Ribosome Display and Photo-Cross-Linking Techniques for In Vitro Identification of Target Proteins of Bioactive Small Molecules

• Published in: Analytical chemistry (Washington) 2014-07, Vol.86 (14), p.6768-6773
• Main Authors: Wada, Akira, Hara, Shuta, Osada, Hiroyuki
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Beads; Binding; Binding sites; Biocompatibility; Codon, Terminator; Cross-Linking Reagents - chemistry; Crystallography, X-Ray; Diazomethane - chemistry; Electrophoresis; Humans; In vitro testing; Luminescent Measurements; MicroRNAs; Peptide Library; Photochemistry - methods; Polymerase Chain Reaction; Pools; Proteins; Proteins - analysis; Proteins - metabolism; Recombinant Fusion Proteins - metabolism; Ribonucleic acid; Ribosomes - chemistry; Ribosomes - metabolism; RNA; RNA, Messenger - metabolism; Tacrolimus - metabolism; Tacrolimus Binding Protein 1A - genetics; Tacrolimus Binding Protein 1A - metabolism; Target recognition
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac4030208

The identification of target proteins of bioactive small molecules as bioprobe candidates or drug seeds is indispensable for elucidating their actions and predicting their side effects. To meet the current need, we developed a scheme for detection and identification of target proteins by using ribosome display and photo-cross-linking techniques, and demonstrated the feasibility. The mRNAs encoding full-length human proteins (FHPs) were constructed and translated in vitro to prepare pools of FHP–ribosome–mRNA complexes used for ribosome display selection. Expression levels of the FHPs were confirmed by Western blot analysis, and photo-cross-linked small-molecule beads were assessed through cell-free synthesized FHP binding assay. After ribosome display selection against photo-cross-linked small-molecule beads, RT-PCR using mRNAs recovered from the selected ternary complexes and electrophoresis of the PCR products allowed specific detection of the target proteins binding to the beads. In addition, a repeat of ribosome display selection enabled us to identify the target proteins even if the molar quantity was one ten-thousandth of that of the other proteins in a cell-free synthesized FHP pool. Therefore, these results showed that ribosome display using photo-cross-linked small-molecule beads and further extended FHP pool could be one of the powerful techniques for identification of unknown target proteins of bioactive small molecules.