Protein-protein interaction studies based on molecular aptamers by affinity capillary electrophoresis.
Protein-DNA/protein-protein interactions play critical roles in many biological processes. We report here the investigation of protein-protein interactions using molecular aptamers with affinity capillary electrophoresis (ACE). A human alpha-thrombin binding aptamer was labeled with 6-carboxyfluorescein and exploited as a selective fluorescent probe for studying thrombin-protein interactions using capillary electrophoresis with laser-induced fluorescence. A 15-mer binding DNA aptamer can be separated into two peaks in CE that correspond to the linear aptamer (L-Apt) and the thrombin-binding G-quadruplex structure in the presence of K(+) or Ba(2+). In a bare capillary, the peak area of G-quadruplex aptamer (G-Apt) was found to decrease with the addition of thrombin while that of L-Apt remained unchanged. Even though the peak of the G-Apt/thrombin binding complex is broad due to a weaker binding affinity between aptamer and thrombin, we were still able to quantify the thrombin and anti-thrombin proteins (human anti-thrombin III, AT III) based on the peak areas of free G-Apt. The detection limits of thrombin and AT III were 9.8 and 2.1 nM, respectively. The aptamer-based competitive ACE assay has also been applied to quantify thrombin-anti-thrombin III interaction and to monitor this reaction in real time. The addition of poly(ethylene glycol) to the sample matrix stabilized the complex of the G-Aptthrombin. This assay can be used to study the interactions between thrombin and proteins that do not disrupt G-Apt binding property at Exosit I site of the thrombin. Our aptamer-based ACE assay can be an effective approach for studying protein-protein interactions and for analyzing binding site and binding constant information in protein-protein and protein-DNA interaction studies.
Huang
Chih-Ching
CC
Center for Research at Bio/nano Interface, Department of Chemistry and Shands Cancer Center, McKnight Brain Institute, University of Florida, Gainesville, Florida 32611-7200, USA.
Cao
Zehui
Z
Chang
Huan-Tsung
HT
Tan
Weihong
W
D052158:Aptamers, Peptide / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D006801:Humans; D008968:Molecular Conformation; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000737:chemistry*; D013997:Time Factors
D052158:Aptamers, Peptide; D005452:Fluoresceins; C024098:6-carboxyfluorescein; D011092:Polyethylene Glycols; D004247:DNA; D013917:Thrombin