Chemically Accurate Relative Folding Stability of RNA Hairpins from Molecular Simulations

This study describes a comparison between melts and simulated stabilities of the same RNAs that could be used to benchmark RNA force fields, and potentially to determine future melt-ing experiments. Using umbrella sampling molecular simulations of three 12-nucleotide RNA hairpin stem loops, for which there are experimentally determined free energies of unfold-ing, we projected unfolding onto the reaction coordinate of end to end (5′ to 3′ hydroxyl oxygen) distance. We estimate the free energy change of the transition from the native con-formation to a fully extended conformation—the stretched state—with no hydrogen bonds between non-neighboring bases. Each simulation was performed four times using the AM-BER FF99+bsc0+χOL3 force field and each window, spaced at 1 Å intervals, was sampled for 1 μs, for a total of 552 μs of simulation. We compared differences in the simulated free energy changes to analogous differences in free energies from optical melting experiments using ther-modynamic cycles where the free energy change between stretched and random coil sequences is assumed to be sequence independent. The differences between experimental and simulated ΔΔG° are on average 1.00 ± 0.66 kcal/mol, which is chemically accurate and suggests analo-gous simulations could be used predictively. We also report a novel method to identify where replica free energies diverge along the reaction coordinate, thus indicating where additional sampling would most improve convergence. We conclude by discussing methods to more economically perform such simulations.