Use of cDNA Tiling Arrays for Identifying Protein Interactions Selected by In Vitro Display Technologies

Kenichi Horisawa,Nobuhide Doi,Hiroshi Yanagawa

doi:10.1371/journal.pone.0001646

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.

Highlights

Protein display technologies [1], such as phage display [2], ribosome display [3,4,5], DNA display [6] and mRNA display [7,8,9], are powerful tools for construction and in vitro selection of large libraries of genotype-phenotype conjugates
Development of totally in vitro display techniques, such as ribosome display [3,4,5] and mRNA display [7,8,9], 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
We demonstrate a highly sensitive analysis employing a transcriptionfactor 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 superior to the use of cloning and sequencing for decoding genetic information of proteins enriched by in vitro selection

Summary

Introduction

Protein display technologies [1], such as phage display [2], ribosome display [3,4,5], DNA display [6] and mRNA display [7,8,9], 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. We demonstrate a highly sensitive analysis employing a transcriptionfactor 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 superior to the use of cloning and sequencing for decoding genetic information of proteins enriched by in vitro selection

Methods

Results

Discussion