Parallel Screening for Rapid Identification of Orthogonal Bioluminescent Tools

[Image: see text] Bioluminescence imaging with luciferase enzymes and luciferin small molecules is a well-established technique for tracking cells and other biological features in rodent models. Despite its popularity, bioluminescence has long been hindered by a lack of distinguishable probes. Here we present a method to rapidly identify new substrate-selective luciferases for multicomponent imaging. Our strategy relies on parallel screening of luciferin analogues with panels of mutant enzymes. The compiled data set is then analyzed in silico to uncover mutually orthogonal sets. Using this approach, we screened 159 mutant enzymes with 12 luciferins. Thousands of orthogonal pairs were revealed with sufficient selectivity for use in biological environments. Over 100 pairs were validated in vitro, and three were applied in cell and animal models. The parallel screening method is both generalizable and scalable and will streamline the search for larger collections of orthogonal probes.