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 hai rpin 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 coupli ngs ((n)J(CH)) produce ECOSY type cross-peaks, thus facilitating the d etermination 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% i sotopic enrichment. The observed (n)J(CH) couplings were then correlat ed to the known structure. The signs of (2)J(C4'H5'), (2)J(C4'H5'), an d (2)J(C5'H4') can be used for the stereospecific assignment of H5'/(p ro-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 suga r conformation. Comparison of the magnitude of (3)J(C3'H5') and (3)J(C 3'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 i nformation which correlates signs and magnitudes of (n)J(CH) both to d ouble-stranded A-form and nonhelix RNA structure forms a basis from wh ich strategies for the structure determination of larger RNA and RNA-p rotein complexes can be derived.