Molecular interaction studies of vascular endothelial growth factor with RNA aptamers.
With widespread applications in biosensors, diagnostics and therapeutics, unraveling the mechanism of the interactions between aptamers and their targets has become extremely important. In this study, the interaction forces between an aptamer and its protein target were successfully measured via AFM-based force spectroscopy at the molecular level. The angiogenic protein, vascular endothelial growth factor (VEGF(165)) covalently tethered to a mixed self-assembled monolayer surface and an anti-VEGF(165) aptamer attached to an AFM cantilever was used to probe this interaction. By comparing the binding probability and the force distribution of this system in a series of experiments, the interaction between the aptamer and the protein was confirmed and the effect of loading rate on the rupture force was studied. The specificity of the aptamer was investigated by measuring interactions with VEGF(121), an isoform of VEGF missing a critical binding domain, and VEGF(165) isoform blocked with heparin. The lower frequency of binding events compared with that of VEGF(165) reflected that despite a high affinity to its preferential target, the selectivity of this aptamer is impaired to some extent due to the flexible structure of aptamers. By changing the concentration of Mg(2+) ion in the binding buffer, we could verify the effect of metal ions as stabilizers of aptamer conformation. The results provide evidence at the molecular level that the structural stability of aptamer is closely related to higher binding force and that rigid aptamer tertiary structures require higher forces to unbind the aptamer/protein complex.
Zhang
Xiaojuan
X
Department of Chemical and Life Science Engineering, Virginia Commonwealth University, 601 W. Main Street. Richmond, VA, USA.
Yadavalli
Vamsi K
VK
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry*
D052157:Aptamers, Nucleotide; D042461:Vascular Endothelial Growth Factor A