FLEX: genetically encodable enzymatic fluorescence signal amplification using engineered peroxidase.

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.

GEN-Click: Genetically Encodable Click Reactions for Spatially Restricted Metabolite Labeling.

Chemical reactions for the in situ modification of biomolecules within living cells are under development. Among these reactions, bio-orthogonal reactions such as click chemistry using copper(I) and Staudinger ligation are widely used for specific biomolecule tracking in live systems. However, currently available live cell copper(I)-catalyzed azide/alkyne cycloaddition reactions are not designed in a spatially resolved manner. Therefore, we developed the "GEN-Click" system, which can target the copper(I)-catalyzed azide/alkyne cycloaddition reaction catalysts proximal to the protein of interest and can be genetically expressed in a live cell. The genetically controlled, spatially restricted, metal-catalyzed biorthogonal reaction can be used for proximity biotin labeling of various azido-bearing biomolecules (e.g., protein, phospholipid, oligosaccharides) in living cell systems. Using GEN-Click, we successfully detected local metabolite-transferring events at cell-cell contact sites.

A multifunctional peroxidase-based reaction for imaging, sensing and networking of spatial biology.

Peroxidase is a heme-containing enzyme that reduces hydrogen peroxide to water by extracting electron(s) from aromatic compounds via a sequential turnover reaction. This reaction can generate various aromatic radicals in the form of short-lived "spray" molecules. These can be either covalently attached to proximal proteins or polymerized via radical-radical coupling. Recent studies have shown that these peroxidase-generated radicals can be utilized as effective tools for spatial research in biological systems, including imaging studies aimed at the spatial localization of proteins using electron microscopy, spatial proteome mapping, and spatial sensing of metabolites (e.g., heme and hydrogen peroxide). This review may facilitate the wider utilization of these peroxidase-based methods for spatial discovery in cellular biology.

Enzymatic Recording of Local Hydrogen Peroxide Generation Using Genetically Encodable Enzyme.

Reactive oxygen species (ROS) are endogenously generated in live cells and essential for cell signaling. However, excess ROS generation can cause oxidative damage to biomolecules, which are implicated in various human diseases, including aging. Here, we developed an in vivo hydrogen peroxide monitoring method using a genetically encodable peroxidase (APEX2)-based system. We confirmed that APEX2 is activated by endogenous H2O2 and generates phenoxyl radicals to produce biotinylated signals (i.e., biotin-phenol) and fluorescent signals (i.e., AmplexRed), which can be detected using a fluorescence microscope. We observed that all subcellular targeted APEX2s were activated by local H2O2 generation by menadione treatment. Among them, the endoplasmic reticulum lumen and lysosome-targeted APEX2 showed the highest response upon addition of menadione which implies that local H2O2 levels in those spaces are highly increased by menadione treatment. Using APEX2, we also found that a minimum amount of menadione (>10 µM) is required to generate detectable levels of H2O2 in all subcellular compartments. We also checked the local H2O2-quenching effect of N-acetylcysteine using our system. As APEX2 can be genetically expressed in diverse live organisms (e.g., cancer cell lines, mice, fly, worm, and yeast), our method can be effectively used to detect local generation of endogenously produced H2O2 in diverse live models.

A chemical tool for blue light-inducible proximity photo-crosslinking in live cells.

We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Chemical or heat stress-induced dynamic changes in the proteome were also detected, and photo-crosslinking in the mouse brain tissue was enabled. Using Spotlight, we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of N-HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells.