Molecular Aptamers for Real‐Time Protein–Protein Interaction Study

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.