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

Abstract

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.