| Aptamers have been widely used to identify various targets such as small molecules and proteins due to their high affinity and high efficiency,but the specificity of aptamers has been regarded as their inherent property and lacked systematic study,which limits their detection in complex real environments.In this paper,through virtual screening in a large-scale chemical library,we found that aptamers can bind potential targets other than the preset targets,they have the phenomenon of Promiscuous Binding,and have a higher possibility of false positives when identifying targets.In a huge chemical space,the workload of systematically screening targets using traditional experimental methods is huge,so a more efficient computational screening method is introduced in this paper.Ochratoxin A（OTA）aptamer OBA3 was used as an example to study the specificity of the aptamer target.It was found that the aptamer OBA3 actually binds Norfloxacin with higher affinity,so we obtained the best mutation site by virtual calculation of the aptamer OBA3,and the specificity and affinity of the mutated aptamer for the target were greatly improved,and we constructed a dual aptamer biosensor using the aptamer OBA3 and the mutated aptamer,and designed a logic gate（NOR）for simultaneous detection of ochratoxin A and norfloxacin at the molecular level to achieve highly specific detection of OTA and norfloxacin alone and interference-free hybrid detection.The main results of the study are as follows:（1）Virtual screening and experimental validation of potential targets of aptamers.Firstly,a biocomputing platform was built to collate and compare different aptamers of the target OTA,and the aptamer OBA3 of ochratoxin A was selected as the research target,and the base sequence of OBA3 has been optimized in the reference literature.A target library containing1679043 compounds was constructed,including drugs,toxins,metabolites and natural products,was constructed,and the aptamer OBA3 was docked with the small molecule targets in the library,and the compounds with the best docking scores were retained.The potential targets of the aptamer were obtained by unconstrained molecular dynamics（MD）simulations.Three targets,Norfloxacin,Difloxacin and Asenapine,with stronger binding to OBA3 were identified by the fluorescent dye Pico Green labeling method and isothermal titration calorimetry（ITC）experiments.（2）Systematic mutagenesis and experimental validation of aptamers by virtual computation.The first part is based on secondary structure sequence screening,and the second part is based on molecular dynamics simulation for sequence enrichment of the binding target.The isothermal titration calorimetry was used to measure the binding parameters of the mutated aptamers to the targets Norfloxacin and OTA,and the results showed that the modified aptamers could distinguish between the two targets,with TG15binding OTA more strongly and with 10-fold higher affinity than the original aptamer OBA3,and T15C binding only Norfloxacin but not OTA.Norfloxacin does not bind OTA.（3）Construction and application of dual-target,dual-adaptor fluorescent biosensors.The fluorescent biosensor was constructed mainly based on the principles of target-induced conformational change,chain displacement and fluorescence resonance energy transfer.After a series of parameter optimization,the linear detection range for OTA was 25-400 nmol·L-1with a detection limit of 8 nmol·L-1,and for norfloxacin was 25-100 nmol·L-1with a detection limit of 53 nmol·L-1.The detection limit for OTA was 31 nmol·L-1with a detection range of25 nmol·L-1-3μmol·L-1,and for norfloxacin was 33 nmol·L-1with a detection range of 25-400nmol·L-1when mixed.The spiked recoveries of OTA and norfloxacin ranged from 94.06%to100.25%and 96.26%to 100.93%,respectively,when applied to milk samples.This study broadens the versatility of aptamer biosensors to ensure food safety and reduce mycotoxin and antibiotic residues in animal-derived foods.Meanwhile,this study systematically explored the specificity of aptamers,which can promote the aptamers into practical applications. |
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