Reliable Post-SELEX Design of Promising Bivalent Aptamers through the Systematic Capillary Electrophoresis Exploration of Ternary DNA–Protein Complexes

Chemically linking two DNA aptamers (Apts) that recognize different protein epitopes can yield outstanding high-affinity bivalent Apts. However, rationally exploring the combination of two Apts (an Apt pair) with a strong binding affinity due to the allosteric effect is challenging. This challenge arises from the lack of methodologies to differentiate the thermodynamic and kinetic affinities of higher-order biocomplexes in free aqueous solutions─a limitation of solid-phase immobilization methods. The present study introduces a kinetic capillary electrophoresis method to discriminate between the binding sites in 2:1 Apt-protein complexes, evaluating their molecular recognition ability from an equilibrium and kinetic perspective. The proposed method precisely determined the specific binding site, dissociation equilibrium constants, rate constants, and allosteric effects of 2:1 complexes comprising thrombin and 22 Apts. It also elucidated the factors controlling the allosteric effect; these factors exhibit slight differences that significantly affect the inhibitory activity. Multiple regression analysis of the anticoagulant activity and determined parameters yielded one excellent Apt pair among over 100 combinations, which exhibited a 6.7-times higher anticoagulant activity than previously reported Apt pairs (HD1/HD22). The identified Apt pair was linked to synthesize a single-stranded bivalent Apt, which demonstrated unprecedented anticoagulant activity (>80-fold higher than that of HD1 alone). Thus, the proposed post-SELEX strategy, which involves binding-site discrimination, precise thermodynamic and kinetic measurements, correlation analysis between the parameters and activity, and Apt linking, offers a framework for rationally designing highly functional bivalent Apts. It also aids in identifying unknown, highly functional Apt pairs from unstudied aptamers identified through SELEX.