To further test the conformational distribution from the REMD simulations, we used a number of simplifying assumptions to compute RDCs from the REMD trajectory. Snapshots from the REMD simulations were superimposed onto an idealized A-form helix oriented in the principal axis system determined using the experimental RDCs and the order tensor fit. RDCs were then arbitrarily scaled by À82/r 3 , in which r is the C-H bond length and accounts for bond length variations during the dynamics. We find excellent agreement between experimental and computed nucleobase RDCs; however, computed C1 0 H1 0 RDCs fail to reproduce observed RDCs, particularly for A32: while the magnitude is similar (18 Hz compared to À30 Hz) the sign differs, suggesting the orientation of the C1 0 H1 0 bond vector differs between experiment and simulation (Supplementary Figure S5) . C1 0 H1 0 RDCs are generally opposite in sign to base RDCs in a double-stranded A-form helix. However, back-calculated C1 0 H1 0 RDCs from the order tensor analysis assuming a ssRNA A-form helix are positive in sign, suggesting the C1 0 H1 0 orientation in the REMD simulations deviates from an A-form structure (Supplementary Figure S5) .