Design and Evaluation of Azobenzene-Based Photoswitches for Light-Responsive RNA Recognition

Abstract

RNA plays central roles in biology and disease, yet strategies for selective small-molecule recognition remain limited. Unlike proteins, RNAs often lack deep binding pockets, which makes conventional ligand design challenging. Azobenzene photoswitches offer a promising solution, providing reversible, light-induced changes in geometry and electronics. This property enables the spatiotemporal control of RNA recognition, allowing for the direct interrogation of RNA structural plasticity.In this work, azobenzene-based small molecules were designed, synthesized, and evaluated as light-responsive RNA ligands. Systematic structural modification revealed how electronic and steric environments govern photophysical performance, including wavelength responsiveness, switching efficiency, and cis-isomer stability. Substituent placement proved critical, with meta-substituted scaffolds exhibiting the most favorable switching behavior under biologically relevant conditions. Attempts to red-shift absorption spectra highlighted trade-offs between synthetic accessibility, photochemical efficiency, and stability, underscoring the importance of design rules that balance these factors. Functional studies demonstrated that azobenzene derivatives bind diverse RNA tertiary motifs, including the MALAT1 triple helix, the MALAT1 G-quadruplex, and the SAM-II riboswitch, with affinities in the low micromolar to sub-micromolar range. Photoisomerization modulated both affinity and functional outcomes, such as the stabilization of the MALAT1 triple helix against enzymatic degradation. These results establish that photoswitchable ligands can influence RNA structure and function in an isomer-specific manner. Overall, this work provides proof-of-concept that azobenzenes can serve as tunable, light-responsive RNA binders. This work establishes the feasibility of using azobenzene scaffolds as light-responsive RNA binders, highlights how structural modifications influence photophysical and biological behavior, and demonstrates their capacity to modulate biologically relevant RNA tertiary structures.