The application of double-quantum and spin echo nuclear magnetic resonance spectroscopy to the assignment of 1H chemical shifts of RNA oligomers

Specific assignment of the non-exchangeable base and ribose anomeric protons is difficult for oligoribonucleotides with a high pyrimidine content. Problems arise from the narrow range of pyrimidine H-6 chemical shifts and the overlap of pyrimidine H-5 resonances with the ribose anomeric proton signals. To solve these problems two multiple pulse methods were used which depend on the scalar coupling between H-6 and H-5. The first method was double-quantum nmr which established the connectivity between specific pyrimidine H-6 and H-5 resonances. A refocussing pulse was added to the end of the double-quantum pulse sequence to improve the identification of several H-6 and H-5 connectivities in a single experiment. The second procedure involved homonuclear decoupled spin echo nmr to remove H-5 signals selectively from the ribose anomeric proton region. These techniques are illustrated by the assignment of the spectra of the oligoribonucleotides UpUpC, UpUpU, GpCpUpC, and UpApGpCpUpU.