Abstract
Cyclic diadenosine monophosphate (C-di-AMP) synthases are key enzymes for synthesizing c-di-AMP, a potent activator of the stimulator of interferon genes (STING) immune pathway. However, characterizing these enzymes has been hampered by the lack of effective sensors. While c-di-AMP riboswitches, as natural aptamers, hold the potential as RNA biosensors, their poorly comprehended structural dynamics and inherent "OFF" genetic output pose substantial challenges. To address these limitations, we synthesized over 10 fluorophore-labeled samples to probe the conformational changes of the riboswitch at the single-molecule level. By integrating these dynamic findings with steady-state fluorescence titration, mutagenesis, in vivo assays, and strand displacement strategy, we transformed the natural aptamer into a c-di-AMP biosensor. This engineered biosensor reversed its genetic output from "OFF" to "ON" upon c-di-AMP binding, exhibiting a 50-fold improvement in the c-di-AMP detection limit. Leveraging this refined biosensor, we developed a robust strategy for high-throughput in vivo evolution of c-di-AMP synthases.