UC Irvine

Optical reporters have revolutionized our ability to visualize biological mechanisms in action. Among the most popular techniques for imaging in whole tissues and living organisms is bioluminescence. One of the most widely used bioluminescent systems comprises firefly luciferase (Fluc) and the small molecule D-luciferin. These components produce photons that can be detected with sensitive cameras. Since mammalian tissues produce little to no photons endogenously, bioluminescence is well suited for imaging in whole organisms. Consequently, Fluc and D-luciferin have been widely used to track cell movements, gene expression patterns and other activities in a variety of preclinical models. Bioluminescence has been largely limited to imaging one cell type at a time, though, due to a lack of distinguishable luciferase-luciferin pairs.

To address this void, my thesis work focused on developing spectrally resolved for multicomponent imaging. I generated both electronically modified luciferins and mutant luciferases. Isomeric luciferin analogs bearing a pyridone moiety were designed and synthesized. These analogs exhibited distinct bioluminescence spectra that could be distinguished using optical filters. However, the analogs were weak emitters compared to the native substrate. Mutant luciferases with improved photon outputs were identified via library screening and directed evolution. Significant improvements in luciferase activity could be achieved in only 1-2 rounds of screening.

While spectrally resolved probes can be readily distinguished in transparent media, discriminating wavelengths through thick tissues is challenging. To address this issue, I developed a mutually orthogonal luciferase-luciferin pair. Pi-extended luciferin analogs were synthesized, and a complementary luciferase was engineered using computational enzyme design. This custom luciferase-luciferin pair was compatible with three existing bioluminescent tools, enabling selective imaging of four luciferases based on substrate preference.

Finally, I expressed and purified orthogonal luciferases for X-ray crystallography. Luciferase purity and fidelity were verified using a variety of analytical techniques. However, when the luciferases were subjected to published crystallization procedures, only protein precipitation was observed. Ligands were shown to stabilize the enzyme, and crystallization attempts with these molecules yielded several promising “hits”. Collectively, my thesis work expands the toolset for multicomponent imaging and builds towards an understanding of luciferase substrate specificity.