LucID: A Self-Activating Bioluminescent Biotin Ligase for Light-Free Proximity Labeling in Deep Tissues

Mapping protein-protein interactions (PPIs) in living systems is critical for understanding dynamic biological processes. While proximity-labeling enzymes like LOV-TurboID and APEX2 are widely used, their dependence on external light, reactive chemicals, or large fusion domains limits in vivo applications, especially in deep tissues. Here, we introduce LucID (Light-free Unifying Catalytic Integrated Domain) - a compact, self-contained proximity-labeling system that functions independently of light or external ROS. LucID was engineered by embedding a minimal catalytic motif derived from TurboID into a NanoLuc scaffold, preserving bioluminescence while enabling spatially confined biotinylation. Upon luciferin addition, LucID leverages BRET (bioluminescence resonance energy transfer) to uncage the catalytic site and activate biotin transfer. With a molecular weight of approximately 23.7 kDa (40% smaller than TurboID), LucID is optimized for delivery via mRNA-LNP platforms. Its modularity and light-independent activation make it a unique tool for real-time interactome mapping in live cells and deep tissues. LucID introduces a new paradigm in proximity labeling: compact, luminescent, and chemically programmable. By integrating catalytic motifs into a light-producing scaffold, LucID enables temporally precise, spatially confined interactome mapping in challenging biological contexts. This innovation holds promise for single-cell proteomics, in vivo interaction studies, and next-generation bioimaging tools. Graphical Abstract | LucID enables light-free, ultra-compact biotinylation via BRET-triggered ROS uncagingLucID is a next-generation proximity labeling tool that combines NanoLuc bioluminescence, engineered TurboID motifs, and a ROS-cleavable thioketal cage to perform spatially precise biotinylation without external light. Upon luciferin addition, BRET triggers localized ROS generation, rapidly cleaving the cage (2.4-3.6 ns) and activating the catalytic site. This compact, self-contained platform enables deep-tissue labeling with high temporal control, making it ideal for in vivo proteomics, CRISPR fusions, and light-inaccessible systems.LucID is a next-generation proximity labeling tool that combines NanoLuc bioluminescence, engineered TurboID motifs, and a ROS-cleavable thioketal cage to perform spatially precise biotinylation without external light. Upon luciferin addition, BRET triggers localized ROS generation, rapidly cleaving the cage (2.4-3.6 ns) and activating the catalytic site. This compact, self-contained platform enables deep-tissue labeling with high temporal control, making it ideal for in vivo proteomics, CRISPR fusions, and light-inaccessible systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/658826v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@54c2dorg.highwire.dtl.DTLVardef@1cc3b16org.highwire.dtl.DTLVardef@13b5489org.highwire.dtl.DTLVardef@2b2a80_HPS_FORMAT_FIGEXP M_FIG C_FIG