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.
The light emission from firefly luciferase is widely used as an optical reporter to help identify new therapeutic molecules. Increasingly, the glow of light from luciferase is being used to image disease progression in living organisms. The work we propose will greatly improve the sensitivity and applicability of luciferase for both the identification of new drug candidates and the low-cost imaging of living organisms.
|Adams Jr, Spencer T; Mofford, David M; Reddy, G S Kiran Kumar et al. (2016) Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain. Angew Chem Int Ed Engl 55:4943-6|
|Mofford, David M; Adams Jr, Spencer T; Reddy, G S Kiran Kumar et al. (2015) Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity. J Am Chem Soc 137:8684-7|
|Mofford, David M; Reddy, Gadarla Randheer; Miller, Stephen C (2014) Latent luciferase activity in the fruit fly revealed by a synthetic luciferin. Proc Natl Acad Sci U S A 111:4443-8|
|Adams Jr, Spencer T; Miller, Stephen C (2014) Beyond D-luciferin: expanding the scope of bioluminescence imaging in vivo. Curr Opin Chem Biol 21:112-20|
|Evans, Melanie S; Chaurette, Joanna P; Adams Jr, Spencer T et al. (2014) A synthetic luciferin improves bioluminescence imaging in live mice. Nat Methods 11:393-5|
|Mofford, David M; Reddy, Gadarla Randheer; Miller, Stephen C (2014) Aminoluciferins extend firefly luciferase bioluminescence into the near-infrared and can be preferred substrates over D-luciferin. J Am Chem Soc 136:13277-82|
|Godinat, AurÃ©lien; Park, Hyo Min; Miller, Stephen C et al. (2013) A biocompatible in vivo ligation reaction and its application for noninvasive bioluminescent imaging of protease activity in living mice. ACS Chem Biol 8:987-99|
|Harwood, Katryn R; Mofford, David M; Reddy, Gadarla R et al. (2011) Identification of mutant firefly luciferases that efficiently utilize aminoluciferins. Chem Biol 18:1649-57|