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

• Published in: Bioconjugate chemistry 2013-11, Vol.24 (11), p.1917-1927
• Main Authors: De Tito, Stefano, Morvan, François, Meyer, Albert, Vasseur, Jean-Jacques, Cummaro, Annunziata, Petraccone, Luigi, Pagano, Bruno, Novellino, Ettore, Randazzo, Antonio, Giancola, Concetta, Montesarchio, Daniela
• Format: Article
• Language: English
• Subjects: Aptamers, Nucleotide - chemistry; beta-Cyclodextrins - chemistry; Biophysics; Chemical Sciences; Dansyl Compounds - chemistry; DNA - chemistry; Fluorescence; G-Quadruplexes; Humans; Integrated approach; Measurement; Models, Molecular; Molecular Structure; NMR; Nuclear magnetic resonance; Nucleic Acid Conformation; Organic chemistry
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc400352s

A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3′-end and a β-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 us 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 chairlike, 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 β-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.