Detection of proteins based on amino acid sequences by multiple aptamers against tripeptides.
A number of different ligands have been tested in the course of the development of protein array technology. The most extensively studied example of protein ligands has been based on antibody-antigen interaction. Other examples include protein-protein, protein-nucleic acid, and protein-small molecule interactions. All these ligands can recognize and specifically bind to protein epitopes. In this study, we have developed a novel technology using DNA-based aptamers to detect proteins based on their amino acid sequences. Mouse cathepsin D was used for the proof of principle experiment. Four tripeptides, Leu-Ala-Ser, Asp-Gly-Ile, Gly-Glu-Leu, and Lys-Ala-Ile, were selected based on the published amino acid sequence of mouse cathepsin D. DNA aptamers against the tripeptides were isolated using the systematic evolution of ligands of exponential enrichment method. We have demonstrated that the aptamers specifically interacted with mouse cathepsin D using the structure-switch method. We further performed a proximity-dependent ligation assay to demonstrate that multiple aptamers could specifically detect the protein from cell extracts. In principle, one library containing 8000 aptamers should be enough to detect almost all proteins in the whole proteome in all organisms. This technology could be applied to generate a new generation of protein arrays.
Niu
Wenze
W
Key Lab of Brain Functional Genomics, MOE and STCSM, Shanghai Institute of Brain Functional Genomics, East China Normal University, 3663 Zhongshan Road N., Shanghai 200062, China.
Jiang
Nan
N
Hu
Yinghe
Y
D000595:Amino Acid Sequence; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D002402:Cathepsin D / Q000378:metabolism; D002846:Chromatography, Affinity; D006801:Humans; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism
D052157:Aptamers, Nucleotide; D009842:Oligopeptides; D011506:Proteins; D002402:Cathepsin D