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Nucleic Acids Research 2006 34(1):e7; doi:10.1093/nar/gnj003
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Published online 10 January 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
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Methods Online

Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids

Norihito Muranaka1,2, Takahiro Hohsaka2,3,* and Masahiko Sisido1

1Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University 3-1-1 Tsushimanaka, Okayama 700-8530, Japan 2School of Materials Science, Japan Advanced Institute of Science and Technology 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan 3PRESTO, Japan Science and Technology Agency 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

*To whom correspondence should be addressed. Tel: +81 761 51 1681; Fax: +81 761 51 1149; Email: hohsaka{at}jaist.ac.jp

Received May 2, 2005. Revised September 22, 2005. Accepted December 7, 2005.

In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target biomolecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four-base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid may be introduced by suppressing an amber codon that may appear from a (NNK)n nucleotide sequence on the mRNA. The mRNA display was expressed in an Escherichia coli in vitro translation system in the presence of three types of tRNAs carrying different four-base anticodons and a tRNA carrying an amber anticodon, the tRNAs being chemically aminoacylated with different nonnatural amino acids. The complexity of the starting mRNA-displayed peptide library was estimated to be 1.1 x 1012 molecules. The effectiveness of the four-base codon mediated mRNA display method was demonstrated in the selection of biocytin-containing peptides on streptavidin-coated beads. Moreover, a novel streptavidin-binding nonnatural peptide containing benzoylphenylalanine was obtained from the nonnatural peptide library. The nonnatural peptide library from the four-base codon mediated mRNA display provides much wider functional and structural diversity than conventional peptide libraries that are constituted from 20 naturally occurring amino acids.


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