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The development of mRNA display : synthesis of peptides containing unnatural amino acids

University of British Columbia

Currently, one of main challenges in drug discovery is the generation of diverse compound libraries that can be easily screened to identify potential inhibitors of therapeutic targets. mRNA display is a technique to create a vast library of unique peptide molecules that can be easily screened for this purpose. mRNA display generates peptides that are covalently linked to their encoding mRNA templates. The utilization of a reconstituted translation system makes it possible to incorporate unnatural amino acids with various structures into peptides. In this project, mRNA display and an E. coli-based reconstituted translation system are combined to create peptides comprised of unnatural amino acids that are covalently attached to their encoding nucleic acid. The incorporation of N-methyl or cyclic unnatural amino acids into peptides are believed to contribute to their resistance to proteolytic degradation. My project has two main objectives. The first one is to use the mRNA display and reconstituted translation system to assemble hexa-peptides with N-methyl or cyclic unnatural amino acids. The compatibilities of these amino acids with the translation system are individually tested. The second objective is to optimize the DNA linker length for an increase in full-length translation product from what is achieved with a standard linker. These efforts will enable the synthesis of peptide-like libraries using this drug discovery platform. N-Methyl or cyclic amino acid-pdCpA conjugates were synthesized as building blocks of E. coli reconstituted translation. The following unnatural amino acids, N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-L-phenylalanine, L-homoproline, L-octahydro- indole-2-carboxylic acid, N-Me-L-aminohexanoic acid, and L-proline, were chemically acylated to tRNAGCC and translated into hexa-peptide with biocytin as the last amino acid. A full-length translation product of each amino acid was observed using the streptavidin-binding assay. The successful incorporation of N-methyl or cyclic amino acids into a peptide were indirectly shown by some resistance to proteinase K treatment as compared to control (natural L-alanine). The DNA linker was then optimized to increase the portion of full-length translation product in the translation mixture. A DNA linker bearing a 26mer chain of polyadenosine provided the highest observed percentage of translated full-length material.

Thesis/Dissertation

application/pdf

2009-06-09

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/8883

Pharmaceutical Sciences

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/