Binding of voriconazole aptamers and modulation of binding by cyclodextrins

Voriconazole is a broad-spectrum antifungal drug widely used to treat life-threatening fungal infections, including invasive aspergillosis and severe candidiasis. Despite its efficacy, large inter- and intra-patient pharmacokinetic variability and frequent drug-drug interactions can lead to subtherapeutic or toxic plasma concentrations, making therapeutic drug monitoring essential. Recently, several aptamer selections for voriconazole have been reported, including two independent selections from our laboratory that yielded multiple aptamer families. In this work, we systematically characterize these aptamers by measuring their binding affinities under various conditions using thioflavin T (ThT) fluorescence spectroscopy and evaluating their selectivity against structurally related antifungal drugs. A new aptamer named VO3 with a dissociation constant (K_D) value of 26μM and high selectivity was identified. The effect of the buffer components such as NaCl and MgCl₂was assessed, and VO3 maintained binding activity even in up to 1 M NaCl and in the absence of Mg²⁺ions. Furthermore, the interaction ofα- andβ- cyclodextrins with voriconazole was studied using a competitive titration method, and onlyβ-cyclodextrin could compete with the aptamer for binding to voriconazole. This study shows that ThT fluorescence is a useful label-free method to study voriconazole aptamers and that aptamers can be used to probe target binding to nanoscale host molecules.