Regioisomeric and Substituent Effects on the Photophysics of Stilbene RNA Dyes

Fluorogenic small-molecule probes are indispensable for visualizing RNA localization and dynamics in live cells, owing to their cell permeability, reversibility, and high brightness.Heterocyclic stilbenes are particularly attractive due to their structural simplicity and pronounced fluorescence turn-on upon RNA binding.However, inconsistent experimental datasets have hindered 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 structurally diverse panel of heterocyclic stilbene RNA dyes encompassing both regioisomers and non-regioisomeric analogs.By varying substitution patterns on the indole donor and introducing isosteric and ringmodifying changes to both donor and acceptor motifs, we broadened the chemical space of this scaffold and identified new derivatives with improved optical properties.In parallel, we benchmarked ab initio excited-state calculations against experiment and distilled the governing charge-transfer photophysics into an improved, computationally efficient Hückel model that reproduces excitation-energy trends across the series.Together, these results establish a comprehensive SAR for heterocyclic stilbene dyes and clarify how structural features dictate 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.