Chemical and Protein Engineering of Nature's Living Light: Bioluminescence

The bioluminescence imaging toolkit has continuously evolved biomedical research. Due to the fact that fluorescence measurements require external excitation light, the uses of fluorescence in thick tissues and live animals are limited. Bioluminescence imaging, on the other hand, overcomes this hurdle since they use enzyme-catalyzed exothermic biochemical reactions to generate excited-state emitters. However, the in vivo performance of ATP-independent marine luciferases is relatively poor due to their blue emission. Therefore, the development of red-shifted bioluminescent reporters is especially desirable for highly sensitive in vivo bioluminescence imaging (BLI). We integrated several approaches, including the chemical synthesis of red-shifted substrates, protein engineering of luciferases via directed evolution, and bioluminescence resonance energy transfer (BRET) with red fluorescent proteins. Our engineered luciferase-luciferin pairs emit bright and red-shifted bioluminescence to achieve superior in vitro and in vivo sensitivity over commonly used bioluminescent reporters.
To further expand the color palette and improve biocompatibility, we developed a family of pyridyl coelenterazine analogs that exhibit different colors of emission and enhanced water solubility. We paired them with our further evolved luciferase, and the resultant luciferase-luciferin pair can highlight early tumors in xenograft models. Furthermore, we demonstrated that it is possible to evolve multiple spectrally resolved and orthogonal luciferase¬-luciferin pairs for multiplexed bioassays. Our new tools offer new opportunities for designing bioluminescent biosensors to non-invasively uncover complex signaling in live¬ cells and live animals.