DNA Nanostructure-Mediated Bispecific Aptamers Enable Multiepitope Recognition and Robust Viral Neutralization.

Viral evolution through mutation often enhances infectivity and facilitates escape from existing therapeutics, highlighting the urgent need for robust strategies that can neutralize key viral proteins despite sequence variation. Here, we present a bispecific neutralizing aptamer, Aptx2-L, which simultaneously engages two distinct epitopes on the receptor-binding domain (RBD) of SARS-CoV-2. This dual-epitope recognition confers robustness against single-point mutations, reducing the risk of viral escape, while cooperative binding at separate sites synergistically enhances thermodynamic affinity. Anchoring the aptamers on a DNA framework further allows precise control of interaptamer spacing and spatial orientation, ensuring optimal geometric presentation and maximal binding efficiency. By synergistically binding, Aptx2-L demonstrates nanomolar binding affinity to RBD and potent inhibition of both wild-type and mutant SARS-CoV-2 pseudovirus infection of cells, outperforming cocktails of monospecific aptamers. Beyond SARS-CoV-2, this bispecific, multiepitope recognition strategy establishes a generalizable platform for targeting mutable proteins and other therapeutically relevant biomolecules where multisite engagement is essential, offering broad potential for molecular diagnostics and therapeutic development.