Aptamers enable a distinct mode of conformational DNA recognition

Abstract Watson–Crick base-pairing defines the classical paradigm of nucleic-acid interactions, but growing research reveals noncanonical modes beyond this framework. Here we uncover a mode of interactions by which two DNA strands associate through a ligand-induced conformational rearrangement. Upon binding a small molecule, a structure-switching primary aptamer folds into a conformation that exposes a previously inaccessible docking interface. An independently evolved secondary aptamer recognizes this interface primarily through noncanonical interactions rather than sequence complementarity, reporting the ligand-bound state of the primary aptamer. This interaction is conformation-sensitive: single base-pair changes in the primary aptamer modulate secondary aptamer recognition. Across four chemically distinct small-molecule targets—serotonin, dopamine, testosterone and deoxycorticosterone 21-glucoside—secondary aptamers were identified for three, and the serotonin system was translated into surface-based sandwich assays. These results demonstrate that a ligand-induced conformational state, rather than sequence complementarity or the chemical identity of the ligand, can serve as the epitope for nucleic-acid recognition, expanding the molecular features that DNA can be evolved to detect.