Screening And Application Of Broad-spectrum Aptamers Against Acyclic Guanosine Analogues

Acyclic guanosine analogues are often illegally added to livestock and poultry farming and produce drug residues,which can enter the human body through the food chain and seriously harming human health.Therefore,it is of great significance to construct a rapid and sensitive detection method for acyclic guanosine analogues.Since acyclic guanosine analogs have small molecular weight and weak immunogenicity,it is difficult to prepare antibodies.Therefore,a novel molecular recognition element needs to be developed for rapid detection.Aptamer is a single-stranded oligonucleotide molecule obtained by in vitro screening technique.Aptamer has distinct advantages,including small batch-to-batch variation,wide range of targets,good stability,easy in vitro synthesis and modification.Its affinity and specificity are similar to antibodies.Studies about the screening and application of acyclic guanosine analogs aptamers have not been reported.In this paper,acyclovir,ganciclovir,penciclovir,famciclovir and valaciclovir were used as positive targets.The broad-spectrum aptamers binding against acyclic guanosine antivirals were obtained by Capture-SELEX technique.The affinity and specificity were characterized.The aptamer affinity column was constructed for enrichment of acyclic guanosine analogues based on selected aptamer with good performance.Firstly,Capture-SELEX technology was used to screen the aptamers against acyclic guanosine analogues.The hybrid library was formed through complementation between the biotin-modified short chain(Capture-P3)and ssDNA library.ssDNA library was indirectly immobilized on the avidin-coated magnetic carrier through the binding interaction between biotin and avidin.Acyclovir,ganciclovir,penciclovir,famciclovir,and valacyclovir were used as positive targets.The exponential enrichment cycle screening was performed through each round of steps of library fixation,magnetic separation,PCR amplification and ssDNA preparation.The solubilization temperature and peak changes of homologous and heterologous sequences were analyzed through real-time fluorescence quantitative PCR technique,which can be used to monitor the screening process of aptamers and determine that the aptamers against acyclic guanosine analogues have been enriched effectively after the 16 th round of screening.The PCR product obtained in the 16 th round was analyzed by high-throughput sequencing.Nine candidate aptamers were selected for subsequent verification of affinity and specificity based on high-throughput sequencing results,nucleotide sequence homology and ssDNA secondary structure.Secondly,the affinity and specificity of the aptamer were characterized and the binding mechanism was preliminarily studied.The detection scheme for acyclic guanosine analogs in chicken samples was constructed based on the optimal aptamer.The affinity and specificity of the nine candidate aptamers were compared using graphene oxide(GO)fluorescence polarization and GO fluorescence competition to determinate the optimal aptamer.The broadspectrum aptamer CIV6,which could recognize five targets simultaneously,was selected.The three-dimensional conformation of aptamer CIV6 was constructed by molecular mock docking and the molecular docking between five targets and aptamer CIV6 was performed.The results showed that acyclic guanosine analogs and aptamer CIV6 were mainly bound by hydrogen bonds.The method based on aptamer CIV6 recognition and GO fluorescence competition was constructed for the detection of acyclic guanosine analogs.The linear range of the method was2?100 ng/m L.The detection limits of acyclovir,famciclovir,ganciclovir,penciclovir and valaciclovir were 0.48,0.53,0.50,0.56,0.38 ng/m L,respectively.The recoveries of above acyclic guanosine analogs in chicken samples were 93.42%?104.60%,96.50%?110.35%,99.07%?109.80%,94.75%?101.82%,96.20%?100.97%,respectively.The above results indicated that the aptamer was feasible for the detection assay.Finally,the aptamer affinity column for enrichment of acyclic guanosine analogs was prepared based on the aptamer CIV6.The aptamer affinity column was prepared by coupling a streptavidin agarose gel with biotin-modified aptamer at a mass ratio of 100:1 and a reaction time of 40 min.Aptamer affinity column was prepared after sealing the surface-active substance with bovine serum protein.The single column capacity of aptamer affinity column was about200 ng.The enrichment ratio of aptamer affinity column was above 70% at no more than 11 times of use.Aptamer affinity column had good specificity because the enrichment ratio of aptamer affinity column to coexisting substances were both below 5%.The results of spiked recovery experiments indicated that the aptamer affinity column could be used for the retention and enrichment of acyclic guanosine analogues in real samples.