RNA simulations: probing hairpin unfolding and the dynamics of a GNRA tetraloop.

Simulations of an RNA hairpin containing a GNRA tetraloop were conducted to allow the characterization of its secondary structure formation and dynamics. Ten 10 ns trajectories of the folded hairpin 5'-GGGC[GCAA]GCCU-3' were generated using stochastic dynamics and the GB/SA implicit solvent model at 300 K. Overall, we find the stem to be a very stable subunit of this molecule, whereas multiple loop conformations and transitions between them were observed. These trajectories strongly suggest that extension of the C6 base away from the loop occurs cooperatively with an N-type-->S-type sugar pucker conversion in that residue and that similar pucker transitions are necessary to stabilize other looped-out bases. In addition, a short-lived conformer with an extended fourth loop residue (A8) lacking this stabilizing 2'-endo pucker mode was observed. Results of thermal perturbation at 400 K support this model of loop dynamics. Unfolding trajectories were produced using this same methodology at temperatures of 500 to 700 K. The observed unfolding events display three-state behavior kinetically (including native, globular, and unfolded populations) and, based on these observations, we propose a folding mechanism that consists of three distinct events: (i) collapse of the random unfolded structure and sampling of the globular state; (ii) passage into the folded region of configurational space as stem base-pairs form and gain helicity; and (iii) attainment of proper loop geometry and organization of loop pairing and stacking interactions. These results are considered in the context of current experimental knowledge of this and similar nucleic acid hairpins.