Fluorescence Enhancement upon G-Quadruplex Folding: Synthesis, Structure and Biophysical Characterization of a Dansyl/Cyclodextrin-tagged Thrombin Binding Aptamer

A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environment-sensitive dansyl probe at the 3'-end and a beta-cyclodextrin residue at the 5'-end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide-alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence and DSC studies. ITC measurements have allowed to analyze in detail its interaction with human thrombin. All the collected data show that this bis-conjugated aptamer fully retains its G-quadruplex formation ability and thrombin recognition properties, with the terminal appendages only marginally interfering with the conformational behavior of TBA. Folding of this modified aptamer into the chair-like, antiparallel G-quadruplex structure, promoted by K+ and/or thrombin binding, typical of TBA, is associated with a net fluorescence enhancement, due to encapsulation of dansyl, attached at the 3'-end, into the apolar cavity of the beta-cyclodextrin at the 5'-end. Overall, the structural characterization of this novel, bis-conjugated TBA fully demonstrates its potential as a diagnostic tool for thrombin recognition, also providing a useful basis for the design of suitable aptamer-based devices for theranostic applications, allowing simultaneously both detection and inhibition or modulation of the thrombin activity