To scan the conformational landscape of 2×2 GG/GG internal loops, we employed an advanced computational sampling method, temperature replica exchange MD (T-REMD). In T-REMD, a series of replicas of the same system are created, each running at a different temperature with exchange attempts periodically done between replicas. As implemented in AMBER 18, the acceptance or rejection of exchange attempts is performed using the Metropolis Monte Carlo criterion such that the frequencies of the observed conformations approach to the equilibrium conditions, within the limits of substantial number of simulation steps . A model RNA, r(UCUGGGGCCAGA)2, was utilized for this purpose, where internal loop residues are underlined to highlight the 2×2 GG/GG motif. At the start of the simulations, all the guanosine loop residues were modeled to be in syn orientations, r(UCUGGGsynGsynCCAGA)2, which displays the least stable conformation adopted by 2×2 GG/GG residues, thereby facilitating conformational transitions and avoiding the system to be trapped in local minima. As noted above, CoSSMos database did not display any syn-syn orientations for 1×1 G/G mismatches (Table ), which rationalizes our choice of the initial structure, which has all loop residues in syn. We used 40 replicas each running for 43 µs with a cumulative time of ~1.7 ms. We then performed cluster analyses to assess the different configurations adopted by the 2×2 GG/GG loop and calculated the observed percentages for each cluster. Over a dozen of clusters with different orientations were identified with two major configurations comprising more than 78% of the total population (Figure ). The major configuration adopted by the 2×2 GG/GG loop was GsynGanti/GantiGsyn observed 42.5% of the population (Figure ) followed by GantiGsyn/GantiGsyn observed 35.8% (Figure ). Two minor clusters were also observed adopting GantiGanti/GantiGanti and GantiGsyn/GsynGanti at 4% and 3.4%, respectively (Figure ).