Use of cDNA Tiling Arrays for Identifying Protein Interactions Selected by In Vitro Display Technologies Kenichi Horisawa, Nobuhide Doi, Hiroshi Yanagawa* Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama, Japan Abstract In vitro display technologies such as mRNA display are powerful screening tools for protein interaction analysis, but the final cloning and sequencing processes represent a bottleneck, resulting in many false negatives. Here we describe an application of tiling array technology to identify specifically binding proteins selected with the in vitro virus (IVV) mRNA display technology. We constructed transcription-factor tiling (TFT) arrays containing ,1,600 open reading frame sequences of known and predicted mouse transcription-regulatory factors (334,372 oligonucleotides, 50-mer in length) to analyze cDNA fragments from mRNA-display screening for Jun-associated proteins. The use of the TFT arrays greatly increased the coverage of known Jun-interactors to 28% (from 14% with the cloning and sequencing approach), without reducing the accuracy (,75%). This method could detect even targets with extremely low expression levels (less than a single mRNA copy per cell in whole brain tissue). This highly sensitive and reliable method should be useful for high-throughput protein interaction analysis on a genome-wide scale. Citation: Horisawa K, Doi N, Yanagawa H (2008) Use of cDNA Tiling Arrays for Identifying Protein Interactions Selected by In Vitro Display Technologies. PLoS ONE 3(2): e1646. doi:10.1371/journal.pone.0001646 Editor: Stephen W. Michnick, Universite´ de Montre´al, Canada Received October 21, 2007; Accepted January 28, 2008; Published February 20, 2008 Copyright: 2008 Horisawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by a Grant-in-Aid for The 21st Century Center of Excellence (COE) Program entitled ‘‘Understanding and Control of Life’s Function via Systems Biology (Keio Univ.)’’ of the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT); The Keio University Global Center of Excellence (G-COE) Program entitled ‘‘Center for Human Metabolomic Systems Biology’’ of the MEXT; and The Research for Promoting Technological Seeds Program of the Japan Science and Technology Agency (JST). Competing Interests: The authors have declared that no competing interests exist. *E-mail: hyana@bio.keio.ac.jp Introduction Protein display technologies [1], such as phage display [2], ribosome display [3–5], DNA display [6] and mRNA display [7–9], are powerful tools for construction and in vitro selection of large libraries of genotype-phenotype conjugates. These libraries can be affinity-screened via the protein moiety (phenotype) followed by decoding of the nucleic acid moiety (genotype) to identify the selected proteins. These display technologies have been employed not only for directed evolution of novel proteins and antibodies [10–12], but also for the screening of protein-protein [13,14], protein-drug [15], and protein-DNA [16] interactions from randomly fragmented cDNA libraries. Development of totally in vitro display techniques, such as ribosome display [3–5] and mRNA display [7–9], based on cell-free translation systems has extended the scope of previous techniques for protein interaction analysis using living cells, such as the yeast two-hybrid method [17] and biochemical methods coupled with mass spectrometry [18], because the variety of testable interaction conditions is greater, and the in vitro techniques are applicable to cytotoxic proteins. However, the display technologies have a common bottleneck in the final step of identifying the specifically selected protein sequences. The decoding is usually achieved by cloning and DNA sequencing, but the following difficulties arise: 1) Only a limited number of clones can be analyzed, and thus positive candidates whose contents in the selected library are less than a threshold determined by the number of analyzed clones are lost as false negatives. 2) Positive sequences with low contents in a library can be enriched by iterative rounds of affinity-selection, but lower- affinity binders compete with higher-affinity binders and therefore drop out of the screening. 3) DNA fragments which are injurious to cloning hosts, e.g., cytotoxic sequences, may be lost. 4) Cloning and sequencing of a huge number of copies of selected sequences is redundant, cost-ineffective, and time-consuming. Although novel high-throughput DNA sequencing methods that require no bacterial cloning process have recently been reported [19], these techniques are not yet widely available. To overcome the above limitations, we examined the use of a DNA microarray technique as an alternative to the cloning and sequencing processes (Figure 1). The combinatorial use of a tiling array [20] representing ORF sequences with in vitro display technology would provide a completely in vitro platform for highly sensitive and parallel analysis of protein interactions. It should be possible to detect enrichment of cDNA fragments of selected candidates even with low contents or low affinity. In this report, we demonstrate a highly sensitive analysis employing a transcription- factor tiling (TFT) array for identifying Jun-associated proteins selected with an mRNA display technology, in vitro virus (IVV) [21,22], and show that the use of tiling arrays is indeed superior to the use of cloning and sequencing for decoding genetic information of proteins enriched by in vitro selection. Methods IVV screening Preparation of bait template and IVV template libraries, and the procedure of IVV screening were described in detail in our previous report [21]. Details are also given in Methods S1 online. PLoS ONE | www.plosone.org 1 February 2008 | Volume 3 | Issue 2 | e1646