Taken together, the data show that the polyadenine tract is relatively ordered at 298 K, with a gradual reduction in order approaching the termini and that the base stacking interactions are the guiding force behind this order. To determine whether disrupting the polyadenine tract will destabilize the global structure, we substituted A29 within the polyadenine tract with a cytosine residue (referred to as A29C). Other types of mutations involving placements of uridine were not explored as these were expected to yield partially base paired conformations. As with the WT construct, we observed no imino protons, indicating the absence of any detectable base pairing and secondary structure (Supplementary Figure S7) . The 2D C-H spectra for the A29C mutant remain highly disperse, and the chemical shift perturbations relative to WT are clustered around the site of mutation (A28 and A30) ( Figure 4A and Supplementary Figure S6) . However, small but significant chemical shift perturbations relative to WT are also observed at more distant residues, including A27, A31, C33 and U34. These perturbations diminish when moving away from the center of the ssRNA and are basically absent in the highly flexible terminal residues ( Figure 4A and Supplementary Figure S6 ). Such longer-range perturbations suggest that the mutation may have a long-range effect possibly by influencing the stacking interactions of several nucleobases. A perturbation to stacking interactions is also supported by distinct NOE connectivities in A29C, which show weakened cross peaks to C29, and new crosspeaks between A28 (H2) and A30 (H1 0 ) that indicate C29 partially loops out to allow A28 to stack onto A30 (Supplementary Figure S7) . The melting temperature of the mutant is reduced by $5 C, and the base stacking energies are computed to be $2 kcal/mol lower compared to WT, indicating that the mutation likely destabilizes the stacking interactions (Supplementary Figure S6) .