Engineering ligands for theophylline riboswitches expands its regulatory dynamic range in prokaryotic and eukaryotic systems.

The theophylline riboswitch has been a foundational tool in synthetic biology for three decades, yet its regulatory performance remains constrained by the modest affinity of its native ligand. Enhancing the dynamic range of riboswitches is critical for precise gene regulation in biotechnological applications. Here, we show that synthetic 4-quinazolinone derivatives, designed through a structure-based approach, are significantly better than theophylline in both binding and functional activation across multiple biological systems. We demonstrate that these derivatives bind the theophylline aptamer with up to 30-fold higher affinity, thereby expanding regulatory performance. In the bacterial system, these ligands enhance "ON" gene expression by up to 380-fold, compared to 75-fold with theophylline. This superior control extends to diverse organisms; in mycobacteria, the activation ratio reached 20-fold, and in eukaryotes, expression increased 11-fold. Furthermore, in riboswitch-mediated conditional CRISPR-Cas9 applications, these ligands achieve 70% genome editing efficiency at 10-fold lower concentrations than theophylline. These results demonstrate that ligand optimization is a crucial driver for enhancing riboswitch performance for advanced biomedical engineering.