Site‐Specific Isotope‐Labeling of Inosine Phosphoramidites and NMR Analysis of an Inosine‐Containing RNA Duplex

Abstract

Structural features and internal dynamics of inosine-containing RNAs are poorly understood. NMR studies of such RNAs require C, N-labeling, which cannot be achieved using in vitro transcription as inosine and guanosine are not distinguished by RNA polymerase. Herein, we report the synthesis of an inosine phosphoramidite with selective C8 and N7-isotope incorporation in the base and uniform C-labeling of the ribose. Chemical synthesis of an RNA duplex containing four consecutive IU base pairs with this optimized isotope-labeling scheme greatly simplifies NMR spectra and resolves signal overlap. The absence of detectable NMR signals of imino protons and unusual inter-residue NOE correlations in this RNA indicate deviations from standard A-form geometry, consistent with reduced stability of this duplex seen in UV melting studies compared to its nonedited RNA counterparts. These studies indicate that the introduction of IU base pairs distorts and destabilizes RNA helices significantly compared to the also noncanonical GU base-pairs. Our optimized isotope-labeling scheme enables high-resolution NMR studies of inosine-edited RNAs.

The synthesis of 15N(7)-Hoogsteen face-labeled adenosine phosphoramidite for solid-phase RNA synthesis

“…N1-, N3-, N7-, and/or O6-glycosylated isomers form as byproducts in minor amounts, consistent with previous reports [10, 11] and in accordance to the qualitative comparison of H-C8 1 H NMR resonances observed in the product mixture (Supplementary Material). The integrity (C1′–N9 connectivity) of compound 6 was further supported by NMR spectroscopic comparison of 6 with an authentic sample that was prepared by direct benzoylation of commercial inosine (Supplementary Material).…”

“…To achieve 15 N(7)-labeled adenosine amidite 12 , we conceived a strategy that employs a silyl-Hilbert-Johnson nucleosidation [9–11] and a recently introduced azido-to-acetamido purine transformation [4] as key steps. Therefore, 15 N(7)-hypoxanthine 5 was synthesized following the protocol by Jones and coworkers (Scheme 1) [12].…”

RNA structure refinement using NMR solvent accessibility data

“…For large RNAs, specific isotope labeling schemes are required to reduce signal overlap 9, 10, 53 . In a simple and straight-forward approach, only one or two nucleotide types are enriched in NMR active nuclei.…”

Self‐Assembly of DNA and RNA Building Blocks Explored by Nitrogen‐14 NMR Crystallography: Structure and Dynamics

“…However, the isotopic 15 N/ 13 C enrichment of nucleosides incorporated into oligonucleotides by solid‐phase synthesis is complex, labor‐intensive and expensive, particularly in longer sequences. Indeed, the introduction of these isotopes into nucleosides requires either modified phosphoramidite building blocks for solid phase synthesis or nucleoside triphosphate analogs for enzymatic DNA or RNA production . Since the preparation of these isotope‐enriched building blocks requires multistep synthetic protocols, the possibility of characterizing nitrogen environments by exploiting the naturally abundant 99.9 % 14 N isotope without recourse to isotopic enrichment is particularly appealing.…”