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

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 complexity. 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 (Delta X-F(TS)) changes dramatically as the loading rate is varied. At loading rates comparable to those used in laser optical tweezer experiments, the hairpin is plastic, with Delta X-F(TS) being midway between folded and unfolded states; whereas at high loading rates, DXFTS 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 intermediates. The most stable force-stabilized intermediate represents straightening of the L-shaped structure. For these structures, the unfolding pathways can be predicted using the contact map of the native structures. Unfolding of a RNA motif with internal multiloop, namely, the 109-nucleotide prohead RNA that is part of the phi 29 DNA packaging motor, at constant value of r(f) occurs with three distinct rips that represent unraveling of the paired helices. The rips represent kinetic barriers to unfolding. Our work shows 1), the response of RNA to force is largely determined by the native structure; and 2), only by probing mechanical unfolding over a wide range of forces can the underlying energy landscape be fully explored.