Identification of mutant firefly luciferases that efficiently utilize aminoluciferins

Firefly luciferase-catalyzed light emission from D-luciferin is widely used as a reporter of gene expression and enzymatic activity both in vitro and in vivo. Despite the power of bioluminescence for imaging and drug discovery, light emission from firefly luciferase is fundamentally limited by the physical properties of the D-luciferin substrate. We and others have synthesized aminoluciferin analogs that exhibit light emission at longer wavelengths than D-luciferin and have increased affinity for luciferase. However, although these substrates can emit an intense initial burst of light that approaches that of D-luciferin, this is followed by much lower levels of sustained light output. Here we describe the creation of mutant luciferases that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells, allowing the use of aminoluciferins for cell-based bioluminescence experiments.