Screening,Binding Mechanism,and Preliminary Application Of Aptamers For Gymnodimine-A

Gymnodimines（GYMs）belong to Cyclic imines（CIs）,which are widely distributed marine biotoxins produced by dinoflagellates.GYMs have potent toxicity with characteristics of"fast-acting",which can enter organisms through the food chains or aerosols,causing great harm to human health and aquaculture.Currently,the developed detection methods include mouse bioassay,liquid chromatography-tandem mass spectrometry and receptor-binding method,etc.These methods have defects of high cost,complicated operation,and low specificity.A specific,sensitive,and efficient detection method is urgently needed.Aptamers are novel molecular recognition elements known as"chemical antibodies".They are single-stranded oligonucleotides that can specifically bind to the target.They are screened by systematic evolution of ligands by exponential enrichment（SELEX）.They have merits of good specificity,high stability,easy synthesis,easy modification,and low immunogenicity.No GYMs-related aptamers have been reported.Conventional aptamer screening adopts the strategy of immobilizing the target.However,GYMs are lipidsoluble molecules with no charge,small molecular weight,limited number of chemical groups,and lack of groups required for immobilization.They are difficult to immobilize onto solid medium,and screening their aptamers is challenging.This research screened aptamers targeting gymnodimine-A（GYM-A）by Capture-SELEX with the strategy of immobilization of libraries instead of immobilization of toxins,and aptamers were identified and optimized.The structural basis of binding between aptamer and GYM-A was explored by molecular structure prediction,molecular docking,and molecular dynamics experiment.At the same time,with the help of a sensitive,fast,high-throughput,and low-cost technology-biolayer interferometry（BLI）,an aptamer-based biosensor was developed.The main results were as follows.Firstly,Capture-SELEX was used to screen aptamers targeting GYM-A.About 10¹⁵ss DNA oligonucleotides were contained in a randomly synthesized initial library,and each round of Capture-SELEX included four steps:library immobilization,target incubation,PCR amplification and single-strand preparation.In the screening process,microcystin-LR（MC-LR）was used as the counter target.The recovery rate of ss DNA in each round was monitored.After 12 rounds of screening,the recovery rate was no longer increased,and the ss DNA enriched in this round was cloned and sequenced.Clustal X 2.1 software was used for cluster analysis of the obtained sequences,and these sequences were divided into 6families.The secondary structure and Gibbs free energy of the sequences were predicted by mfold software,and these sequences with higher similarity or lowest Gibbs free energy in each family were synthesized as candidate aptamers.The K_Dvalue of aptamer G48（K_D=288 n M）was the lowest which was obtained by measuring the affinity with the toxin by BLI technology.G48 was optimized by truncation,and the aptamer G48nop（K_D=95.30n M）with two times higher affinity towards the toxin was obtained.Circular dichroism experiments were used to analyse G48nop,and the results showed that positive peaks existed at 216 nm and 273 nm,and a negative peak existed at 245 nm which indicated that the secondary structure of G48nop contained two structures-a parallel G-quadruplex and a B-form double-stranded DNA.There was a shift in the spectrum after the addition of GYM-A,indicating that GYM-A bound with G48nop.In the BLI experiment,G48nop had no affinity with other ten typical marine biotoxins,but it had affinity with samples containing only GYM-A alone or GYM-A mixed with other toxins,which indicated that G48nop had good specificity.In the microscale thermophoresis experiment,the K_Dvalue of G48nop binding to GYM-A was 34.50±1.72 n M,and G48nop had no or low affinity with other four typical marine biotoxins,which further verified that G48nop had good binding affinity and specificity with GYM-A.Secondly,the structural basis of binding between aptamer GYM-A and G48nop was characterized,and the possoble binding mode of GYM-A and G48nop was deduced.QGRS mapper online software,3D-Nus website,RNA composer website,Discovery studio 4.5 software,Py MOL 2.52 software,and NAMD 2.10 software were used to construct nine kinds of three-dimensional structure models of G48nop based on G-quadruplex.Gromacs 5.1.4 software was used for molecular kinetic simulation,and the results showed that the three-dimensional structure of the parallel G-quadruplex was of minimum change within 10 ns,and the structure was the most stable,which indicated G48nop formed three-dimensional structure based on the parallel G-quadruplex.This structure was named Q3.Q3 was formed by stacking two layers of ring planes byπ-πstacking,and four guanines in each ring plane were connected by Hoogsteen hydrogen bonds.Ox DNA 2.4 software,Discovery studio 4.5 software,and Gromacs 5.1.4 software was used to construct a three-dimensional structure model of G48nop containing hairpin structure.This structure was named H1.H1 consisted of two parts-a loop and a stem.Stable base pairs were formed by nine pairs of bases in the stem by hydrogen bonding.Auto Dock 4.2 software and Auto Dock Vina 1.2.0 software were used to perform molecular docking and calculation of binding energy of Q3 and H1 with GYM congeners,respectively.The results showed that GYM-A could bind to the groove at the top of Q3 by12 CH…πbonds or could bind to the H1 in the the cavity of stem by 2 hydrogen bonds and7 CH…πbonds.The binding energiesΔG of Q3 and H1 toward GYM-A were-9.5kcal/mol and-12.1 kcal/mol,respectively,and the binding energies were low,while the binding energy of H1 was lower,indicating that H1 was more stable.The binding energies of GYM-B and GYM-C toward Q3 or H1,respectively,were higher than that of GYM-A,indicating that either Q3 or H1 could specifically bind to GYM-A.Molecular dynamics results showed that in 100 ns,the root mean square deviation（RMSD）fluctuation of Q3was greater than that of H1,and the RMSD fluctuation of the complex formed by Q3 and GYM-A was greater than that of the complex formed by H1,indicating that the monomer H1 was more stable,and the complex formed by H1 and GYM-A was more stable which further verified the correctness of the molecular docking results.Therefore,GYM-A could bind to the stem of H1,and this binding mode was more stable.Finally,an aptamer-based biosensor for detecting GYM-A was developed based on BLI technology.The detection steps of BLI technology were set as baseline,aptamer coupling,re-baseline,association,and dissociation.Refer to the international harmonization conference standards,the performance and feasibility of the sensor were characterized.The characterization contents included linear range,limit of detection,limit of quantification,specificity,precision,repeatability,and accuracy.The aptasensor for the detection of GYM-A had a linear range of 55-875 n M,a detection limit of 6.21 n M,and a quantification limit of 20.72 n M.There was no cross-reaction with the typical toxins from four categories of marine biological toxins,and GYM-A could be detected in a complex environment where a variety of toxins existed.The inter-assay and intra-assay relative standard deviations were 0.34-4.57%.Each sensor could be reused at least 20 times.In the real sample detection,the recovery rate was 96.65-109.67%,and the recovery was high.Thus,the aptasensor was of good sensitivity,specificity,precision,repeatability,and accuracy.In conclusion,the aptamer G48nop of high affinity and high specificity targeting GYM-A was screened for the first time at home and abroad in this study.Molecular computing found that G48nop could form a hairpin three-dimensional structure when binding with GYM-A.Besides,an aptamer-based biosensor with good performance was developed.The research provides a successful case of aptamer screening for small molecules lacking the required immobilization groups,provides ideas for exploring the mechanism of small molecules and aptamers,and provides technical reserve for further promoting the application of aptasensors in marine biotoxin detection.