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Journal of Nucleic Acids
Volume 2012 (2012), Article ID 538129, 7 pages
http://dx.doi.org/10.1155/2012/538129
Research Article

A Concept for Selection of Codon-Suppressor tRNAs Based on Read-Through Ribosome Display in an In Vitro Compartmentalized Cell-Free Translation System

1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
2Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan
3INAMORI Frontier Research Center, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
4Department of Molecular Chemistry and Biochemistry, Faculty of Science and Engineering, Doshisha University, 1-3 Tatara-Miyakodani, Kyotanabe, Kyoto 610-0321, Japan

Received 30 May 2012; Accepted 29 June 2012

Academic Editor: Masayasu Kuwahara

Copyright © 2012 Atsushi Ogawa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Here is presented a concept for in vitro selection of suppressor tRNAs. It uses a pool of dsDNA templates in compartmentalized water-in-oil micelles. The template contains a transcription/translation trigger, an amber stop codon, and another transcription trigger for the anticodon- or anticodon loop-randomized gene for tRNASer. Upon transcription are generated two types of RNAs, a tRNA and a translatable mRNA (mRNA-tRNA). When the tRNA suppresses the stop codon (UAG) of the mRNA, the full-length protein obtained upon translation remains attached to the mRNA (read-through ribosome display) that contains the sequence of the tRNA. In this way, the active suppressor tRNAs can be selected (amplified) and their sequences read out. The enriched anticodon (CUA) was complementary to the UAG stop codon and the enriched anticodon-loop was the same as that in the natural tRNASer.