Unfolding and conformational variations of thrombin-binding DNA aptamers: synthesis, circular dichroism and molecular dynamics simulations.

Thrombin-binding DNA aptamer (TBA), with a consensus 15-base sequence: d(GGTTGGTGTGGTTGG), can fold into an antiparallel unimolecular G-quadruplex structure that is necessary for its interaction with thrombin. For the first time, using steered molecular dynamics (SMD) simulations, we have successfully simulated the unfolding process of native TBA G-quadruplex. The unfolding pathway proposed is in agreement with previously reported experimental NMR data. Moreover, the critical intermediate structure in the unfolding pathway, predicted by the NMR results, was identified. The structural characteristics of several TBA oligonucleotides modified with locked nucleoside (LNA) or 2'-O-methyl-nucleoside (MNA) at different positions and number were also investigated by CD spectroscopy. An oligonucleotide substituted with either LNA or MNA at position 2 folds into a native-like G-quadruplex, while doubly substituted derivatives of TBA where LNA or MNA is incorporated at positions 11 and 14 are no longer able to form a G-quadruplex. Starting from the same initial intermediate structure, we successfully overcame sampling limitations, and simulated the large conformational variations in structures of native TBA and modified TBAs by classic MD. Analysis of the models showed that inversion of the glycosyl orientation at position 14 contributes significantly to the disruption of G-quadruplex formation in both of the di-substituted modified TBA systems. Our calculations provide a simple and reliable theoretical model that can be used to investigate and predict the effects of the modifications of an oligonucleotide on the resultant G-quadruplex structure. In addition, the computational protocol described can also be applied for other G-quadruplex systems.