A High-Throughput Platform for Rapidly Adapting DNA Aptamers to SARS-CoV-2 Evolution

Rapid pathogen evolution, exemplified by SARS-CoV-2 during the COVID-19 pandemic, threatens public health by eroding the effectiveness of vaccines, therapeutics, and diagnostic tools through continuous viral mutation. 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 novel platform that integrates a random-rational hybrid library diversification with high-throughput MiSeq screening to evolve aptamers as highly versatile recognition elements that can be easily reprogrammed to bind the spike proteins of emerging SARS-CoV-2 strains. Using a repurposed next-generation sequencing (NGS) platform, interactions between 3 different spike proteins and 11,806 unique aptamer variant designs can be effectively screened within a few days. Our starting point is a 40-nt aptamer that binds strongly to the wild-type (WT) spike protein but shows reduced and no affinity toward its Delta and Omicron strains, respectively. With this starting aptamer diversified, our rapid screening method yielded one double mutant that exhibits 4-fold improvement in binding to the Delta spike protein and another double mutant that converts its binding to the Omicron spike protein from no detectable affinity to the kd of nanomolar range. A selective WT binder was also identified with no binding the two variants of interest. Using this pipeline, we identified bases not previously recognized as part of the motif that contribute critically to spike protein binding. Moreover, our pipeline integrates screening data analysis with molecular dynamics simulations, providing insights into aptamer-protein binding interactions. A sensor was developed based on the identified WT-selective binder, enabling highly specific detection of the WT spike protein with minimal cross-reactivity and robust performance in 40% serum. Together, these results demonstrate that aptamers can be rapidly optimized to bind new variants or selectively recognize a specific strain using the repurposed NGS platform. This work highlights the platform as a highly adaptive technology capable of obtaining aptamers within days to keep pace with rapidly evolving pathogens in future pandemics. TeaserA novel high-throughput MiSeq screening platform to rapidly evolve spike-protein-binding aptamers to keep pace with viral evolution.