Strategic aptamer evolution for SARS-CoV-2 to enable the detection in human saliva.

In this study, we enhanced the affinity and specificity of an aptamer for SARS-CoV-2 detection in human saliva. Aptamers are promising recognition elements in biosensors; however, their performance in biological samples such as human saliva, is sometimes limited by insufficient affinity and specificity, even if they are obtained through conventional systematic evolution of ligands via exponential enrichment (SELEX). Here, we introduced inosine mutation into in silico maturation (ISM) process as a post-SELEX optimization. Inosine incorporation expands sequence diversity through non-Watson-Crick base pairing and increases local hydrophobicity at the aptamer-protein interface, enabling structural and physicochemical variation that cannot be achieved by SELEX alone. After inosine-incorporated ISM, we identified an optimized SARS-CoV-2 aptamer, WiS-914, which exhibited improved affinity and specificity for inactivated SARS-CoV-2. This aptamer showed a distinct and more stable topology than the original aptamer. When it was applied to inactivated SARS-CoV-2 detection in actual human saliva using our convenient and on-site virus detection platform, more sensitive detection with lower limit of detection was achieved compared with the original aptamer. These results demonstrate that inosine-integrated ISM is an effective post-SELEX optimization for generating high-performance aptamers suitable for detecting targets in biological samples.