Variable helix elongation as a tool to modulate RNA alignment and motional couplings

The application of residual dipolar couplings (RDCs) in studies of RNA structure and dynamics can be complicated by the presence of couplings between collective helix motions and overall alignment and by the inability to modulate overall alignment of the molecule by changing the ordering medium. Here, we show for a 27-nt TAR RNA construct that variable levels of helix elongation can be used to alter both overall alignment and couplings to collective helix motions in a semi-predictable manner. In the absence of elongation, a four base-pair helix II capped by a UUCG apical loop exhibits a higher degree of order compared to a six base-pair helix I (ϑI/ϑII=0.56±0.1). The principal Szz direction is nearly parallel to the axis of helix II but deviates by ∼40° relative to the axis of helix I. Elongating helix I by three base-pairs equalizes the alignment of the two helices and pushes the RNA into the motional coupling limit such that the two helices have comparable degrees of order (ϑI/ϑII=0.92±0.04) and orientations relative to Szz (∼17°). Increasing the length of elongation further to 22 base-pairs pushes the RNA into the motional decoupling limit in which helix I dominates alignment (ϑII/ϑI=0.45±0.05), with Szz orientated nearly parallel to its helix axis. Many of these trends can be rationalized using PALES simulations that employ a previously proposed three-state dynamic ensemble of TAR. Our results provide new insights into motional couplings, offer guidelines for assessing their extent, and suggest that variable degrees of helix elongation can allow access to independent sets of RDCs for characterizing RNA structural dynamics.