Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR

NMR approaches using
nucleotide-specific deuterium labeling schemes
have enabled structural studies of biologically relevant RNAs of increasing
size and complexity. Although local structure is well-determined using
these methods, definition of global structural features, including
relative orientations of independent helices, remains a challenge.
Residual dipolar couplings, a potential source of orientation information,
have not been obtainable for large RNAs due to poor sensitivity resulting
from rapid heteronuclear signal decay. Here we report a novel multiple
quantum NMR method for RDC determination that employs flip angle variation
rather than a coupling evolution period. The accuracy of the method
and its utility for establishing interhelical orientations are demonstrated
for a 36-nucleotide RNA, for which comparative data could be obtained.
Applied to a 78 kDa Rev response element from the HIV-1 virus, which
has an effective rotational correlation time of ca. 160 ns, the method
yields sensitivity gains of an order of magnitude or greater over
existing approaches. Solution-state access to structural organization
in RNAs of at least 230 nucleotides is now possible.