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.
Cao
Zehui
Z
Department of Chemistry and Shands Cancer Center, McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA.
Tan
Weihong
W
D001483:Base Sequence; D017931:DNA Primers; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D011506:Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship
D017931:DNA Primers; D011506:Proteins