Dynamics Of Thrombin-binding DNA Aptamer Studied By NMR Techniques And The Application Of The Diagonal-Free COSY

The thrombin binding aptamer d(GGTTGGTGTGGTTGG),with the short name TBA, is a 15-mer DNA, which specifically binds to the thrombin protein and inhibits thrombin-catalyzed fibrin during clot formation. Therefore, TBA could be used as a potential anticoagulant reagent for patients with thrombosis and embolism. TBA can form an intramolecular G-quadruplex in the presence of certain metal ions, with the ion fitted into the space in the quadruplex core. In this dissertation the dynamics of TBA in the presence of K+ have been studied. The exchange rates of the eight imino protons in the G-quadruplex structure are measured and the unfolding mechanism of TBA is discussed. At the same time, the Vogel-Fulcher-Tammann (VFT) equation can better fit the dynamic data of TBA, and this is the first time for VFT equation to be applied in biological kinetics. In addition, the diagonal-free COSY (DF-COSY) pulsed sequence is modified using pulse field gradient. When the DF-COSY pulse sequence was used, COSY-like spectrum without any diagonal peaks can be obtained.TBA can form a chair-like conformation in the presence of various metal ions, however, their structures are different. The exchange rates of the eight imino protons of K+-TBA complex have been measured at different temperatures by NMR techniques. These dynamic data showed that the stability of the TGT loop is similar to the two TT loops in the K+-TBA complex, and that the bases T4, T13 and T9 play more important roles in keeping the hydrogen bond stable in the K+-TBA complex than in Sr2+-TBA complex. These data further support the unfolding mechanism of the transition of TBA from the G-quadruplex to random coil. However, the unfolding process of K+-TBA complex has its unique characteristics, because its structure is not exactly the same as others.The Vogel-Fulcher-Tammann(VFT),which is widely applied in condensed mater physics for analysis of temperature-dependent dynamical data, is for the first time applied to fit the hydrogen exchange rates of TBA as a function of temperature. The VFT equation for the hydrogen exchange should read k ex= A exp[ ? ( E / R )/(T ? T0)], which contains a constant temperature T0. The VFT equation can better interpret these dynamic data, and the constant temperature T0 of the VFT equation happens to be the melting temperature of TBA. The relationship between the VFT equation and Arrhenius equation has been quantitatively discussed. Under suitable conditions the VFT equation can be reduced to the Arrhenius eqution. After successfully analyzing the data of TBA, the VFT equation was further applied in biological molecules. Considering the mechanism of the hydrogen exchange of the biological molecules, the VFT equation and the Arrhenius equation have combined to form a new equation, which can be written as: ln ke x = C ? ( EV FT / R) /(T ? T0 ) ? ( Ea / R) /T. The new equation can better interpret the data of the hydrogen exchange of the biological molecules, such as protein, DNA, RNA, etc. Under some condition, the Arrhenius term in the new equation could be left out and the new equation could be replaced by the VFT equation.The general COSY spectrum is the most useful and widely applied in the studies of molecular structures, because the cross peaks of COSY spectrum can provide with the network information of the spin system which have J-coupling. However, many cross peaks, which are very close to diagonal, often overlap with the diagonal peaks which have dispersive line shape, and the information from cross peaks is lost. In order to avoid this, a pulse sequence was modified using pulse field gradint, and obtained diagonal-free COSY spectrum (DF-COSY), which is just COSY-like spectrum and has no diagonal peaks. Since the information of all cross peaks are retained. DF-COSY spectrum can be used as a quick tool for molecular studies, especially for large molecules and biological samples, such as protein, urea, blood serum, etc.