Enhanced synthetic luciferin substrates for firefly luciferase

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Bioluminescence assays and imaging methods have been widely adopted for drug discovery because of their simplicity, robustness, and low cost. The inherently low background and lack of need for excitation light makes bioluminescence superior to fluorescence methods for many applications. Nevertheless, luciferase has yet to reach its full potential. Light emission from luciferase is limited by 1) access to the luciferin substrate (modulated by affinity, cell-permeability, and active transport by drug pumps), and 2) the photophysical properties of the luciferin (efficiency and wavelength of emission). To maximize the power of luciferase for biological applications we have synthesized new luciferin substrates that enhance luciferase light emission. We propose the following specific aims: 1) synthesis of new luciferin substrates with enhanced light emission properties; 2) identification of mutant luciferases that efficiently utilize synthetic luciferins; 3) evaluation of synthetic luciferins and mutant luciferases for live cell bioluminescence imaging.