Bioluminescence imaging (BLI) is a powerful tool for monitoring cellular events in real time. BLI relies on light production from a chemical reaction: the luciferase-catalyzed oxidation of a small molecule substrate (luciferin). Luciferases and luciferins have been used for decades to image processes in complex tissues and preclinical models. However, few distinct bioluminescent probes are available for in vivo work, precluding many studies of multicellular interactions. We have addressed this limitation by engineering new luciferases that can selectively process synthetic luciferin analogs. The resulting orthogonal pairs can be used in tandem for multi-component imaging. In this work, we describe heterocyclic luciferins as new probes for orthogonal bioluminescence applications. We synthesized several analogs, and characterized their light-emitting properties. The luciferins are competent light emitters with a variety of known luciferases in vitro and in cellulo. We further screened the analogs against libraries of luciferase mutants. Computational algorithms were used to identify unique enzyme-substrate pairs that could be used in conjunction with existing bioluminescent tools. The structural diversity of the luciferins enabled more selective orthogonal pairs to be identified and used for substrate-resolved imaging.

High-resolution orthogonal imaging with diverse bioluminescent substrates

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.