For the sake of completeness, we also subjected another singly modified RNA construct to NMR studies, one that recapitulates both states 2 and 4. Modification of G20 resulted in the most exchanged broadened spectrum of all m 1 G constructs, with significant broadening occurring at ~10.6 ppm (non-canonically paired nucleotides), 12 ppm, and 13.2 ppm (Figure ). However, it is important to note that the GNRA tetraloop remained well formed, with a sharp, albeit overlapping peak, appearing at 10.6 ppm. When subjected to 2D NMR studies (Figure ), no off-diagonal peaks were observed between the lower stem, G4:G19, and the bottom of the modified 2×2 GG/GG in ternal loop (G5/m 1 G20). This spectrum differs from the other singlymodified constructs, where both m 1 G19 and m 1 G5 residues, within the internal loop, had offdiagonal peaks with the neighboring upper or lower stems, which is consistent with stable formation of base pairs when modified nucleotides were utilized (Figure ). These differences in stability reveal that attempts to enforce G5 to adopt the anti-conformation (Figure , states 2 and 4) are disfavored. In this instance, these states are rapidly interconverting, causing exchange broadening in the NMR spectra that makes these states undetectable by traditional 1D and 2D imino NMR methods. This conclusion is consistent with the m 1 G5 modified RNA, which enforces G5 to syn conformation and very closely resembles the NMR spectrum of the unmodified RNA (Figures and).