(260f) A Novel Method for the Construction of Custom Peptide Libraries From Oligonucleotide Arrays

Peptide libraries have been successfully used for structural studies and drug discovery to identify bioactive and affinity peptides, to generate synthetic vaccines, and for epitope mapping. While solid phase peptide synthesis is commonly used to prepare peptide libraries, its high cost and low production yields limit the size of the each library to be analyzed. On the other hand, large libraries can be produced by expressing genes coding for peptides and small proteins in live organisms at a much-reduced cost. This study demonstrates the use of a maskless light-directed parallel oligonucleotide synthesis to create peptide-coding gene libraries in a variety of host systems. This technology allows us to synthesize tens of thousand of oligonucleotides up to 200mer in length with any desired sequence in a single run. After synthesis, these oligonucleotides are cleaved off the substrate and amplified by polymerase chain reaction to convert them into double stranded DNA. Our studies show that emulsion PCR is required to ensure equal amplification of oligonucleotides, and more importantly prevent cross-recombination of highly similar sequences as seen in conventional PCR. Following amplification and purification, these PCR products can be cloned in any host system (eukaryotic, prokaryotic, or viral) for the expression of peptides. To demonstrate the feasibility of this method, we have designed and synthesized a gene library coding for mutants of the antimicrobial peptide Pediocin, successfully cloned and expressed it both in S. cerevisiae and E. coli to study the effect of amino acid mutations on antimicrobial activity. We have also used this approach to build custom phage display libraries. By combining our massively parallel in situ oligonucleotide synthesis and the use of live organisms as peptide factories, we hereby report an efficient and inexpensive method to construct custom peptide libraries which will allow researchers to study these molecules for their different characteristics (antimicrobial, ligand-binding, etc.) without the limitation on quantity and the high costs of other techniques.