Aptamer microarray as a novel bioassay for protein-protein interaction discovery and analysis

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 mu M 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. (C) 2012 Elsevier B.V. All rights reserved.