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第二信使的新型遗传编码的基于FRET的传感器。这些传感器将在体内用于获得各种重要信号转导途径的分子水平信息。最初，该设计策略将用于产生一种新的Ca2+传感器，该传感器不受与细胞组分的背景相互作用的影响。寡核苷酸定向诱变将用于进化一对改变的传感蛋白（钙调蛋白和M13），其在Ca2+存在下彼此相互作用。噬菌体展示将用于筛选大型突变体文库，其中将实施阳性和阴性选择以优化所需的相互作用。由两侧为两个荧光蛋白的两个传感蛋白组成的融合蛋白将在哺乳动物和细菌细胞中表达。使用定向诱变将四种蛋白质之间的边界区域随机化，并使用荧光激活细胞分选来选择具有高体内FRET响应的突变体。分选将在哺乳动物细胞中进行，并且表现出所需荧光性质的那些细胞将在越来越严格的条件下进行重复的分选轮次。然后，成功的传感器将被用于研究钙离子依赖的过程，通过将它们靶向不同的细胞器。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.