High-Throughput On-Chip Screening Enables Rapid Adaptation of DNA Aptamers to SARS-CoV-2 Evolution

Rapid pathogen evolution threatens public health by eroding the effectiveness of vaccines, therapeutics, and diagnostic tools. Although spike-protein-targeting monoclonal antibodies (mAbs) were developed within 10-12 months of the initial outbreak to serve as key theranostic agents, their redesign has struggled to keep pace with viral evolution, rendering many neutralizing antibodies ineffective. Here, we demonstrate a high-throughput aptamer engineering platform that combines a random-rational hybrid library diversification with repurposed MiSeq screening to rapidly reprogram aptamers against emerging SARS-CoV-2 spike variants. Interactions between 3 different spike proteins and 11,792 unique aptamer variant designs were profiled within days (a single run from pool amplification to screen analysis). Starting from a 40-nt aptamer originally selected against wild-type (WT) spike protein, our screen identified a Delta-binding mutant with a 4-fold affinity improvement and an Omicron-binding mutant that converted undetectable binding into nanomolar affinity. We also identified a WT-selective mutant with substantially reduced affinity for Delta as well as bases that contribute to spike recognition. Integrating high-throughput binding data with molecular dynamics simulations further helped to rationalize the sequence-dependent structural features underlying variant-specific aptamer-spike interactions. Finally, we developed fluorescent strand-displacement sensors based on both WT- and Omicron-selective mutants, enabling highly specific detection of spike protein variants with robust performance. Together, these findings demonstrate a rapid and sequence-resolved aptamer engineering platform for adapting aptamers to evolving pathogens.