Screening And Application Of Aptamers Against Amantadine And Rimantadine

With the continuous expansion of the scale of aquaculture,the problems of drug abuse,illegal use and drug residue have gradually attracted people’s attention.The Ministry of Agriculture of China and the FDA of the United States have issued regulations to prohibit the addition of amantadine and rimantadine into veterinary drugs,but there are still a small number of businesses illegally adding these compounds into animal feed.The traditional detection methods such as chromatography,spectroscopy and enzyme-linked immunoassay for amantadine drugs are time-consuming and labor-intensive.The detection equipment is expensive,the detection is easily affected by environmental factors,and it is difficult to obtain antibodies against small molecular targets.In recent years,aptamer,as a new type of molecular recognition probe,has been widely used because of its advantages of convenient synthesis,easy modification and high stability.However,the study on aptamer screening and application of amantadine drugs has not been reported.In this study,for the first time,non-immobilized Capture-SELEX technology was used to successfully screen the aptamer with high affinity and strong specificity for amantadine and rimantadine,which solved the screening problem caused by the difficulty of immobilization of small molecules.A fluorescence analysis method based on the aptamer was constructed to realize the rapid and sensitive detection of amantadine and rimantadine residues without labeling,and the binding mechanism between the aptamer and the target was also studied.The details are as follows:Firstly,amantadine and rimantadine aptamers were screened based on Capture-SELEX technology.Aminated magnetic beads coated with avidin were used as the screening matrix.We designed a biotinylated capture probe and a full-length 80 nt DNA library with abundant library capacity.The library was immobilized to the magnetic bead through the specific binding of biotin-avidin,and after incubation,separation,elution,PCR amplification,PCR product purification,enzyme digestion and other steps.The aptamer pools of amantadine and rimantadine were obtained through 16 rounds of selection.After TA cloning and sequencing,38 and 34 aptamer sequences were obtained for amantadine and rimantadine,respectively.The homology and secondary structure of the sequences were analyzed,and 4 candidate aptamers were selected from amantadine and rimantadine aptamer pools.Secondly,the binding properties of the candidate adapters were analyzed based on the method constructed by SYBR Green I（SGI）dye and graphitic carbon nitride（g-C₃N₄）material.Combining the results of affinity and specificity analysis of these two methods,Am-20 and Rim-17 were obtained with high affinity and specificity for amantadine and rimantadine,respectively.The best dissociation constants of Am-20 and Rim-17 were 33.60±9.79 n M and38.06±8.00 n M respectively,and the aptamer showed low cross binding with its structural analogues and coexisting compounds.Thirdly,based on the screened aptamers combined with SGI dye,a fluorescent biosensor was constructed to detect amantadine and rimantadine.We optimized the SGI-aptamer binding time and aptamer-target incubation time.Under the optimal conditions,the relative fluorescence intensity had a good linear relationship with the target concentration within 1～100 ng/m L.The limits were 0.87 ng/m L and 0.96 ng/m L,respectively.The constructed method was applied to chicken samples,and the recoveries of amantadine and rimantadine were 94.68%～108.67%and90.53%～102.10%,respectively.The relative standard deviation（RSD）were 1.23%～4.18%and1.46%～3.55%,respectively.Finally,the binding mechanism of aptamer-target was explored.The binding sites of aptamer and target were determined by molecular simulation docking technology.Base mutation was used to predict binding patterns and the results of molecular simulation were verified.According to the simulation results of molecular docking and the secondary structure of the aptamer,the full-length sequence was tailor-designed to determine its key binding domain.Finally,the stem loop structure formed at the binding site was determined as the action unit for aptamer-target recognition.