where the Hoogsteen face of the modified guanosine base pairs with the Watson-Crick side of the unmodified guanosine located on the opposite strand (Figure ). Modifications of G5 and G19 folds the RNA construct into the predicted most stable state, GsynGanti/GantiGsyn, as predicted by T-REMD simulations and as evidenced by the sharp line width in the imino region of the 1D NMR spectrum (Figure ). Furthermore, two peaks, which form due to non-canonical base pairs denoted with '*', appeared around 10.2 ppm, presumably due to stabilization of the 2×2 GG/GG internal loop (Figure ). Peaks in the region where canonical base pairs appear were also sharp between 12 and 14ppm, indicating that this RNA adopted a single folded conformation in solution (Figure ). This and). This modification afforded a very similar spectrum to the unmodified system, with a single non-canonical peak appearing around 10.6 ppm, consistent with the formation of the GNRA tetraloop (Figure ). In addition, lower intensity peaks denoted with '*' appeared at ~10.0, 10.3, and 11.0 ppm in the non-canonical region as Gsyn/Ganti can be seen both upfield and downfield of this GNRA peak, although these resonances are significantly broadened by NMR exchange (Figure ). Characterization of this construct by 2D NMR studies revealed two sets of non-canonical peaks, which overlay with the NMR results of the unmodified RNA (Figure ). The remarkable match in both peak intensity and location conclusively shows that this singly modified construct has a 'near' identical conformational ensemble to the unmodified construct. The presence of two sets of non-canonical peaks in the NOESY spectrum collected at a longer mixing time (300 ms) confirmed that both states 1 and 3 predicted by T-REMD are the most stable states in solution (Figure ).