FLEX: genetically encodable enzymatic fluorescence signal amplification using engineered peroxidase

Nirmali Sharma; Minkyo Jung; Pratyush Kumar Mishra; Ji Young Mun; Hyun-Woo Rhee

doi:10.1016/j.chembiol.2024.02.007

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. Online ahead of print.

Authors

Nirmali Sharma¹, Minkyo Jung², Pratyush Kumar Mishra³, Ji Young Mun⁴, Hyun-Woo Rhee⁵

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: jymun@kbri.re.kr.
⁵ Department of Chemistry, Seoul National University, Seoul 08826, Korea; School of Biological Sciences, Seoul National University, Seoul 08826, Korea. Electronic address: rheehw@snu.ac.kr.

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.