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.

C-Glycoside analogs of bioactive glycans and glycoconjugates are resistant to glycosidic hydrolysis and thus represent valuable scaffolds for drug discovery and chemical biology. We previously found that CH₂-linked isomaltose (CH₂-IM) exhibits potent amylase-inducing activity in Aspergillus nidulans compared to its native and linkage-edited counterparts. Here, we report a streamlined synthesis of CH₂-IM via photoredox-catalyzed vinyl-C-glycosylation using N,N-dialkylbenzylamine derivatives as efficient halogen-atom transfer reagents. This approach enables concise access to CH₂-IM and also facilitates the synthesis of 6′-modified analogs, providing a versatile platform for probing structure–activity relationships of IM derivatives. Notably, functionalization at the 6′- and anomeric positions completely abolishes the amylase induction activity.