A general strategy to enhance aptamer affinity by suppressing dissociation through symmetric assembly

Aptamers are programmable molecular recognition elements with broad utility in diagnostics, therapeutics, and synthetic biology. However, many aptamers suffer from insufficient affinity due to rapid target dissociation, and no general strategy currently exists to overcome this limitation. Here, we report a symmetry-guided assembly approach that enhances aptamer affinity by suppressing the dissociation rate constant (koff). Three identical aptamer units are spatially organized into a flexible trivalent assembly to enable kinetic cooperativity through rapid rebinding. Applied to aptamers targeting SARS-CoV-2 spike (both trimeric and monomeric S1 subunit), VEGF165 (dimeric), and cardiac troponin I (monomeric), the resulting trimers exhibited dissociation constants (Kd) in the low pM range and koff values in the 10-6 s-1 range, over 100-fold improvements relative to monomers. In a serum-based VEGF165 assay, the trimeric aptamer improved detection sensitivity by 30-fold. This modular, chemistry-based strategy is applicable to existing aptamers and establishes dissociation suppression as a general principle for engineering ultrahigh-affinity aptamers.