Molecular dynamics and free energy study of the conformational equilibria in the UUUU RNA hairpin

A series of molecular dynamics (MD) simulations was performed to elucidate the thermodynamic basis for the relative stabilities of hairpin, duplex, and single stranded forms of the 5'-CGC(UUUU)GCG-3' oligonucleotide. According to a recent NMR study this sequence exhibits dynamic conformational equilibrium in aqueous solution in the vicinity of room temperature. Free energy calculations using the molecular mechanics-Poisson Boltzmann-surface area (MM-PB/SA) approach support a shift in the conformational equilibrium from duplex to hairpin as the temperature is increased from 276 to 300 K, in agreement with the NMR results. The effect of added salt on the relative stabilities of RNA conformers is also reproduced by our calculations. The calculated Delta H degrees for the equilibrium between hairpin and single stranded forms is estimated to be -23.4 kcal/mol, in reasonable agreement with experimental values. Our results reveal that the conformational equilibrium strongly depends on the solute entropy and the electrostatic interactions modulated by added salt. Simulations of hairpin loop conducted at two different temperatures converged to the same lowest energy loop conformation. This conformer is stabilized by favorable van der Waals interactions as a result of U5-U6-U7 base stacking, a hydrogen bond between the U4 base and the phosphate linking U6 and U7, and hydrogen bonds involving the 2'OH groups at U4 and U6. However, the sugar pucker of the four uridines in the lowest energy conformer is different from that reported by a NMR study. While the NMR study found that U5 and U7 adopt the C2'-endo conformation, the simulation results suggests that overall the structure with the U5 and U7 in the C3'-endo conformation is thermodynamically more stable than the structure containing the C2'-endo pucker by approximately 8 kcal/mol. Calculations based on the MM-PB/SA scheme show that although the electrostatic solvation free energy favors the C2'-endo conformation for the U5 and U7 riboses, it is offset by the less favorable intramolecular electrostatic and van der Waals energies. To enhance the conformational sampling, a replica exchange molecular dynamics (REMD) simulation was conducted in a generalized Born (GB) continuum solvent for the hairpin loop. This simulation indicates that the stable loop structure observed in the explicit solvent simulations corresponds to the free energy minimum. It also reveals that while the U4, U5, and U6 sugar rings are predominantly in the C3'-endo conformation, there is considerable variation in the sugar pucker of the U7 ribose ring.