Mutant Firefly Luciferases Catalyze Light Emission with Complementary Analog Luciferins

Bioluminescence imaging (BLI) is among the most dynamic imaging modalities for visualizing whole cells and gene expression patterns in vivo. This technique captures the light-emission from the luciferase-catalyzed oxidation of small molecule luciferins with highly sensitive CCD cameras. While powerful, current options for multiplexed BLI in mice are limited by the number of native luciferase/luciferin pairs found in nature, as well as the narrow window of wavelengths of light that can permeate tissue. Our lab aims to expand the bioluminescent toolkit by pairing mutant luciferases with synthetic luciferin analogs, to obtain a biochemical resolution of multiple targets by imaging sequentially. Several generations of luciferase mutant libraries were screened against sterically and electronically modified families of luciferins, in order to find orthogonal pairs. Promising luciferases were expressed recombinantly for biochemical analysis with their respective luciferins. These pairs were evaluated for their potential for multicomponent in vivo imaging using protein and tissue culture models.