Reversible Folding of Hyperstable RNA Tetraloops Using Molecular Dynamics Simulations

Abstract

Structured RNAs exhibit a distinct preference for loops of precisely 4 nucleotides. Approximately 70% of these “tetraloops” are comprised of just three specific loop sequences: UUCG, GCAA, or CUUG. The abundance of these sequences is thermodynamic in origin, as each motif forms a unique network of non-canonical interactions within their loops that stabilize the folded state. Modification to the Amber force field enables the de-novo folding of three hyperstable RNA tetraloops to 1-3 A RMSD from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. To study the thermodynamics and kinetics of folding on an RNA tetraloop we simulated the (rGCAA) tetraloop with stem lengths of two (octamer), and four (dodecamer) C-G base pairs. The thermodynamics is obtained from replica exchange molecular dynamics (REMD) simulations with overall sampling exceeding 300 microseconds. The kinetics of the octamer was studied in a 100 microseconds molecular dynamics simulation using the Anton supercomputer. The thermodynamics reveal that the octamer folds and unfolds reversibly. However, the dodecamer behaves glassy and adopts multiple metastable loop configurations that do not unfold in extensive REMD simulations. The ability to recapitulate the signature non-canonical interactions of the three most abundant hyperstable stem-loop motifs represents a significant step towards the accurate description of nucleic acid tertiary structures, dynamics and stability using unbiased all-atom molecular dynamics simulations.