FLEX: genetically encodable enzymatic fluorescence signal amplification using engineered peroxidase

  • Cell Chem Biol. 2024 Mar 8:S2451-9456(24)00081-3. doi: 10.1016/j.chembiol.2024.02.007.
Nirmali Sharma  1 ,  Minkyo Jung  2 ,  Pratyush Kumar Mishra  3 ,  Ji Young Mun  4 ,  Hyun-Woo Rhee  5
Affiliations
  • 1. Department of Chemistry, Seoul National University, Seoul 08826, Korea; Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea.
  • 2. Neural Circuits Research Group, Korea Brain Research Institute, Daegu 41062, Republic of Korea.
  • 3. Department of Chemistry, Seoul National University, Seoul 08826, Korea.
  • 4. Neural Circuits Research Group, Korea Brain Research Institute, Daegu 41062, Republic of Korea. Electronic address: [email protected].
  • 5. Department of Chemistry, Seoul National University, Seoul 08826, Korea; School of Biological Sciences, Seoul National University, Seoul 08826, Korea. Electronic address: [email protected].
PMID: 38513646 DOI: 10.1016/j.chembiol.2024.02.007
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 lysosome-mitochondria 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.

Keywords
APEX; CLEM; FLEX; correlative light and electron microscopy; fluorescent peroxidase substrate; fluorescent probe; fluorescent probes; lysosome; mitochondria; organelle communication; proximity labeling.
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