Spatiotemporally Resolved Mapping of Synaptic-to-Nuclear Signaling in Neurons via a Light-Free Tri-BRET Proximity Labeling System

Neurons rely on rapid, compartment-specific signaling cascades—from membrane receptors at synapses to nuclear transcriptional machinery—that are difficult to capture with existing proximity-labeling methods. We introduce the first covalent “sandwich” architecture that locks a photosensitizer between a targeting ligand (antibody, peptide) and a luciferase enzyme, enforcing sub-10 nm spacing for highly efficient Bioluminescence Resonance Energy Transfer (BRET)–driven radical generation. Our tri-modular system comprises three orthogonal constructs: 1. RLuc8–Ce6, which decorates the synaptic surface via antibody coupling and produces singlet oxygen (^1O₂) upon coelenterazine-h activation; 2. NanoLuc–Ru(II), directed to the cytosol or nucleus with TAT/NLS peptides, generating both ROS and carbon-centered radicals under furimazine; 3. Antares/AkaLuc–Ir(III), targeted to endosomes, releasing carbon radicals in hypoxic or acidic microenvironments upon AkaLumine addition By covalently tethering photosensitizer and luciferase, each module achieves exceptional BRET efficiency and confines radical chemistry to a nanometer-scale radius around the enzyme. The use of three spectrally distinct luciferins (CTZ-h, furimazine, AkaLumine) allows sequential or simultaneous multi-compartment labeling without spectral crosstalk or external illumination. In neuronal models and tissue slices, this platform delivers deep (>3 mm) penetration, high proteomic coverage (>1,000 proteins), and true spatial–temporal control of reactive-radical proximity labeling. We present a modular BRET-based proximity labeling system that enables spatially controlled protein tagging across cellular compartments. Moreover, all labeling reactions are bioorthogonal, relying on catalyst-initiated radical generation exclusively triggered via luciferin–luciferase BRET activation. This ensures minimal background, no cross-reactivity with native biomolecules, and compatibility with live-cell systems. This work establishes a robust, chemically defined toolkit for mapping dynamic protein interactions across subcellular domains in neurons—and, by extension, any complex tissue—overcoming the penetration, specificity, and phototoxicity limits of existing technologies.