Two- and three-dimensional HCN experiments for correlating base and sugar resonances in 15N,13C-labeled RNA oligonucleotides.
New 2D and 3D 1H-13C-15N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide H1'-H8(H6) connectivities in 13C- and 15N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the H1'-C1'-N9/N1 and H8/H6-C8/C6-N9/N1 connectivities, respectively. The correlation of H1' protons to glycosidic nitrogens N9/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective 13C-refocusing and 15N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D 1H-15N and 3D 1H-13C-15N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra- and internucleotide H1'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV.
Sklenár
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V
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Peterson
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RD
Rejante
M R
MR
Feigon
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J
D001483:Base Sequence; D002241:Carbohydrates / Q000737:chemistry*; D002247:Carbon Isotopes; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D009705:Nucleosides / Q000737:chemistry*; D009843:Oligoribonucleotides / Q000737:chemistry*; D011522:Protons; D012313:RNA / Q000737:chemistry*
D002241:Carbohydrates; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D009705:Nucleosides; D009843:Oligoribonucleotides; D011522:Protons; D012313:RNA; D006859:Hydrogen