Potential of mean force (PMF) is a very useful tool in studying free energy changes as a function of reaction coordinates especially in biological systems . We predicted the PC1 and PC2 of the loop and the immediate neighboring base-pairs, yielding four base-pairs in total, and used them as reaction coordinates to generate the free energy landscapes for these two constructs (Figure ). PMF analyses with respect to PC1 and PC2 showed that the dynamics experienced in GCCC is different from CCCG. While GCCC displayed multiple distinct minima, CCCG showed just one distinct minimum (Figure ). We further investigated the MD trajectories using cluster analyses, and observed that the MD trajectory of GCCC can be clustered into two major and one minor clusters (Figure ), where either one or both of the C/C mismatches were forming a single hydrogen bond in these clusters. In CCCG construct, trajectory was clustered into one main cluster, where hydrogen bonding pattern was alike to GCCC (Figure ). Similar hydrogen bonding patters has been observed in X-ray crystal structures, too . It is noteworthy to highlight that MD calculations predicts atomistic details of 2×2 CC/CC internal loops where cluster analyses can resolve even small details, which are not always possible in NMR experiments.