Screening,Characterization,and Preliminary Applications Of Aptamers For Domoic Acid

Amnesic shellfish toxin domoic acid,a potent neurotoxin and glutamate agonist,is a marine biotoxin produced primarily by diatoms of the genera Pseudo-nitzschia and Nitzschia.DA is widely distributed in global oceans.It is high toxicity,acts quickly,lacks antidote,and can cause human vomiting,diarrhea,confusion,memory loss and even death.With the frequent occurrence of harmful algal bloom（HAB）,the risk of DA accumulation in seawater and seafood increases,posing a serious threat to the ecological environment,human health,and social economy.Currently,the developed DA detection methods mainly include high-performance liquid chromatography,immunoassay,capillary electrophoresis,etc.Although each has its own advantages,they still have limitations such as complex equipment,complex operation,long time consuming and poor specificity.It is still necessary to establish more rapid,efficient,and specific methods for DA detection.The novel molecular recognition element aptamer brings enlightenment to the analysis and detection of DA.Aptamers are generally obtained by systematic evolution of ligands by exponential enrichment（SELEX）.It has many advantages such as high affinity,high specificity,being easily modified and fixed,low synthesis cost.Currently,there are few reports on DA aptamers.In view of their very small molecular weight and incompatibility with classical target-immobilized SELEX,two library-immobilized SELEX techniques were parallelly used in this study to screen aptamers for DA.High affinity aptamers were selected through cloning sequencing,high-throughput sequencing（HTS）,bioinformatics analysis and biolayer interferometry（BLI）.Based on the results of secondary structure prediction,three-dimensional structure modeling,molecular docking,and molecular dynamics（MD）simulation,the binding mechanism of aptamers was explored,and truncated optimization was operated.Finally,two optimal aptamers with high affinity and high specificity were used respectively to prepare biosensors based on BLI,and the aptasensors were preliminary applied to the detection of real samples.The main results of this study are as follows.In this research,Capture-SELEX and newly developed MnO₂-SELEX were parallelly used in DA aptamer screening.It not only helped maintain the natural structure of target DA,but also avoided the non-specific enrichment of oligonucleotides caused by background adsorption.At the same time,kainic acid（KA）,a structural analogue of DA,was introduced as the counter target to enhance the screening specificity.The ssDNA library was immobilized on streptavidin（SA）magnetic beads with the help of biotin-labeled capture sequence.The DA dissolved in the selection buffer was incubated with the immobilized library,and the DA binding sequences were eluted.After 12 rounds of screening,the recovery ratio entered the plateau period,indicating the completion of screening.In addition,this study utilized the physical adsorption of MnO₂ nanosheets on ssDNA to achieve efficient immobilization of ssDNA library and established a novel MnO₂-SELEX technology.The ssDNA immobilization efficiency of this method is more than 99%.The extremely high library immobilization efficiency of this method enhanced the screening pressure,which could not only reduce the interference caused by trace free ssDNA in the system,but also avoid the eluting of oligonucleotides with no affinity or low affinity as far as possible.With higher screening efficiency,the end point was reached after 9 rounds of screening.Cloning sequencing and HTS analysis were performed on the products obtained by the two screening methods.The results showed that the sequences were effectively enriched by SELEX process and the sequence diversity decreased.Clustal X 2.1 and mfold were used for homology analysis,secondary structure prediction,and Gibbs free energy prediction,respectively.Using BLI technology,C1(KD value 1.69×10^-6 M)and M70(KD value4.82×10^-7M)with the highest affinity among the candidate aptamers were selected as the optimal aptamers obtained by two screening methods.After the primer sequence of aptamer C1 was removed,C1-s was obtained,and its affinity was only slightly improved.Based on the secondary structure prediction by mfold,C1-s was truncated to obtain four potential aptemers,in which C1-d(KD value 1.09×10^-7 M)showed the most significant affinity improvement.The primer sequence of aptamer M70(KD value 4.82×10^-7 M)was truncated to obtain M70-s,and its affinity(KD value 1.26×10^-7 M)was significantly improved.In addition,QGRS mapper shows that M70-s can form G-quadruplex structure,so it is difficult to optimize M70-s based on truncation process by mfold.Through BLI verification,C1-d and M70-s showed high affinity and specificity for DA and were the best choice after preliminary truncation optimization.The mechanism and critical sites of the interaction between aptamers and DA were explored to provide reference for further optimization of aptamers.Three-dimensional structural modeling of aptamers C1-s,C1-d and M70-s was performed,and molecular docking and MD simulation were performed with DA.The results of molecular calculation showed that the binding free energy of C1-s-DA was-13.51 kcal/mol.Five bases in C1-d formed hydrogen bond interactions with DA,and the binding free energy of C1-d-DA complex was-21.90 kcal/mol.The binding free energy of C1-s-DA was significantly lower than that of C1-s-DA.The lower the binding free energy,the higher the affinity of the aptamer to the target.Root mean square deviation（RMSD）showed that complex C1-d-DA reached a stable state faster than C1-s-DA.The data of conformation change,gyration radius and mass distance of C1-d-DA showed that C1-d gradually gathered and closely combined with DA to form more hydrogen bonds during the simulation.The affinity of truncated aptamer C1-d was significantly improved compared with that of original aptamer C1-s.In the aptamer M70-s,three G-tetrads,G8-G13-G30-G35,G9-G14-G31-G36,G10-G15-G32-G37,were stacked to form a G-quadruplex structure.There were five G bases in M70-s that form hydrogen bond interaction with DA,and the binding free energy of M70-s-DA is-20.98kcal/mol.The RMSD value,gyration radius and mass distance of M70-s-DA have little fluctuation overall,which reflected the highly stable combination of M70-s-DA.In contrast,M70-s-DA and C1-d-DA have similar binding free energies and form the same number of hydrogen bonds,but the distance between C1-d and DA to form hydrogen bond was shorter,so the binding free energy of C1-d-DA was slightly lower than M70-s-DA.Based on the results of molecular calculation and the structural information provided by mfold,the core sequences of aptamers C1-d and M70-s were preserved and some unnecessary nucleotides were removed.Further truncated aptamers C1-f(KD value 9.46×10^-8 M)and M70-b(KD value 8.31×10^-8 M)with increased association rate,decreased dissociation rate and improved affinity were obtained,which was conducive to the subsequent application research.The high affinity aptamers C1-d and M70-s were used to construct the biosensor for DA detection based on the sensitive and real-time BLI molecular interaction platform.The aptamers C1-f and M70-b were respectively coupled to the super streptavidin（SSA）sensor,and the sensor balancing,target binding and target dissociation procedures were set to realize sample detection.The limit of detection（LOD）and the limit of quantitation（LOQ）of the aptasensor C1-f were 7.23 n M and 24.10 n M,respectively,and the linear range was from200～4000 n M.Intra-group relative standard deviation（RSD）was 0.60%～2.83%and inter-group RSD were 1.60%～3.62%.The LOD of the aptasensor M70-b was 5.79 n M,the LOQ was 19.31 n M,and the linear range was 100～4000 n M.The intra-group and inter-group RSD of aptasensor M70-b were 0.53%～2.25%and 0.78%～3.39%.Both two aptasensors showed low LOD,LOQ,wide linear range,high precision,and high specificity,and could be used for DA detection in environments with interference from other types of marine biotoxins.At the same time,the aptasensor also has good repeatability and could be reused at least 10 times.The recovery rates of aptasensor C1-f in seawater and shellfish samples were 90.33%～100.74%and 92.42%～103.27%,respectively,and the recovery rates of aptasensor M70-b in seawater and shellfish samples were 96.22%～111.46%and 97.30%～113.80%,respectively.The aptasensor prepared in this study had good performance,high recovery rate and high accuracy in the real sample detection,which could realize the rapid,real-time,sensitive,and specific detection of DA.In conclusion,this research provided two molecular recognition components with high affinity and high specificity for DA,establishes a novel,efficient aptamer screening method,explored the binding mechanism of DA and aptamers for the first time,and provided a new idea and theoretical basis for the screening and optimization of aptamers for small moleculars.In this study,the BLI aptasensor of DA was prepared for the first time,which provided technical reserve for the rapid detection and analysis methods of DA,and was expected to contribute to the ecological environment,food safety,and human health.