Detection of Scalar Couplings Involving 2‘-Hydroxyl Protons Across Hydrogen Bonds in a Frameshifting mRNA Pseudoknot

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Abstract
The −1 frameshift-stimulating mRNA pseudoknot from pea enation mosaic virus-1 (PEMV-1) is composed nearly entirely of RNA triple helix.4 The 2‘-OH hydroxyl protons of riboses C15 and C16 are hydrogen bond donors to the N1 atoms of adenosines A27 and A25, respectively, positioned in the minor groove of pseudoknot stem S1. In this paper, a nonrefocused 1H,15N CPMG HSQC of uniformly 13C,15N-labeled 33-mer PEMV-1 RNA has been tailored to reveal a correlation of the 2‘-OH hydroxyl proton of C15 to the N1 nitrogen resonance of A27 mediated by a cross hydrogen bond scalar coupling. The 1hJ(2‘OH,N) cross hydrogen bond scalar coupling constant determined from a quantitative 1D {15N} spin−echo difference experiment for the C15/A27 interaction is 1.7 ± 0.1 Hz, while that for the C16/A25 interaction appears larger, 3.5 ± 0.3 Hz, despite the fact that the corresponding direct correlation between the 2‘-OH hydroxyl proton of C16 and the N1 of A25 is missing due to unfavorable solvent exchange properties. These findings reveal a detailed picture of critical noncanonical O−H···N hydrogen-bonding loop−stem interactions in an RNA triple helical structure.