Ketal-Bridged Rhodamines as a New Scaffold for Near-Infrared Chemigenetic Indicators with Enhanced Fluorogenicity

Chemigenetic indicators could combine the spectral tunability and photostability of small-molecule dyes with precise genetically encoded labels and provide crucial tools for biological imaging, but current indicators still have limitations in fluorogenicity, photostability and synthetic complexity. We report a new type of NIR chemigenetic indicators via engineering ketal-bridged rhodamines. We demonstrate that the ketal-bridged rhodamines exhibit distinct NIR excitation and emission, superior brightness, improved photostability and enhanced fluorogenicity upon binding to self-labeling protein HaloTag. We also introduce a new "bridge addition" strategy for facile synthesis of these ketal-bridged rhodamines with high overall yields. The ketal-bridged rhodamine based chemigenetic indicators show high fluorescence activation ratios in live cell imaging, allowing highly fluorogenic imaging of proteins in varying subcellular organelles and super-resolution imaging of subcellular structures and dynamics via structured illumination microscopy (SIM). The versatility of the chemigenetic indicator also enables imaging of RNA and translocation into phase granules and kinase activity through a phase separation approach. The ketal-bridged rhodamine fluorophores could enrich the chemigenetic toolboxes, affording a promising scaffold for new indicators with improved contrast in bioimaging and biosensing applications.