Regioisomeric and substituent effects on the photophysics of stilbene RNA dyes

Y. Li, G. Giudetti, M. Jung Kim, R. Zhu, A.I. Krylov, and C. Zhang
Biochemistry , submitted (2026)

Fluorogenic small-molecule probes are indispensable for visualizing RNA localization and dynamics in live cells, owing to their cell permeability and low cytotoxicity. Heterocyclic stilbenes are particularly attractive because they exhibit pronounced fluorescence “turn-on” upon RNA binding. However, inconsistent experimental datasets have limited the development of robust structure–activity relationships (SAR), and the mechanistic basis of their photophysical behavior remains incompletely understood. Here, we report the systematic synthesis, photophysical characterization, and live-cell imaging of a panel of indole-containing stilbene regioisomers. By surveying diverse substitution patterns on the indole ring, we expanded the chemical space of these dyes and identified new derivatives with improved optical properties. In parallel, we benchmarked ab initio excited-state calculations against experiment and distilled the underlying charge-transfer photophysics into an improved, computationally efficient Hückel model that captures excitation-energy trends; this model was further validated through the synthesis and characterization of additional stilbene analogs beyond the indole regioisomer series. Collectively, these results establish a comprehensive SAR for indole–stilbene dyes and delineate how structural features govern emission wavelength, quantum yield, and RNA-induced fluorescence enhancement. This integrated experimental–computational framework provides a foundation for the rational design of next-generation, RNA-selective fluorogenic probes.

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