Two-and three-dimensional HCN experiments for correlating base and sugar resonances in 15N, 13C-labeled RNA oligonucleotides |
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Authors: | Vladimír Sklenář Robert D. Peterson Marita R. Rejante Juli Feigon |
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Affiliation: | (1) Department of Chemistry and Biochemistry, University of California, 90024 Los Angeles, CA, USA;(2) Present address: Molecular Biology Institute, University of California, 90024 Los Angeles, CA, USA;(3) Institute of Scientific Instruments, Academy of Sciences of the Czech Republic, 612 64 Brno, Czech Republic |
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Abstract: | Summary 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 3D1H-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. |
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Keywords: | Rev response element HIV NMR Three-dimensional NMR Labeled RNA |
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