Morpholino Spin-Labeling for Base-Pair Sequencing of a 3’-Terminal RNA Stem by Proton Homonuclear Overhauser Enhancements: Yeast Ribosomal 5S RNA

Base-pair sequences for 5S and 5.8S RNAs are not readily extracted from proton homonuclear nuclear Overhauser enhancement (NOE) connectivity experiments alone, due to extensive peak overlap in the downfield (11–15 ppm) proton NMR spectrum. In this paper, we introduce a new method for base-pair proton peak assignment for ribosomal RNAs, based upon the distance-dependent broadening of the resonances of base-pair protons spatially proximal to a paramagnetic group. Introduction of a nitroxide spin-label covalently attached to the 3’-terminal ribose provides an unequivocal starting point for base-pair hydrogen-bond proton NMR assignment. Subsequent NOE connectivities then establish the base-pair sequence for the terminal stem of a 5S RNA. Periodate oxidation of yeast 5S RNA, followed by reaction with 4-amino-2,2,6,6-tetramethylpiperidinyl-l-oxy (TEMPO-NH₂) and sodium borohydride reduction, produces yeast 5S RNA specifically labeled with a paramagnetic nitroxide group at the 3’-terminal ribose. Comparison of the 500-MHz ₁H NMR spectra of native and 3’-terminal spin-labeled yeast 5S RNA serves to identify the terminal base pair (GpC₁₂₀) and its adjacent base pair (G₂·U₁₁₉) on the basis of their proximity to the 3’-terminal spin-label. From that starting point, we have then identified (G·C, A·U, or G·U) and sequenced eight of the nine base pairs in the terminal helix via primary and secondary NOE’s.