Engineered NanoBiTs for multicomponent bioluminescence imaging

Bioluminescent enzymes are widely used as genetically encoded reporters in a variety of imaging contexts, but they are still limited in their capacity for multicomponent imaging. Though prior success has been found in development of new substrate-enzyme pairs, this process is time-consuming and requires complex chemistry. Here we introduce a new approach via engineering of NanoBiT, a split bioluminescent reporter. NanoBiT is comprised of an 11-amino-acid peptide (SmBiT) and a, 158 amino acid protein half (LgBiT). We evaluated two, 400-member libraries of mutations to LgBiT paired with various SmBiT sequences. We discovered a number of SmBiT-LgBiT pairings that enable multicomponent imaging, offering a complementary approach to existing substrate-enzyme pairs, as well as a new paradigm for multicomponent bioluminescence imaging.