Ribonucleic acid structure determination by NMR spectroscopy relies primarily on local structural restraints provided by 1H-1H NOEs and J-couplings. When employed loosely, these restraints are broadly compatible with A- and B-like helical geometries and give rise to calculated structures that are highly sensitive to the force fields employed during refinement. A survey of recently reported NMR structures reveals significant variations in helical parameters, particularly the major groove width. Although helical parameters observed in high-resolution X-ray crystal structures of isolated A-form RNA helices are sensitive to crystal packing effects, variations among the published X-ray structures are significantly smaller than those observed in NMR structures. We have shown that restraints derived from aromatic 1H-13C residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) can overcome NMR restraint and force field deficiencies and afford structures with helical properties similar to those observed in high-resolution X-ray structures. Multiple new methods for accurate measurement of residual dipolar couplings in larger nucleic acids have also been developed. In particular, analogous to the recently introduced ARTSY method for measurement of one-bond 1H-15N residual dipolar couplings (RDCs) in large perdeuterated proteins, we have developed new methods for measurement of base 13C-1H and 15N-1H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in 1H-15N and 13C-1H TROSY-HSQC spectra, and have been demonstrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for 15N-1H and 65/(S/N) Hz for 13C-1H. The signal-to-noise ratio of both 1H-15N and 1H-13C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with 1H-1H NOE transfer, allows for fast repetition of the experiment. By working in a mixed D2O/H2O solvent, we find that considerable enhancement in resolution of the imino proton resonances can be achieved, at a cost in signal to noise. However, when measuring RDCs, the increase in resolution more than offsets the lower sensitivity. Different optimal solvent compositions apply for G:C versus A:U basepairs. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6 relative to the X-ray structure of the native riboswitch.

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