Molecular Design of RNA-Light Probes for Super-Resolution Fluorescence Imaging of Nucleolar Heterogeneity and Dynamics

Super-resolution imaging has emerged as an indispensable tool for uncovering dynamic fine structures and complex functions of the nucleolus. However, current approaches heavily rely on covalent protein labeling, which is unable to capture nucleolar RNA for revealing the complete morphological structure of the nucleolus. Here, we address this gap by introducing a novel RNA-light probe engineered using a naphthalimide fluorophore scaffold, optimized through synergistic donor substituent and side chain modifications. This design achieves enhanced molecular planarity, fluorescence responsiveness, and precise nucleolar targeting in living cells. Among 24 synthesized candidates, probe 7f demonstrated exceptional performance, featuring a 3-pyrrolidine donor group and a polyamine side chain that enable selective RNA recognition, superior brightness, and robust nucleolar labeling. Utilizing 7f and nucleolar marker proteins fused to fluorescent proteins or protein tags, we performed multicolor 3D structured illumination microscopy (SIM) to visualize the heterogeneity and dynamics of the nucleolus in real time. This study establishes a new benchmark for RNA-targeted super-resolution imaging and offers unprecedented insights into nucleolar biology.