Orthogonal Luciferase-Luciferin Pairs for Bioluminescence Imaging.
Orthogonal Luciferase-Luciferin Pairs for Bioluminescence Imaging.
复制标题
DOI：
10.1021/jacs.6b11737
复制
发表时间：
2017-02-15
影响因子：
15
通讯作者：
Prescher JA
中科院分区：
文献类型：
--
作者：
Jones KA;Porterfield WB;Rathbun CM;McCutcheon DC;Paley MA;Prescher JA
使用荧光素酶-荧光素对的生物发光成像广泛应用于生物医学研究。自然界中已鉴定出几种荧光素酶，其中许多已适用于追踪整个动物的细胞。不幸的是，用于体内成像的最佳荧光素酶利用相同的底物，因此不能轻易区分单个受试者中的多种细胞类型。为了开发更广泛的可区分探针，我们精心制作了可以由工程荧光素酶选择性加工的定制荧光素。用空间修饰的荧光素迭代筛选突变酶文库，并鉴定出正交酶-底物“命中”。当互补的酶-底物伙伴在体外和培养细胞模型中相互作用时，这些工具会产生光。基于他们的选择性，这些设计师对将支持多组件成像，并能够直接询问目前现有工具无法实现的细胞网络。我们的筛选平台也是通用的，将加快鉴定更独特的荧光素酶和荧光素，进一步扩展生物发光工具包。
Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate, and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate “hits” were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multi-component imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.