Together, these NMR studies confirmed that the G5 syn conformation was the most stable state, in agreement with T-REMD simulations (Figure , states 1 and 3). Concurrently, this data also revealed that the NMR signals for G6:G19 base pair was broadened due to rapid interconversion between syn and anti states in all the constructs, including those with the m 1 G modifications as evidenced by exchange broadened NMR spectra (Figures S4CE). The observable differences in dynamics between the G/G base pair neighboring the lower stem (G5:G20) and the G/G base pair neighboring the upper stem (G6:G19) were not expected, as the internal loop and neighboring nucleotides are symmetric. However, there was potential for energetic differences in stacking between the lower and upper stems based off: (i) A/U flanking both the 5′ and 3′ the internal loop causing asymmetry between the strands 5′-AGGGCA-3′ versus 5′-UGGGCU-3′ and (ii) the contribution of a 3 base pair stem versus a 3 base pair stem and hairpin for the bottom and top, respectively (Figure ). In order to interrogate the contribution of further neighboring nucleotides on the 2×2 GG/GG loop dynamics, we designed an additional construct by swapping the base pairing location of the A-U pair in the upper stem (A8/U17 to U8/A17, termed "AU-swap"). This construct improved symmetry, with 5′-AGGGCU-3′ for both strands. NMR studies confirmed that this construct was highly symmetric (Figure ), with resonances for the upper and lower stem highly overlapped due to the similarity in their local chemical environment and resulting NMR chemical shifts. Non-canonical peaks, again appeared in the 10-11 ppm region, with two distinct sets of off-diagonal peaks in the NOESY highlighted with a red box that represent imino signals for the 2×2 GG/GG loop. Assignments and characterization were not possible due to such highly overlapped peaks and will remain the subject of future studies. The resulting changes in 2D NMR spectra after a small change in nucleotide location confirmed that the 2×2 GG/GG internal loop is exquisitely sensitive to stacking interactions, and suggests that small changes in stacking energies may propagate into differences in dynamics observed between the two non-canonical G/G pairs of the 2×2 GG/GG internal loop.