Screening,Characterization And Preliminary Application Of Nucleic Acid Aptamer For Saxitoxin

Saxitoxin(STX)is one of the potent marine biotoxins that has high rate of lethality.STX has been studied by researchers since 1932,however,there are no effective treatments at present,and the existing detection methods have some defects such as poor reproducibility,cumbersome operation,high cost and time-consuming.Therefore,the emergence of nucleic acid aptamers has brought new opportunities for the detection and treatment of STX.Aptamers,as a new molecular recognition element,has extensive applications ranging from analysis,diagnosis to treatment due to various advantages such as high affinity and specificity,low batch to batch variation,freedom to incorporate chemical modification and immobilization.Although various screening techniques have emerged to optimize and accelerate aptamer screening over the past decades,there are few simple and efficient screening methods at present,especially for small molecular targets such as STX,which have low molecular weights and limited moieties for modification,immobilization and recognization.In this study,the library was immobilized through the novelly simple and efficient immobilized metal chelate-systematic evolution of ligands by exponential enrichment(IMC-SELEX)and the reported Capture SELEX for performing STX aptamer screening.After the binding mechanism between aptamers and STX was analyzed through molecular docking and dynamics simulation,the two aptamers obtained from the screening and optimization were constructed into aptasensors to detect STX.Moreover,the circular ss DNA aptamers were constructed by recombinant expression of ss DNA Ligase,and the biological activity of the circular aptamer was verified in mice.The main research contents and results are as follows:1.Screening of STX nucleic acid aptamer with immobilized library and preliminary identification of affinity.A simple and efficient technology has been proved in protein purification,which was first introduced into the screening of aptamers,namely IMC-SELEX.IMC-SELEX was established to immobilize the screening library by the efficient adsorption capacity of Ni-NTA column on ss DNA.The complex of ss DNA and STX was efficiently separated without complicated operation guidelines and techniques.Aptamer STX-R-75 and aptamer STX-G4-45 with high affinity for STX were screened from the random ss DNA library and the G-quadruplex ss DNA library by only 6 rounds of screening,with the K_dvalues of 210 n M and 42.6 n M,respectively.library and STX was generally higher than that obtained from random library.Immobilized ss DNA library by IMC-SELEX could effectively avoid the problem of affecting the natural conformation of target molecule STX.Compared with immobilized STX(10 rounds screening)and graphene oxide-SELEX(GO-SELEX,9 rounds screening),the screening efficiency of IMC-SELEX was significantly improved(6 rounds screening)and the screening operation was simple and convenient.The development of IMC-SELEX expands new approach for the aptamers screening of target molecules,especially small molecules.Furthermore,a loop ss DNA library was constructed based on the principle of Capture SELEX without the addition of T4 DNA ligase,so that the library interacted with STX on the basis of loop structure for avoiding the possible structural effects of cyclization and other modifications on linear aptamers.A high affinity aptamer 73for STX was obtained after 12 rounds of screening,with a K_d value of 55.7 n M.This result confirmed the feasibility of constructing a loop ss DNA library based on the Capture SELEX for aptamers screening of target molecules.2.Optimization of STX nucleic acid aptamer and studying of binding mechanism.Referring to the prediction results of three aptamers secondary structure by NUPACK software,the part region of primers and the hairpin structure of were truncated,and the core sequences 45e,73a and 75a were obtained.These three aptamers specifically bound to STX with the the K_d values of 21.2 n M,23.6 n M and 136 n M,respectively.The binding mechanism of the higher affinity aptamers 45e and 73a to STX was analyzed by molecular docking and molecular dynamics simulation.The simulation results showed that the target STX was mainly bound to the top of the aptamer by hydrogen bonding,van der waals interaction and electrostatic forces,and the binding free energies of 45e-STX and 73a-STX were-23.38 kcal/mol and-20.44 kcal/mol,respectively.These results reasonably explained that both aptamers 45e and 73a could bind to STX with high affinity.Based on this,the aptamers 45e-1(K_d=19 n M)and 73a-1(K_d=21.4 n M)were obtained by further optimization,which could be used as potential molecular recognition elements for the subsequent research.3.Preparation and application of STX nucleic acid aptamer sensors.Two STX aptamer biosensors were prepared with aptamers 45e-1 and 73a-1 as molecular recognition elements based on biolayer interferometry(BLI)for analyzing molecule interactions.The rapid detection and analysis of STX could be completed in 10 min,and the operation was relatively simple.Traditional hydrophilic interaction liquid chromatography coupled to mass spectrometry(HILIC-MS)takes 20 min and requires professional operators.Enzyme-linked immunosorbent assay(ELISA)takes at least 1 h,which is cumbersome and prone to cross-reaction.The detection limits of these two aptamer sensors were both 0.5 ng/m L without cross-reaction with other similar toxins,which were significantly lower than the STX safety standard(3 ng/m L)stipulated by WHO at home and abroad.Furthermore,the detection of different concentrations of STX in seawater was realized,and the recovery rate and coefficient of variation values(CV)fluctuated within the normal range.These results indicated that the aptasensors constructed based on the two aptamers both had excellent stability and reproducibility,and had the potential to be applied to the detection of STX in complex systems.4.Construction of STX circular nucleic acid aptamer and exploration of biological activity.The ss DNA Ligase was successfully expressed after constructing the plasmid vector.A large number of circular ss DNA aptamers were prepared that could resist to the degradation of ExoⅠ.BLI and ELISA were used to analysis the binding affinity of the circular aptamer to STX.These results showed that the binding affinity of STX to circular aptamer was improved,especially the affinity of the aptamer 73a-1 obtained from the loop ss DNA library increased by 1.6 times.Subsequently,it was verified that the circular aptamer itself had no obvious toxicity to mice.When the purified circular aptamer was incubated with STX and injected into mice,the average death time of mice was doubled,indicating that the circular aptamer could significantly delay the time of death of mice compared with the linear aptamer.This result showed a good prospect of increasing the stability of aptamers through cyclizing the aptamer with ss DNA Ligase,which expanded new ideas for the further study of aptamers.In conclusion,a novel screening method was introduced and a screening library was designed that could form a characteristic spatial structure.The simple and efficient IMC-SELEX and the reported Capture SELEX were used to immobilize the library for screening aptamers of STX.After analyzing the binding mechanism of aptamer and STX,two high affinity and specific aptamers were obtained through screening and optimization,and the aptamer sensor was constructed to achieve rapid detection and analysis of STX in complex samples.Furthermore,the circular ss DNA aptamers were constructed by recombinant expression of ss DNA Ligase,and the protective effect of circular ss DNA aptamers on mice was verified in vivo.This study laid a favorable foundation and provided a promising new approach for the aptamers screening,characterization and subsequent in-depth study of other target molecules,especially small molecules.