Fluorescent proteins enable researchers to track multiple targets over extended periods of time in cellulo. However, these tools are not easily transitioned in vivo, owing to the need for excitation light and autofluorescence complications. A complementary optical imaging technology, bioluminescence, does not require incident light and is often more suitable for imaging in thick tissues and whole organisms. This modality relies on enzymes (luciferases) that catalyze the oxidation of small molecules (luciferins), with concomitant photon release. Unfortunately, the optimal luciferases for in vivo imaging utilize the same substrate, hindering efforts to visualize multiple targets simultaneously. Herein we present a method for crafting new luciferase-luciferin pairs using a combination of chemical synthesis and genetic engineering. Analogs of firefly luciferin were designed, synthesized, and assayed for photon production. Among these scaffolds, brominated luciferins emerged as promising leads for new tool development. The luciferin analogs were screened against libraries of mutant firefly luciferases, and the data were mined using a custom computer algorithm to identify unique enzyme-substrate pairings. Lead enzyme “hits” were expressed and purified for further study. The bioluminescent probes developed through this work will enable a variety of multi-component imaging studies not possible with existing toolsets.

Constructing new bioluminescent tools with minimally perturbed luciferins

251st National Meeting (2016)

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