Preparation and Validation of Ultra-sensitive Aptamers by Computer Simulation and Site-Specific Mutation

Aptamer is widely used in the detection field as a new type of probe due to their advantages of easy synthesis and modification. Due to the traditional aptamer screening method, the aptamers that are screened by SELEX technology generally have redundant sequences, which affect the binding affinity and detection sensitivity. In this study, a structural transformation strategy for rationally shortening was established by analyzing the secondary structure, finding the sequence of the binding site by base mutation, and double cloning the binding sites. We obtained a 27-mer new aptamer SEQ.A52 with the dissociation constants (Kd) of 10.74 nM, which is 21 times higher than the original aptamer. When used to detect enrofloxacin (ENR) with the fluorescent method, the sensitivity could reach 50 to approximately 1000 pM, and the limit of detections (LOD) was only 41.35 pM, which was 88 times higher than the original aptamer. All the results implied that the strategy could be used for structural modification of some other aptamers to increase affinity and sensitivity.