UBC Theses and Dissertations
Development of peptide-like library Zhou, Miao
Peptide libraries are important in peptide based drug discovery and investigations of biological targets. Different chemical and biological methods have been developed to screen and decode peptide libraries. mRNA display technology is a biological method that utilizes in vitro translation systems to build peptide libraries, and the screened peptides are decoded by their covalently attached oligonucleotide templates. Like other biological methods, mRNA display technology can generate peptide libraries with high fidelity and speed but the chemical repertoire is limited mostly to natural amino acids. Our research goal is to expand the chemical repertoire of mRNA display technology by using it in combination with an in vitro reconstituted translation system, and build peptides containing stabilizing chemical features such as N-methyl amino acids that would make peptides more proteolytic resistant and drug-like. Using a streptavidin-dependent gel shift assay, the unnatural amino acids, N-methyl-L-alanine, N-methyl-L-glutamate, N-methyl-L-ornithine, L-abrine, t-butyl-L-glycine, biocytin, and sarcosine were shown to be able to incorporate successively into hexa-peptides in the mRNA display format. The peptides containing unnatural amino acids were shown to be more resistant to proteinase K proteolysis than peptides containing only the natural amino acid L-alanine. Several mRNA libraries were made in accordance with the mRNA display format. Repeated E. coli in vitro reconstituted translations of these mRNA libraries with varying translation conditions fail to yield peptides of significant lengths. However, a library of tetra-peptides was synthesized in the absence of mRNA display format. Thus, the puromycin linker required by mRNA display technology seemed to interfere with proper translations in the E. coli in vitro reconstituted translation system. Experiments showed that E. coli in vitro reconstituted translation system is slow at making full-length peptides in the context of mRNA display technology and a majority of products are prematurely terminated translation products, which could be due to slow peptidyl formation in the E. coli in vitro reconstituted translation system, or the result of undesirable interaction between the E. coli ribosome and puromycin linker that interfere with normal translation. This slow translation kinetic issue needs to be addressed with further experiments in order to successfully pursue the development of this technology.
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