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

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• 作者：Stefano De Tito ; Fran莽ois Morvan ; Albert Meyer ; Jean-Jacques Vasseur ; Annunziata Cummaro ; Luigi Petraccone ; Bruno Pagano ; Ettore Novellino ; Antonio Randazzo ; Concetta Giancola ; Daniela Montesarchio
• 刊名：Bioconjugate Chemistry
• 出版年：2013
• 出版时间：November 20, 2013
• 年：2013
• 卷：24
• 期：11
• 页码：1917-1927
• 全文大小：524K
• 年卷期：v.24,no.11(November 20, 2013)
• ISSN：1520-4812

文摘

A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3鈥?end and a 尾-cyclodextrin residue at the 5鈥?end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide鈥揳lkyne 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鈥?end, into the apolar cavity of the 尾-cyclodextrin at the 5鈥?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.