Mechanical unfolding of RNA: from hairpins to structures with internal multiloops

ABSTRACT Mechanical unfolding of RNA structures, ranging from hairpins to ribozymes, using laser optical tweezer experiments have begun to reveal the features of the energy landscape that cannot be easily explored using conventional experiments. Upon application of constant force (f), RNA hairpins undergo cooperative transitions from folded to unfolded states whereas subdomains of ribozymes unravel one at a time. Here, we use a self-organized polymer model and Brownian dynamics simulations to probe mechanical unfolding at constant force and constant-loading rate of four RNA structures of varying com-plexity. For simple hairpins, such as P5GA, application of constant force or constant loading rate results in bistable cooperative transitions between folded and unfolded states without populating any intermediates. The transition state location (DxTSF) changes dramatically as the loading rate is varied. At loading rates comparable to those used in laser optical tweezer experi-ments, the hairpin is plastic, with DxTSF being midway between folded and unfolded states; whereas at high loading rates, Dx TS F moves close to the folded state, i.e., RNA is brittle. For the 29-nucleotide TAR RNA with the three-nucleotide bulge, unfolding occurs in a nearly two-state manner with an occasional pause in a high free energy metastable state. Forced unfolding of the 55 nucleotides of the Hepatitis IRES domain IIa, which has a distorted L-shaped structure, results in well-populated stable inter-mediates. The most stable force-stabilized intermediate represents straightening of the L-shaped structure. For thes