Ligand binding and catalysis in an RNA aptamer

In vitro selection of aptamer sequences has demonstrated RNAs ability to recognize multiple classes of ligands, from anionic species to whole cells. In the vast majority of these studies the structural and biophysical properties of the RNA are monitored during the ligand binding event, and the ligand is treated as a rigid body. The adaptive binding nature of RNA, in which the RNA is largely unstructured in the absence of the ligand and only folds into a compact structure in the presence of the ligand, helps to facilitate this bias due to the drastic structural changes that take place in the RNA upon ligand recognition. The landscape of the ligand binding pocket of aptamers is rich in delocalized electron systems and electrostatic fields that are generated by the extensive aromatic base stacking and the phosphate backbone of the aptamer. Taking these factors into account, the environment of the ligand binding pocket has the potential to modulate the overall physical properties of the bound ligand.; Nuclear Magnetic Resonance studies of the malachite green aptamer in complex with malachite green (MG) reveal an asymmetric shift in the carbon chemical shifts of bound MG. Computational studies verified that the ligands physical properties, specifically the planarity and charge distribution of the cationic dye, are altered by association with the RNA. The findings presented here demonstrate that changes in the physical properties experienced by the bound ligand are sufficient to modulate reactivity of a modified ligand specifically engineered to be susceptible to the environment of the binding pocket. The aptamer's ligand binding pocket also has a drastic effect on the pKa of a modified ligand, demonstrating further the effect the aptamer has on the physical properties of the bound ligand.; These results suggest that detailed knowledge of the ligand binding event can be used to determine the intrinsic catalytic potential of RNA systems. With respect to the RNA World theory, these findings provide a possible mechanism by which ribozyme sequences may have evolved from purely ligand binding sequences.; The binding capacity of the aptamer for structurally modified ligands will also be discussed.