Implementation and Testing of PURE mRNA Display

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DESCRIPTION (provided by applicant): There is a pressing need to develop new molecular tools that recognize protein surfaces for uses ranging from imaging probes to systems biology (blocking or modulating protein-protein interactions, serving as sensors) to proteomics (affinity reagents for mass spectrometry or chip-based diagnostics) to novel therapeutic leads. Here, we propose to implement and test a new combinatorial approach for constructing both natural and unnatural mRNA display libraries-PURE mRNA display (PURE = Protein synthesis Using Recombinant Elements). In this approach, the protein synthesis machinery will be assembled from purified components, providing complete experimental control of both concentrations and constituents. PURE mRNA display thus has several potential advantages relative to the classical mRNA display, most importantly in providing a facile route to construct designer display libraries bearing unnatural amino acids. Our specific aims are: 1. To test if it is possible to construct mRNA-peptide fusions using the PURE protein synthesis system. 2. To Compare and contrast PURE vs. retic-based mRNA display for in vitro directed evolution experiments.