title: Aptamer microarray as a novel bioassay for protein-protein interaction discovery and analysis.
abstract: Aptamer microarray is investigated as a novel bioassay for protein-protein interaction (PPI) discovery and analysis. Assaying a mixture of fluorescence-labeled thrombin and Escherichia coli proteins with an aptamer microarray, we found that thrombin and an unknown protein of E. coli (protein X) formed a complex of PPI, which was captured by an anti-thrombin aptamer probe. The PPI observed on the microarray was double-checked by protein microarrays and confirmed by aptamer-baited co-immunoprecipitation (Co-IP) assays. Characterizing the Co-IP products, we identified protein X as an E. coli Dps protein (DNA-binding protein from starved cells). A SDS-PAGE analysis suggested that Dps should be a substrate for thrombin, a trypsin-like serine protease. A dose-response microarray experiment predicted an apparent dissociation constant of 1.33 μM for the PPI. Moreover, an on-microarray competition assay revealed that the capture of the PPI by the anti-thrombin aptamer probe would be blocked by an E. coli aptamer via complementary base pairing. Thus, a network of protein-protein, protein-DNA, and DNA-DNA interactions and their interaction orders could be addressed in addition to simple PPI discovery.
authors.affiliation: Department of Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei 10617, Taiwan. chenlinchi@ntu.edu.tw
authors.forename: Lin-Chi
authors.initials: LC
authors.lastname: Chen
authors.forename: Shin-Cheng
authors.initials: SC
authors.lastname: Tzeng
authors.forename: Konan
authors.initials: K
authors.lastname: Peck
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001681:Biological Assay; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification*; D009841:Oligonucleotides / Q000737:chemistry; D060066:Protein Interaction Maps
chemical_list: D052157:Aptamers, Nucleotide; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; C081676:thrombin aptamer; D004247:DNA