Molecular and Organismal-Level Studies of Bioluminescence

 描述（由申请人提供）：萤火虫荧光素酶的生物发光是一种功能强大的方法，可用于从体外药物结合试验到实时体内生物发光成像的广泛应用。该方法相对便宜且易于使用。生物发光利用化学将小分子酶促转化为光发射体，从而消除了其他成像平台所见的背景信号。改变荧光素酶或修饰小分子荧光素底物可以调节体内光输出的分布和强度。尽管生物发光试剂能够在体内产生可测量的光子通量，但一些光在到达检测器之前被血红蛋白吸收衰减。这突出了对更亮的底物和/或可以更好地接近整个动物内的荧光素酶的底物的需要。荧光素酶对D-荧光素酶的接近是生物发光成像的实验限制，并且D-荧光素酶具有已知的生物分布模式。然而，荧光素的修饰可以调节这种分布，并使特定组织能够更好地成像。事实上，来自我们实验室的D-glycin类似物已被证明在活小鼠脑中优于标准D-glycin底物，即使该类似物以低20倍的浓度提供。此外，荧光素酶的突变使得能够选择性地使用这些荧光素类似物。 扩大荧光素酶的范围，甚至来自果蝇的酶（CG 6178）也显示出与我们的荧光素类似物的潜在荧光素酶活性。来自我们实验室的一些Escherin类似物是不同荧光素酶突变体的选择性底物 并且可以提供成对的酶-底物的途径，其可以用于相同的动物或样品而没有交叉反应性。在交叉反应性最小化到报告子能够报告不同事件的程度的情况下，报告子是正交的。 我们假设：1）Bisphin类似物与优化的突变体Bisphase或甚至潜在的荧光素酶的组合可以在感兴趣的组织内产生更稳健的光输出，为测量生物事件提供更大的动态范围; 2）选择的突变的Bisphase和Bisphin类似物对能够正交，改善体内生物发光用于测量一个动物内的单独事件的效用; CG 6178的结构将增强我们对生物发光的理解，并为设计能够与内源酶一起生物发光的底物提供可能性。为了检验这些假设，我们将使用表达荧光素酶、荧光素酶突变体和CG 6178的活小鼠来测定我们的荧光素类似物的生物分布、强度和光输出的选择性。我们将联合收割机将不同的荧光素酶突变体在单独的组织中与单个小鼠组合，并测试与单独的荧光素底物的正交性。我们还将解决CG 6178与底物结合的晶体结构，以了解这种潜在荧光素酶发光的分子基础。该奖学金的任何目标的成功都将改善生物发光，供生物医学研究界使用。

 DESCRIPTION (provided by applicant): Bioluminescence from firefly luciferase is a powerful method used in a wide-range of applications ranging from in vitro drug-binding assays to real-time in vivo bioluminescence imaging. The method is relatively inexpensive and easy to use. Bioluminescence uses chemistry to enzymatically convert a small-molecule to a light emitter, which eliminates the background signal seen with other imaging platforms. Either altering the luciferase enzyme or modification to the small-molecule luciferin substrate can modulate the distribution and intensity of light output in vivo. Despite the ability of bioluminescent reagents o produce measurable photon flux in vivo, some of the light is attenuated by hemoglobin absorption before reaching the detector. This highlights the need for brighter substrates and/or substrates that can better access luciferase within whole animals. Access of luciferin to luciferase is an experimental limitation of bioluminescent imaging and D-luciferin has a known biodistribution pattern. However, modification of luciferin can modulate this distribution and enable better imaging in specific tissues. Indeed, a luciferin analog from our lab has been shown to outperform the standard D-luciferin substrate in live mouse brain, even though the analog was supplied at 20-fold lower concentration. Further, mutation of luciferase enables selectivity for these luciferin analogs. Extending the scope of what a luciferase is, even an enzyme from Drosophila (CG6178) revealed latent luciferase activity with our luciferin analogs. Some of the luciferin analogs from our lab are selective substrates for distinct luciferase mutants and may provide a path to luciferase-luciferin pairs that can be used in the same animal or sample without cross-reactivity. In the case where cross-reactivity is minimized to a point where reporters are able to report on distinct events, the reporters are orthogonal. We hypothesize that 1) the combination of luciferin analogs with optimized mutant luciferases or even latent luciferase enzymes can generate more robust light output within the tissue of interest, providing a greater dynamic range for measuring biological events; 2) select pairs of mutated luciferases and luciferin analogs are capable of orthogonality, improving the utility of bioluminescence in vivo for measuring separate events within one animal; and 3) CG6178's structure will enhance our understanding of bioluminescence and open the possibility to designing luciferin substrates capable of bioluminescence with endogenous enzymes. To test these hypotheses, we will use live mice expressing luciferase, luciferase mutants, and CG6178 to assay the biodistribution, intensity, and selectivity of light output with our luciferin analogs. We will combine distinct luciferase mutants within individual mice in separate tissues and test for orthogonality with separate luciferin substrates. We will also solve crystal structures of CG6178 with substrate bound to understand the molecular basis of light emission with this latent luciferase. The success of any of the aims in this fellowship will improve bioluminescence for use by the biomedical research community.