We then used our REMD simulations to explore the temperature dependence of base stacking compared to the described UV/Vis melting curves. Base stacking energies from the REMD simulation between temperatures 278-330 K show a similar gradual decrease with increasing temperature and a similar, although reduced, T m value (experimental 31.7 ± 1.90 C compared to computed 20-25 C, as estimating the T 50 value from melting curve, Figure 2A) . However, the calculated base stacking energy plateaus around 320 K while the experimental slope begins to plateau around 330 K, indicating that stacking energies may be under-estimated in the REMD simulation or that additional unaccounted-for factors contribute to the ssRNA stability. Nevertheless, our data suggest that base stacking is the guiding force behind ssRNA stability, consistent with previous studies.