Library-based development:optical imaging probes (RMI)

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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.