Molecular Simulations Studies of RNA Tetraloop Hyperstability: The Effect of Stem Length on Folding Dynamics

Abstract

RNA hairpins, formed by looping oligonucleotides, are among the most common RNA secondary structures and involve conserved interactions that drive hierarchical folding and stabilize folded states. Recent modifications to all-atom molecular dynamics force fields have provided the means to quantitatively assess RNA hairpin folding and stability for comparison with experiment. This study focuses on using these techniques to analyze two hyperstable RNA hairpins, each containing a hyperstable tetraloop sequence (rGCAA) and a G-C stem of two or four basepairs. The systems were simulated for a total of 300 microseconds using replica exchange molecular dynamics, and the resulting structure ensembles were analyzed for common folded states, intra-loop hydrogen bonds, and stem base-pairing retention. We find that the longer stem produces kinetic traps with non-native loop conformations, while offering credence to supposed alternative tetraloop folds from experiment. Shorter stems produce more variable folding behaviors and demonstrate reversible folding/unfolding actions. These results offer an unbiased, thermodynamic characterization of RNA tetraloops, highlight the presence of kinetic traps, and provide critical information for kinetic folding studies of tetraloops as well as additional folding studies of larger RNA molecules.