CARBON-PROTON SCALAR COUPLINGS IN RNA - 3D HETERONUCLEAR AND 2D ISOTOPE-EDITED NMR OF A C-13-LABELED EXTRA-STABLE HAIRPIN

Long range carbon-proton scalar couplings were measured for an RNA hairpin of 12 nucleotides using 3D and C-13-edited 2D NMR. The large one-bond carbon-proton scalar couplings ((1)J(CH)) and small n-bond couplings ((n)J(CH)) produce ECOSY type cross-peaks, thus facilitating the determination of the sign and magnitude of the smaller (2)J(CH) or (3)J(CH). The UUCG RNA hairpin (5'-rGGACUUCGGUCC-3'), whose structure has been determined by our laboratory, was uniformly C-13-labeled at 30% isotopic enrichment. The observed (n)J(CH) couplings were then correlated to the known structure. The signs of (2)J(C4'H5'), (2)J(C4'H5'), and (2)J(C5'H4') can be used for the stereospecific assignment of H5'/(pro-S)/H5'' (pro-R) protons and for determining torsion angle gamma(O5'-C5'-C4'-C3'). The sign of any one of the (2)J(C1'H2'), (2)J(C2'H3'), (2)J(C3'H2') and (2)J(C4'H3)' can be used to determine the ribose sugar conformation. Comparison of the magnitude of (3)J(C3'H5') and (3)J(C3'H5'') can also be used for the stereospecific assignment of H-5'(pro-S)/H-5''(pro-R). The utility and potential limitations of using (n)J(CH) for RNA structure determination are discussed in-depth. This new information which correlates signs and magnitudes of (n)J(CH) both to double-stranded A-form and nonhelix RNA structure forms a basis from which strategies for the structure determination of larger RNA and RNA-protein complexes can be derived.