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Stability and Binding Properties of a Modified Thrombin Binding Aptamer

Bruno Pagano ^*, Luigi Martino , Antonio Randazzo  and Concetta Giancola 

^* Dipartimento di Scienze Farmaceutiche, Università di Salerno, Fisciano, Salerno, Italy; and Dipartimento di Chimica "P. Corradini" and Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli "Federico II", Naples, Italy

Correspondence: Address reprint requests to Concetta Giancola, Dipto. di Chimica "P. Corradini", Università di Napoli "Federico II", via Cintia, 80126 Naples, Italy. Tel.: 39-081-674266; Fax: 39-081-674257; E-mail: giancola{at}unina.it.

Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), ^3'GGT^5'-^5'TGGTGTGGTTGG^3', which also folds into a quadruplex structure, is more stable than its unmodified counterpart and shows a higher thrombin affinity. The stability of the modified aptamer was investigated using differential scanning calorimetry, and the energetics of mTBA and TBA binding to thrombin was characterized by means of isothermal titration calorimetry (ITC). ITC data revealed that TBA/thrombin and mTBA/thrombin binding stoichiometry is 1:2 for both interactions. Structural models of the two complexes of thrombin with TBA and with mTBA were also obtained and subjected to molecular dynamics simulations in explicit water. Analysis of the models led to an improvement of the understanding of the aptamer-thrombin recognition at a molecular level.