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.