Stability and Binding Properties of a Modified Thrombin Binding Aptamer
Bruno Pagano 1, Luigi Martino 2, Antonio Randazzo 2 and Concetta Giancola 2*
1 University of Salerno
2 University of Naples "Federico II"
* To whom correspondence should be addressed. E-mail: giancola{at}unina.it.
Submitted on July 14, 2007
Revised on July 30, 2007
Accepted on 10 September 2007
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Abstract |
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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'GGT5'-5'TGGTGTGGTTGG3', 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 has been investigated using differential scanning calorimetry (DSC), and the energetics of mTBA and TBA binding to thrombin has been characterized by means of isothermal titration calorimetry (ITC) measurements. 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.
Key Words:
DSC, Docking, ITC, Molecular Dynamics, Thrombin binding aptamer
