Structural and thermodymanic studies of the thrombin-binding aptamer and related quadruplexes.

摘要

Nucleic acid quadruplex structures possess several unique properties compared to the canonical nucleic acid structures. These structures are unique in their sequence dependence and metal ion requirements. Evidence for the presence and functional roles of quadruplexes in cellular systems has been increasing rapidly in the past decade. They have also proven to be useful as diagnostic and chemotherapeutic agents. Understanding of the structure and stability of quadruplexes is essential to elucidation of their biological interactions.; Antiparallel quadruplexes contain guanine stretches that are antiparallel to each other, in which half of the nucleosides have the anti conformations about the glycosidic bonds and half possess the syn conformations. In contrast, the guanine stretches in the parallel topology are parallel to each other with all nucleosides in the anti conformation. Although the antiparallel fold is more prevalent, parallel quadruplexes are becoming increasingly common. The influence of individual nucleoside conformation on the overall folding topology was examined in order to understand the forces that determine the quadruplex fold. Selective substitution of rG for dG allowed us to reverse the antiparallel fold of the thrombin binding aptamer (TBA). In addition, this approach also converts a unimolecular quadruplex into a bimolecular one. The reverse substitutions of dG for rG in the all-RNA analogue of TBA confirmed the change in topology from parallel to antiparallel conformation as well as the change from bimolecular to unimolecular. Changing conformational properties of guanine nucleosides allows us to control the quadruplex folding topology.; Metal ion---nucleic acid interactions are crucial in biological system and have significant impact on structures and functions of macromolecules. Quadruplexes are particularly unique in their unusual dependence on specific metal ions for their formation and stabilization through binding via inner sphere coordination. The thermodynamic behavior of quadruplexes stabilized by monovalent and divalent cations was studied by Isothermal Titration Calorimetry (ITC) at various temperatures. Results show that the enthalpy of quadruplex formation is temperature dependent and non-zero heat capacities were determined. These changes in the heat capacity are discussed in terms of changes in exposure of polar and nonpolar groups upon quadruplex formation.