Rational Development of a Ligand for Theophylline Riboswitch Aptamer Expands Its Regulatory Dynamic Range in Prokaryotic and Eukaryotic Systems

The theophylline riboswitch has served as a foundational tool in synthetic biology for three decades, but efforts to optimize its regulatory performance are limited. Here, we report the development of 4-quinazolinone derivatives, a new class of synthetic ligands designed using a structure-based approach. The biophysical techniques, including Fluorescence assay, ITC, SPR, RNase footprinting, and inline probing, were used to investigate aptamer-ligand interaction; developed ligands interact with the theophylline aptamer with 30-fold higher affinity (KD = 31-96 nM) than the native theophylline ligand (KD = 919 nM). The higher association rate (kon) and lower rate of dissociation (koff) resulted in the enhancement of binding affinity. The synthetic compounds also showed up to 3-fold increase in cellular penetration. These enhancements in binding affinity and superior cellular penetration (~3-fold) of the developed ligand resulted in expanding the riboswitch's regulatory capabilities. We validated the riboswitch activity using fluorescence imaging, spectrofluorometry, flow cytometry, western blotting, and qRT-PCR. In bacterial systems, our 4-quinazolinone ligands enhanced "ON" gene expression by up to 157-fold (compared to 47-fold with theophylline), while dramatically improving "OFF" switch suppression to 81% (relative to 28% with theophylline) at the same ligand concentration. In mycobacteria, 4-quinazolinone derivatives demonstrated an activation ratio of 20.45-fold (versus 6.09-fold with theophylline), further demonstrating the superior performance of our synthetic ligands across diverse bacterial systems. In eukaryotes, these ligands increased gene expression up to 11.27-fold, compared to 2.93-fold with theophylline. Furthermore, Docking and inline-probing data confirmed that the 4-quinazolinone derivatives bind to the same pocket as the native theophylline ligand. Our findings expanded the utility and dynamic range of the theophylline riboswitch, providing a more robust platform for diverse biomedical applications.