0 kcal. mol--1) Energy barrier (-4.0 < binding energy ≦ 0 kcal. mol.)-1) And energy mountain range (binding energy is more than or equal to 0 kcal. mol)-1). Also defined, the combination of aptamer, GTX1/4 complex, was considered successful binding only when GTX1/4 entered the deep potential well. By comparing the energy profiles of the two aptamers GO18-T-d and GO18-T-d _ S, as shown in FIG. 7, GO18-T-d contains one deep potential well and two shallow potential wells while GO18-T-d _ S contains two deep potential wells and one shallow potential well, with fewer grids in the shallow potential wells. The deep well of GO18-T-d contains 167 cells in total, which is 117 cells less than the 284 cells of GO18-T-d _ S. This indicates that GTX1/4 is more likely to bind to GO18-T-d _ S and is less likely to be trapped in the shallow well, and GO18-T-d _ S has a higher affinity.
The fifth step: MST experimental verification
This experiment used a microcalorimetric electrophoresis (MST) assay to determine the binding affinity of aptamers to
1. Sample preparation: the buffer solution used for the MST experiment of the invention is consistent with the simulation condition and is 150mmol/LMgCl2And 20 mmol/L Tris-HCl was added to maintain the pH of the system at 7.5. 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 20 mu mol/L by using buffer solution, 200 mu L of nucleic acid sample is placed in a heater to be heated for 10 minutes at 95 ℃, the heater is closed, and then the temperature is reduced to room temperature at 2 ℃/min.
2. MST experiment detection: MST experiments for measuring KDIn this case, the GTX1/4 solution is diluted sequentially into 16 sets by using a gradient dilution method, specifically, ① prepares 16 PCR tubes numbered 1 to 16, 20. mu.L of 8. mu. mol/LGTX1/4 solution is added to the first tube, 10. mu.L of buffer solution is added to the 2 nd to 16 th tubes, ③ transfers 10. mu.L of the previous tube to the next tube in sequence and mixes them well, 10. mu.L is sucked from the 20. mu.L solution of the 16 th tube, 10. mu.L of 16 tubes are finally obtained, and the concentration of the GTX1/4 solution is reduced in sequence, ④ adds 10. mu.L of 0.4. mu. mol/L of fluorescence labeled aptamer solution to each tube in sequence, molecular dynamics simulation shows that the binding site of GTX1/4 is above G-tetrad, so that the fluorescence label (6-FAM) is linked to the 3' -end of the aptamer, the initial concentration of GTX 864/L of the aptamer in the system is 364. mu. mol/L of the initial concentration of the diluted nucleic acid aptamer, and the concentration of the aptamer is 0.2. mu. mol/L of the concentration of the MST aptamer for incubation at room temperature after the light protection.
After the concentrations of aptamer and GTX1/4 were determined, the sample was placed in the capillary and in turn placed in the groove of the capillary holder, which was then closed after the MST instrument was loaded. Fill out the final concentration of GTX1/4 in 16 capillaries in sequence on the operating interface, then set the MST Power toAnd 40%, clicking the Start Cap Scan of the operation interface again to check whether the initial fluorescence values of the scanned 16 capillaries are consistent (ensuring the consistency of the concentration of the fluorescence labeling molecules). 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. And when the result is displayed normally, clicking a Start MST Measurement button to carry out an MST experiment. And after the experiment is finished, importing the data into MO. The measurement results are shown in FIG. 8, K of GO18-T-dDK with a value of 75.63nM, GO18-T-d _ SDThe value was 3.60 nM. This indicates that the newly designed aptamer, GO18-T-d _ S, has a significantly improved affinity (>20 times). The structure-based aptamer post-SELEX optimization method proved to be reasonable and effective.
Reference to the literature
1. Ellington, A.D. and Szostak, J.W. (1990) In vitro selection of RNAmolecules that bind specific ligands.Nature, 346, 818-822.
2. Tuerk, C. and Gold, L. (1990) Systematic evolution of ligands byexponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.Science, 249, 505-510.
3. Kim, Y.S. and Gu, M.B. (2014) Advances in aptamer screening and smallmolecule aptasensors.Adv Biochem Eng Biotechnol, 140, 29-67.
4. Parkinson, G.N., Lee, M.P. and Neidle, S. (2002) Crystal structure ofparallel quadruplexes from human telomeric DNA.Nature, 417, 876-880.
5. Webba da Silva, M. (2007) NMR methods for studying quadruplex nucleicacids.Methods, 43, 264-277.
6. Pan, A.C., Borhani, D.W., Dror, R.O. and Shaw, D.E. (2013) Moleculardeterminants of drug-receptor binding kinetics.Drug Discov. Today, 18, 667-673.
7. Salomon-Ferrer, R., Case, D.A. and Walker, R.C. (2013) An overview ofthe AMBER biomolecular simulation package.Wiley Interdisciplinary Reviews Computational Molecular Science, 3, 198-210.
8. Abraham, M.J., Murtola, T., Schulz, R., Páll, S., Smith, J.C., Hess,B. and Lindahl, E. (2015) GROMACS: High performance molecular simulationsthrough multi-level parallelism from laptops to supercomputers.Softwarex, 1-2, 19-25.
9. Darden, T., York, D. and Pedersen, L. (1993) Particle mesh Ewald: AnN-log(N) method for Ewald sums in large systems.The Journal of Chemical Physics, 98, 10089-10092.
10. Hess, B., Bekker, H., Berendsen, H.J.C. and Fraaije, J.G.E.M. (1997)LINCS: A Linear Constraint Solver for molecular simulations.J. Comput. Chem., 18.
11. Liu, Q., Herrmann, A. and Huang, Q. (2019) Surface Binding EnergyLandscapes Affect Phosphodiesterase Isoform-Specific Inhibitor Selectivity.Comput Struct Biotechnol J, 17, 101-109.
12. Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K.,Goodsell, D.S. and Olson, A.J. (2009) AutoDock4 and AutoDockTools4: Automateddocking with selective receptor flexibility.J. Comput. Chem., 30, 2785-2791.
13. Patro, L.P.P., Kumar, A., Kolimi, N. and Rathinavelan, T. (2017) 3D-NuS: A Web Server for Automated Modeling and Visualization of Non-Canonical3-Dimensional Nucleic Acid Structures.J. Mol. Biol., 429, 2438-2448.
14. Humphrey, W., Dalke, A. and Schulten, K. (1996) VMD: visual moleculardynamics.J Mol Graph, 14, 33-38, 27-38.
15. Bhattacharyya, D., Mirihana Arachchilage, G. and Basu, S. (2016)Metal Cations in G-Quadruplex Folding and Stability.Front Chem, 4, 38.
16. Seidel, S.A., Dijkman, P.M., Lea, W.A., van den Bogaart, G., Jerabek-Willemsen, M., Lazic, A., Joseph, J.S., Srinivasan, P., Baaske, P., Simeonov,A.et al.(2013) Microscale thermophoresis quantifies biomolecularinteractions under previously challenging conditions.Methods, 59, 301-315.。