Reliable characterization of binding affinities is crucial for selected aptamers. However, the limited repertoire of universal approaches to obtain the dissociation constant (KD) values often hinders the further development of aptamer-based applications. Herein, we present a competitive hybridization-based strategy to characterize aptamers using DNA origami-based chiral plasmonic assemblies as optical reporters. We incorporated aptamers and partial complementary strands blocking different regions of the aptamers into the reporters and measured the kinetic behaviors of the target binding to gain insights on aptamers' functional domains. We introduced a reference analyte and developed a thermodynamic model to obtain a relative dissociation constant of an aptamer-target pair. With this approach, we characterized RNA and DNA aptamers binding to small molecules with low and high affinities.
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