Screening And Application Of Aptamer Against Ribavirin Based On Capture-SELEX

Ribavirin is an antiviral drug widely used in the treatment of hepatitis C and other diseases,which has a strong inhibitory effect on the replication of the virus.However,many illegal vendors currently use ribavirin as a veterinary drug for disease prevention and treatment in the process of poultry breeding,which not only reduces the quality of meat,but also affects human health.The accumulation of the drug in the human body through the food chain can trigger drug resistance,reproductive toxicity and carcinogenicity.The use of ribavirin in veterinary drugs is explicitly banned by China Ministry of Agriculture and the US FDA.Traditional ribavirin detection methods such as immunoassay and chromatography have the disadvantages of difficulty in antibody preparation,complicated operation,and expensive equipment,which cannot satisfy the needs of rapid detection.Therefore,a new method for rapid,sensitive and accurate detection of ribavirin is urgently needed.As a new type of recognition molecule,aptamer has the advantages of good stability,strong specificity,convenient preparation,etc.,which can realize rapid and sensitive detection of the target.In this study,the high-affinity and specific aptamers of ribavirin were screened for the first time through the Capture-SELEX technology.Based on the screened aptamers,an efficient and sensitive fluorescence analysis method was constructed for the detection of ribavirin,which can realize the rapid detection of ribavirin in actual samples.At the same time we explored the binding sites of aptamers and ribavirin.Details are as follows:Firstly,a screening method for ribavirin aptamers was constructed based on Capture-SELEX.A 80 nt random library was synthesized in vitro,and a 13 nt biotinylated short complementary chain was designed.The interaction force between avidin and biotin was used to immobilize random library on avidin-coated nano-scale magnetic beads.After incubation,magnetic separation,PCR amplification,purification,and enzyme digestion,the secondary library was prepared for cyclic screening,and the screening process was monitored by the melting curve.After 15 rounds of enrichment,PCR products were analyzed by high-throughput sequencing.Seven of the sequences were selected as candidate aptamer by abundance analysis and secondary structure simulation.Secondly,we analyzed the binding characteristics of candidate aptamer sequences.The affinity of candidate aptamers were analyzed by the colorimetric method based on gold nanoparticles and the fluorescence method based on carbon nanoparticles.Two methods were used to verify the specificity of four aptamers with good affinity.APT-1 and ATP-5 showed good specificity.Based on the analysis results of affinity and specificity,APT-1 was the best aptamer for binding to ribavirin.The dissociation constants obtained by the two methods are34.34±6.038 nmol/L,61.19±21.48 nmol/LThirdly,a method for ribavirin detection in chicken samples based on aptamer recognition was constructed.Based on the fluorescence resonance energy transfer between carbon nanoparticles and FAM,fluorescence quenching and recovery were achieved.Within the detection concentration range of 1.0-50 ng/m L,the relative fluorescence intensity and target concentration show a good linear relationship y=21.747x+28.245 ng/m L(R~2=0.9918),the detection limit was 0.67 ng/m L.Different amounts of ribavirin were added in chicken and the recovery rates were calculated respectively.The results showed that the recovery rates ranged from 87.26%to 105.57%.Finally,we studied the binding sites between the aptamer and ribavirin.Based on the simulated secondary structure,the aptamer was truncated in units of stem loops and the affinity of the truncated sequences was verified.The binding region initially located on the stem loops.At the same time,molecular docking simulation was used to analyze the binding site and interaction between ribavirin and aptamer.The results showed that the aptamer interacts with ribavirin through 5 bases(G37,T38,A40,T53,A54).Five hydrogen bonds were formed to achieve stable bond.The key bases were further mutated and the affinity of the sequences after the base mutation was analyzed to verify the results of molecular docking.