Photoaffinity Electrophoretic Mobility Shift Assay for Analyzing the Binding Specificity of DNA Aptamers to α-Thrombin

DNA aptamers are attractive alternatives to antibodies and advances in selection technologies have yielded several candidate sequences, creating a strong need for efficient methods to evaluate their binding specificity. We previously established a photoaffinity electrophoretic mobility shift assay (EMSA) for analyzing DNA-protein interactions. In this study, we investigated whether this method can be applied to assess the binding specificities of α-thrombin-binding DNA aptamers. The binding specificities of a 15-mer and a 29-mer aptamer were evaluated via photoaffinity EMSA using reaction mixtures enriched in zymogen prethrombin-2, whose structure closely resembles α-thrombin but lacks fibrinogen-clotting activity. Photoaffinity EMSA enabled quantification of the relative binding strengths of α-thrombin and prethrombin-2 resolved in the same sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) lane, revealing that the specificity for α-thrombin was approximately 2.5-3.5-fold higher than that for prethrombin-2 for both aptamers. In the case of the 15-mer aptamer, introducing the photoreactive group at the 9th nucleotide reduced its binding specificity for α-thrombin. This finding indicates that the position of the modification affects aptamer-protein recognition and offers clues about the structural basis of binding. Photoaffinity EMSA also enabled us to distinguish the binding sites of the 15-mer and 29-mer aptamers on α-thrombin using competition experiments with dabigatran and heparin, while allowing selective detection of α-thrombin in human plasma. Together, these results demonstrate that photoaffinity EMSA is a simple and informative approach that can evaluate the binding specificities of multiple DNA aptamers for a target protein in a single SDS-PAGE-based assay.