NovaID: photocatalytic proximity labeling with an engineered HyperNova

Photocatalytic proximity labeling has transformed the mapping of local molecular environments in living cells, but many existing platforms depend on flavin, heme, or metal cofactors, limiting their broader deployment. Here we repurpose HyperNova, a monomeric photosensitizer with an autocatalytically formed chromophore, as a genetically encoded photocatalyst for photocatalytic proximity labeling. Using biotin-aniline as the substrate, we show that HyperNova enables 560 nm light-activated proximity labeling across multiple subcellular compartments in living cells. We further engineer a blue-light-activated variant, HyperNova Green, and demonstrate that HyperNova and HyperNova Green can be selectively activated by 560 nm and 460 nm light, respectively, enabling dual-wavelength orthogonal labeling within the same sample and in distinct compartments of the same cell. In addition, fusion of HyperNova to Antares2 enables bioluminescence resonance energy transfer (BRET)-mediated activation, offering an external-illumination-free mode of proximity labeling. Together, these results establish NovaID as a compact and versatile proximity labeling platform with broad potential for spatiotemporally-resolved mapping of local subcellular environments.