Acknowledgments We thank Dr Seiji Tateyama for valuable suggestions, Dr Noriko Tabata for experimental advice, and Genefrontier Corp. and NimbleGen Systems Inc. for the microarray analysis. References 1. Matsumura N, Doi N, Yanagawa H (2006) Recent progress and future prospects in protein display technologies as tools for proteomics. Curr Proteomics 3: 199–215. 2. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315–1317. 3. Mattheakis LC, Bhatt RR, Dower WJ (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci USA 91: 9022–9026. 4. He M, Taussig MJ (1997) Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res 25: 5132–5134. 5. Hanes J, Plu¨ckthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci USA 94: 4937–4942. 6. Doi N, Yanagawa H (1999) STABLE: protein-DNA fusion system for screening of combinatorial protein libraries in vitro. FEBS Lett 457: 227–230. 7. Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H (1997) In vitro virus: bonding of mRNA bearing puromycin at the 39-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett 414: 405–408. 8. Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci USA 94: 12297–12302. 9. Miyamoto-Sato E, Takashima H, Fuse S, Sue K, Ishizaka M, et al. (2003) Highly stable and efficient mRNA templates for mRNA-protein fusions and C- terminally labeled proteins. Nucleic Acids Res 31: e78. 10. Keefe AD, Szostak JW (2001) Functional proteins from a random-sequence library. Nature 410: 715–718. 11. Hanes J, Jermutus L, Weber-Bornhauser S, Bosshard HR, Plu¨ ckthun A (1998) Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc Natl Acad Sci USA 95: 14130–14135. 12. Fukuda I, Kojoh K, Tabata N, Doi N, Takashima H, et al. (2006) In vitro evolution of single-chain antibodies using mRNA display. Nucleic Acids Res 34: e127. 13. Hammond PW, Alpin J, Rise CE, Wright M, Kreider BL (2001) In vitro selection and characterization of Bcl-X(L)-binding proteins from a mix of tissue-specific mRNA display libraries. J Biol Chem 276: 20898–20906. 14. Miyamoto-Sato E, Ishizaka M, Horisawa K, Tateyama S, Takashima H, et al. (2005) Cell-free cotranslation and selection using in vitro virus for high- throughput analysis of protein-protein interactions and complexes. Genome Res 15: 710–717. 15. McPherson M, Yang Y, Hammond PW, Kreider BL (2002) Drug receptor identification from multiple tissues using cellular-derived mRNA display libraries. Chem Biol 9: 691–698. 16. Tateyama S, Horisawa K, Takashima H, Miyamoto-Sato E, Doi N, et al. (2006) Affinity selection of DNA-binding protein complexes using mRNA display. Nucleic Acids Res 34: e27. 17. Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340: 245–246. 18. Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, et al. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24: 218–229. Tiling Arrays for mRNA Display Author Contributions Conceived and designed the experiments: HY KH. Performed the experiments: KH. Analyzed the data: KH. Wrote the paper: KH ND. 19. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376–380. 20. Kapranov P, Cawley SE, Drenkow J, Bekiranov S, Strausberg RL, et al. (2002) Large-scale transcriptional activity in chromosomes 21 and 22. Science 296: 916–919. 21. Horisawa K, Tateyama S, Ishizaka M, Matsumura N, Takashima H, et al. (2004) In vitro selection of Jun-associated proteins using mRNA display. Nucleic Acids Res 32: e169. 22. Horisawa K, Doi N, Takashima H, Yanagawa H (2005) Application of quantitative real-time PCR for monitoring the process of enrichment of clones on in vitro protein selection. J Biochem 137: 121–124. 23. Gunji W, Kai T, Sameshima E, Iizuka N, Katagi H, et al. (2004) Global analysis of the expression patterns of transcriptional regulatory factors in formation of embryoid bodies using sensitive oligonucleotide microarray systems. Biochem Biophys Res Commun 325: 265–275. 24. ’t Hoen PA, de Kort F, van Ommen GJ, den Dunnen JT (2003) Fluorescent labelling of cRNA for microarray applications. Nucleic Acids Res 31: e20. 25. Nuwaysir EF, Huang W, Albert TJ, Singh J, Nuwaysir K, et al. (2002) Gene expression analysis using oligonucleotide arrays produced by maskless photolithography. Genome Res 12: 1749–1755. 26. Kim TH, Barrera LO, Zheng M, Qu C, Singer MA, et al. (2005) A high- resolution map of active promoters in the human genome. Nature 436: 876–880. 27. Chinenov Y, Kerppola TK (2001) Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene 20: 2438–2452. 28. Liberati NT, Datto MB, Frederick JP, Shen X, Wong C, et al. (1999) Smads bind directly to the Jun family of AP-1 transcription factors. Proc Natl Acad Sci USA 96: 4844–4849. 29. Jain J, McCaffrey PG, Miner Z, Kerppola TK, Lambert JN, et al. (1993) The T- cell transcription factor NFATp is a substrate for calcineurin and interacts with Fos and Jun. Nature 365: 352–355. 30. Campbell (1996) Biology, 4th Ed. New York: The Benjamin/Cummings Publishing Company Inc. 31. Schrem H, Klempnauer J, Borlak J (2004) Liver-enriched transcription factors in liver function and development. Part II: the C/EBPs and D site-binding protein in cell cycle control, carcinogenesis, circadian gene regulation, liver regenera- tion, apoptosis, and liver-specific gene regulation. Pharmacol Rev. 56: 291–330. 32. Davydov IV, Bohmann D, Krammer PH, Li-Weber M (1995) Cloning of the cDNA encoding human C/EBP gamma, a protein binding to the PRE-I enhancer element of the human interleukin-4 promoter. Gene 161: 271–275. 33. Lu R, Yang P, O’Hare P, Misra V (1997) Luman, a new member of the CREB/ ATF family, binds to herpes simplex virus VP16-associated host cellular factor. Mol Cell Biol. 17: 5117–5126. 34. Gachon F, Fonjallaz P, Damiola F, Gos P, Kodama T, et al. (2004) The loss of circadian PAR bZip transcription factors results in epilepsy. Genes Dev. 18: 1397–1412. PLoS ONE | www.plosone.org 6 February 2008 | Volume 3 | Issue 2 | e1646