Molecular and Organismal-Level Studies of Bioluminescence

 描述(申请人提供)：萤火虫荧光素酶的生物发光是一种强大的方法，应用范围广泛，从体外药物结合分析到实时体内生物发光成像。这种方法相对便宜，易于使用。生物发光使用化学方法将小分子转化为光发射体，从而消除了其他成像平台所看到的背景信号。改变荧光素酶或修饰小分子荧光素底物都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，但一些光在到达探测器之前会被血红蛋白吸收而衰减。这突显了对更明亮的底物和/或底物的需求，这些底物可以更好地在整个动物体内访问荧光素酶。荧光素与荧光素酶的结合是生物发光成像的实验极限，而D-荧光素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，并在特定组织中进行更好的成像。事实上，我们实验室的一种荧光素类似物在活体小鼠大脑中的表现优于标准的D-荧光素底物，尽管该类似物的浓度要低20倍。此外，荧光素酶的突变使这些荧光素类似物具有选择性。扩大荧光素酶的范围，甚至果蝇的一种酶(CG6178)也显示出与我们的荧光素类似物潜在的荧光素酶活性。我们实验室的一些荧光素类似物是不同的荧光素酶突变体的选择性底物。 并且可以提供一种途径来获得荧光素酶-荧光素对，该荧光素酶-荧光素对可以在相同的动物或样品中使用而没有交叉反应。在交叉反应被最小化到记者能够报道不同事件的点的情况下，记者是正交的。我们假设，1)荧光素类似物与优化的突变荧光素酶甚至潜在的荧光素酶的组合可以在目标组织内产生更强劲的光输出，为测量生物事件提供更大的动态范围；2)选择突变的荧光素酶和荧光素类似物对能够正交性，提高体内生物发光用于测量同一动物内不同事件的效用；3)CG6178的S结构将加深我们对生物发光的理解，并为设计能够与内源酶进行生物发光的荧光素底物打开了可能性。为了验证这些假设，我们将使用表达荧光素酶、荧光素酶突变体和CG6178的活鼠来测试我们的荧光素类似物的生物分布、强度和光输出的选择性。我们将在不同的组织中结合不同的小鼠体内不同的荧光素酶突变，并测试与不同的荧光素底物的正交性。我们还将解算CG6178与底物的晶体结构，以了解这种潜在的荧光素酶发光的分子基础。该奖学金中的任何一个目标的成功都将改善生物发光，供生物医学研究社区使用。

 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.