FLEX: genetically encodable enzymatic fluorescence signal amplification using engineered peroxidase

Abstract

Fluorescent tagging of biomolecules enables their sensitive detection during separation and determining their subcellular location. In this context, peroxidase-based reactions are actively utilized for signal amplification. To harness this potential, we developed a genetically encodable enzymatic fluorescence signal amplification method using APEX (FLEX). We synthesized a fluorescent probe, Jenfluor triazole (JFT1), which effectively amplifies and restricts fluorescence signals under fixed conditions, enabling fluorescence -based detection of subcellularly localized electron -rich metabolites. Moreover, JFT1 exhibited stable fluorescence signals even under osmium -treated and polymer -embedded conditions, which supported findings from correlative light and electron microscopy (CLEM) using APEX. Using various APEX -conjugated proteins of interest (POIs) targeted to different organelles, we successfully visualized their localization through FLEX imaging while effectively preserving organelle ultrastructures. FLEX provides insights into dynamic lysosomemitochondria interactions upon exposure to chemical stressors. Overall, FLEX holds significant promise as a sensitive and versatile system for fluorescently detecting APEX2-POIs in multiscale biological samples.