Free-Energy Landscape of a Thrombin-Binding DNA Aptamer in Aqueous Environment

Thrombin-binding aptamer (TBA-15) is a single-stranded 15-mer oligonucleotide that has a wide range of biomedical applications. In the presence of metal cations of proper sizes, this aptamer displays G-quadruplexes with a single cation enclosed at its central binding site when it is completely folded. To understand how this aptamer folds into its stable three-dimensional structure in the presence of K+ ions, we carried out free-energy calculations using the state-of-art replica exchange molecular dynamics simulation (REMD) at the all-atom level. The resulting free energy map revealed that TBA-15 follows a two-state folding behavior with a substantially large folding barrier of 6 kcal/mol at ambient temperature. Our simulation showed that the intervening TGT-loop, which is located in the middle of the TBA-15 sequence, virtually remains intact regardless of folding and unfolding states. Furthermore, in the conserved TGT-loop structure, the base-pair stacking of G8 and T9 induces the native-like base orientations of G6 and G10 pertaining to the upper G-quadrant. This stacking interaction enhances the loop stability and reduces its dynamic fluctuations. Interestingly, for the G-stem to fold into its native state, the aggregation of the G8 and T9 residues in the TGT-loop is a key step for initiating the folding event of the G-stem by capturing a bulky cation.