Enhanced synthetic luciferin substrates for firefly luciferase

描述(申请人提供)：生物发光分析和成像方法因其简单、可靠和低成本而被广泛采用于药物发现。固有的低本底和不需要激发光使生物发光在许多应用中优于荧光方法。然而，荧光素酶还没有充分发挥其潜力。荧光素酶的光发射受限于1)对荧光素底物的访问(受亲和力、细胞通透性和药物泵的主动运输调节)，以及2)荧光素的光物理性质(发射效率和波长)。为了最大限度地发挥荧光素酶的生物应用能力，我们合成了增强荧光素酶发光的新的荧光素底物。我们提出了以下具体目标：1)合成具有增强发光性能的新的荧光素底物；2)鉴定有效利用合成荧光素的突变的荧光素酶；3)评价合成的荧光素和突变的荧光素酶用于活细胞生物发光成像。 与公共健康相关：萤火虫荧光素酶的光发射被广泛用作光学报告程序，帮助识别新的治疗分子。越来越多地，荧光素酶发出的光被用来成像活着的有机体中的疾病进展。我们提出的工作将极大地提高荧光素酶在识别新的候选药物和低成本生物成像方面的敏感性和适用性。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.