title: Molecular aptamers for real-time protein-protein interaction study.
abstract: Protein-protein interactions play critical roles in cellular functions, but current techniques for real-time study of these interactions are limited. We report the real-time monitoring of protein-protein interactions without labeling either of the two interacting proteins; this procedure poses minimum effects on the binding properties of the proteins. Our strategy uses a protein/aptamer complex to probe the interactions in a competitive assay where the binding of an aptamer to its target protein is altered by a second protein that interacts with the target protein. Two signal transduction strategies, fluorescence resonance energy transfer (FRET) and fluorescence anisotropy, have been designed to study the interactions of human alpha-thrombin with different proteins by using two aptamers specific for two binding sites on alpha-thrombin. Our method has been shown to be simple and effective, does not require labeling of proteins, makes use of easily obtainable aptamers, provides detailed protein-protein interaction information and has excellent sensitivity for protein detection and protein-protein interaction studies. The FRET and the fluorescent anisotropy approaches complement each other in providing insight into the kinetics, mechanisms, binding sites and binding dynamics of the interacting proteins.
authors.affiliation: Department of Chemistry and Shands Cancer Center, McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA.
authors.forename: Zehui
authors.initials: Z
authors.lastname: Cao
authors.forename: Weihong
authors.initials: W
authors.lastname: Tan
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D011506:Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D017931:DNA Primers; D011506:Proteins