A simulated annealing protocol was utilized with NMR restraints imposed on the RNA in 5′-UCUGGGGCCAGA-3′ to create the loop residues in all-syn orientations while keeping the global RNA structure in A-form orientation. The generalized Born implicit solvent model 47 with a 0.3 M NaCl concentration was used in simulated annealing. The system was first heated from 0 to 400K for 5 ps and then cooled to 100K. Force constants of 50 kcal mol -1 Å -2 and 50 kcal mol -1 rad -2 were used for distance and dihedral angle restraints, respectively to create a single structure at the end with loop residues in syn-syn/syn-syn configuration. This final structure, which displayed 2×2 GG/GG loop residues all in syn, was used as initial structure in T-REMD simulations. The initial structure was solvated and, then, equilibrated as described in the previous section. For T-REMD simulations 40 replicas spanning 277-374.5 K were used. A preliminary assessment with an exchange attempt at 10 ps with this temperature ladder resulted in exchange probability of 20-30%. Langevin thermostat with a collision frequency of 2 ps -1 was used to control the temperature in each replica. PME with 8.0 Å cutoff along with periodic boundary conditions and constant volume dynamics (NVT) was used in all T-REMD simulations (Table ). Simulations were performed using pmemd.cuda.MPI of AMBER18. Each replica was run for 43 µs.