Diversifying the Glowing Bioluminescent Toolbox

Advances in chemical design, genetic engineering, and biomedical technologies have contributed to impressive progress in bioluminescence imaging (BLI) of complex biological systems from cells to living mammals. While the technique is prevalent in small animal in vivo molecular imaging applications, most tools have been limited to only a few sets of bioluminescent systems. Now, the Prescher lab at the University of California, Irvine, has introduced an elegant interdisciplinary approach that paves a new path for the diversification of the bioluminescence toolbox. Bioluminescence involves the production of light via the enzymatic oxidation of small-molecule substrates by luciferase enzymes. The process has been exploited for noninvasive and highly sensitive optical imaging of small animal models. Selective integration of luciferase into mammalian systems, either by implanted cells or through linked expression to promoter genes, has allowed for a wealth of applications including tumor cell tracking, monitoring tissue-specific gene expression, and reporting on circadian rhythms. Moreover, chemical modification of the light-emitting substrates have further expanded the range of BLI applications to analyzing small molecules, assessing drug delivery, and studying enzyme activity. Compared with other in vivo imaging modalities, BLI is inexpensive, requires no excitation light source, and is easy to use. These factors coupled with the versatility of its potential applications has resulted in its widespread implementation across biological sciences. However, only a small subset of bioluminescent substrate−enzyme pairs is amenable to in vivo BLI, limiting its use for multicomponent applications. Expanding the bioluminescent toolbox requires optimization of the biochemistry (substrate−enzyme interactions), photophysics, and both the biodistribution and bioavailability of the substrate. Research in recent years has generated modified substrates with improved in vivo emission properties (e.g., red-shifted light which better penetrates through mammalian tissue or brighter emission to improve the signalto-noise ratio) or biodistribution. Yet, the majority of these developments have focused on improved substrates for the widely used firefly luciferase enzyme. Achieving multicomponent imaging poses an added challenge of requiring bioluminescent pairs that are orthogonalmeaning they do not cross-react with one another. To this end, emerging work has reported on new or mutant luciferase enzymes that can act more favorably with unnatural substrates. Efforts to optimize substrate−enzyme interactions have led to discoveries such as the furimazine−Nanoluc pair, a promising system which addresses challenges in stability and brightness that has plagued imidazopyridine−luciferase systems (e.g., coelenterazine−Renilla luciferase). In other pioneering work, Miller and co-workers identified mutant luciferases that more readily interact with substrates based on a cyclized scaffold termed CycLuc over the parent D-luciferin. Interestingly, these mutant luciferases can process CycLuc derivatives more readily than firefly luciferase. These examples highlight the promise of parallel modifications of both enzymes and substrates to achieve multicomponent BLI. The Prescher lab recently introduced a direct approach for finding bio-orthogonal bioluminescent pairs. They generated a panel of derivatives of D-luciferin and screened them against a library of mutant luciferases, identifying functional substrate− enzyme systems. From there, the degree of orthogonality of pairs of enzymes with cognate substrates was assessed with a