Use of cDNA tiling arrays for identifying protein interactions selected by in vitro display technologies.
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 approximately 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 ( approximately 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.
Horisawa
Kenichi
K
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama, Japan.
Doi
Nobuhide
N
Yanagawa
Hiroshi
H
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D005809:Genes, Regulator; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000378:metabolism; D016366:Open Reading Frames; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D009841:Oligonucleotides; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D014157:Transcription Factors