In vitro selection of enzymes by mRNA-Display

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The goal of this project is the selection of Peptides and Proteins with Michaelase and Aldol condensation activities using different mRNA-display libraries. mRNA-Display is a technique that allows to generate covalent fusions between an mRNA, or a library of synthetic RNAs, and the peptide or protein that it encodes. mRNA-display libraries can be generated by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3' end. The stable linkage between the informat ional (nucleic acid) and functional (peptide) domains of the resulting joint molecules allows a specific mRNA to be enriched from a complex mixture of mRNAs based on the properties of its encoded peptide. We will use mRNA-display libraries that are already available in our laboratory, namely a library that is completely randomised without structural bias, except for an increased probability of a-helical amd b-sheet-forming domains. The other library is based on the lipocalin scaffold and consists of random amino acids in the connecting loop domains of the scaffold. The selection scheme will be based on chemical derivatization and tagging of the cDNA that corresponds to each sequence in the library. Those peptide sequences that are active catalysts will introduce a tag onto the cDNA so that it can be separated from inactive sequences, amplified, and be used as the input for the subsequent selection cycle. This strategy should allow to select enzymatic activities of oligopeptides from random sequences without any bias from natural enzymes that catalyse similar reactions.