Conformational switch of insulin-binding aptamer into G-quadruplex induced by K⁺ and Na⁺: an experimental and theoretical approach.

Guanine-rich sequences can form the G-quadruplex structure in the presence of specific metal ions. Here, circular dichroism, UV-vis absorption, fluorescence, and molecular dynamics simulation studies revealed that insulin-binding aptamer (IBA) could form an intramolecular G-quadruplex structure after binding K(+). Circular dichroism (CD) spectra demonstrated that IBA could fold into a parallel G-quadruplex with a strong positive peak at 263 nm. Analysis of equilibrium titration data revealed that cation binding was cooperative with the Hill coefficient of 2.01 in K(+) and 1.90 in Na(+). Thermal denaturation assays indicated that K(+)-induced G-quadruplex is more stable than Na(+)-induced structure. Folding of IBA into G-quadruplex leading to the contact quenching occurs as a result of the formation of a nonfluorescent complex between donor and acceptor. Based on fluorescence quenching of IBA folding, a potassium-sensing aptasensor in the range of 0-1.4 mM was proposed. Since the quenching process was predominantly static, the binding constant and the number of binding sites were determined. In this research, based on the experimental data, the initial model of IBA G-quadruplex was constructed by molecular modeling method. The modeling structure of IBA is an intramolecular parallel-strand quadruplex conformation with two guanine tetrads. The extended molecular dynamics simulation for the model indicated that the G-quadruplex maintains its structure very well in aqueous solution in presence of K(+) in the central cavity. In contrast, it was demonstrated that the G-quadruplex structure of model in the water collapses in absence of this cation.