New Strategy for Development of Novel FRET-based sensors

描述(由申请人提供)：将开发一种通用策略，用于创建用于小分子二级信使的新型可遗传编码的基于FRET的传感器。这些传感器将在体内被用来获得各种重要信号转导途径的分子水平信息。最初，设计策略将用于产生一种新的钙离子传感器，该传感器不受与细胞组件的背景相互作用的影响。寡核苷酸定向突变将被用来进化一对在有钙离子存在的情况下相互作用的改变的传感蛋白(钙调蛋白和M13)，噬菌体展示将被用来筛选大量的突变体文库，在那里将实施正负选择来优化所需的相互作用。一种由两个感应蛋白和两个荧光蛋白组成的融合蛋白将在哺乳动物和细菌细胞中表达。这四种蛋白质之间的边界区域将使用定向突变进行随机化，并将使用荧光激活的细胞分类来筛选体内FRET反应较高的突变体。分选将在哺乳动物细胞中进行，那些表现出所需荧光特性的细胞将在越来越严格的条件下接受反复几轮分选。然后，成功的传感器将被用于研究钙依赖过程，方法是将它们定位于不同的细胞细胞器。最终，定向进化、噬菌体展示和FACS分选的组合使用将扩展到其他小分子传感器的开发。

DESCRIPTION (provided by applicant): A general strategy will be developed for the creation of novel genetically encodable FRET-based sensors for small molecule secondary messengers. These sensors will be utilized in vivo to obtain molecular level information on a variety of important signal transduction pathways. Initially, the design strategy will be used to generate a new Ca2+sensor that is immune to background interactions with cellular components. Oligonucleotide directed mutagenesis will be employed to evolve a pair of altered sensing proteins (calmodulin and M13) that interact with each other in presence of Ca2+ Phage display will be used to screen large libraries of mutants, where positive and negative selections will be implemented to optimize the desired interactions. A fusion protein consisting of the two sensing proteins flanked by two fluorescent proteins will be expressed in both mammalian and bacterial cells. The boundary regions between the four proteins will be randomized using directed mutagenesis and fluorescence activated cell sorting will be used to select mutants with a high in vivo FRET response. The sorting will take place in mammalian cells and those cells exhibiting the desired fluorescent properties will be subjected to repeated rounds of sorting under increasingly stringent conditions. The successful sensors will then be used to study Ca2+dependent processes by targeting them to different cellular organelles. Ultimately, the combined use of directed evolution, phage display and FACS sorting will be extended to the development of other small molecule sensors.