Library-based development:optical imaging probes (RMI)

描述（由申请人提供）：小分子和基于GFP的荧光团在细胞的光学成像中已显示出价值，并已应用于单个生物分子的研究。 然而，严重的限制，其生物物理特性和特异性阻碍了其在高分辨率活细胞成像的应用。 提出了一种基于库的方法，重点是开发具有优化特性的新荧光探针，用于活细胞中的单分子分辨率光学成像。 我们将进行三个平行的努力，以产生小分子和遗传编码的探针，可以针对细胞内的特定RNA或蛋白质序列。 首先，花青、罗丹明和Alexa型荧光团的文库将以组合方式合成，并筛选在体外和活细胞中以高特异性标记小肽基序或RNA适体的能力。 其次，天然细菌生物素转移酶将被重新设计，以催化细胞内一系列小分子荧光团的13个氨基酸基序的共价标记。 第三，绿色荧光蛋白的物理性质将通过合理设计和突变体库筛选的组合来系统地改善，以使它们可用于细胞中的单分子成像。 这些改进的荧光蛋白将被用于构建FRET型指标，用于神经生物化学成像。

DESCRIPTION (provided by applicant): Small molecule and GFP-based fluorophores have demonstrated value in optical imaging of cells and have been applied to the study of individual biomolecules. However, severe limitations with respect to their photophysical properties and their specificity hinder their application in high-resolution live cell imaging. A library-based approach focused on the development of new fluorescent probes with optimized properties for single-molecule resolution optical imaging in living cells is proposed. We will undertake three parallel efforts to generate both small-molecule and genetically-encoded probes that can be targeted to specific RNA or protein sequences inside cells. First, libraries of cyanine, rhodamine, and Alexa-type fluorophores will be synthesized in combinatorial fashion and screened for the ability to label small peptide motifs or RNA aptamers in vitro and in live cells with high specificity. Second, the natural bacterial enzyme biotin transferase will be re-engineered to catalyze covalent labeling of a 13-amino acid motif with a range of small-molecule fluorophores inside cells. Third, the photophysical properties of the green fluorescent proteins will be systematically improved by a combination of rational design and screening of mutant libraries to make them useful for single-molecule imaging in cells. These improved fluorescent proteins will then be used to build FRET-type indicators for imaging neurobiochemistry.