Unfolding of RNA. (A) Free energy surfaces G1(q) (green) and G2(q) (blue) of the RNA-linker construct and the corresponding PMF G0(q) (red). The Inset shows the force-extension curves of two simulated pulling trajectories, a forward trajectory in the unfolding direction (blue) and a reverse trajectory in the folding direction (green). (B) Free energy surfaces reconstructed from 100 trajectories each in the forward and reverse directions at force loading rates of R = 2 (filled circles), 10 (open circles), and 50 pN/s (triangles), analyzed with the quasi-harmonic approximation to the inverse Weierstrass transform Eq. 10. The solid curve is the exact G0(q). For clarity, an artificial force of F = 14 pN is imposed on all curves. The dashed curve at the bottom right indicates the pulling potential with a spring constant of k = 0.1 pN/nm. The Inset shows three extension-versus-time trajectories obtained at constant forces of 14, 14.5, and 15 pN.