2'-hydroxyl proton positions in helical RNA from simultaneously measured heteronuclear scalar couplings and NOEs.

The 2'-hydroxyl group in RNA plays an important structural role; it defines hydration in the minor groove, impacts thermodynamic stability of RNA, and often participates in RNA catalysis. To better study this important functional group in RNA, we describe a constant-time HMQC-IPAP-NOESY 3D NMR experiment. It simultaneously yields highly resolved 13C-separated NOEs from ribose protons to the 2'OH proton, as well as E.COSY-type measurement of JC-OH couplings, thereby permitting a quantitative study of the orientation of the 2'OH proton in RNA. The observed NOE patterns indicate that the 2'OH bonds in A-form helical RNA are primarily oriented toward the base domain, as further supported by small (1.3 +/- 0.7 Hz) 3JC1'-2'OH and relatively large (4.2 +/- 0.7 Hz) 3JC3'-2'OH couplings. The constant-time HMQC-IPAP-NOESY is suitable for measurement of interactions of rapidly exchanging protons in proteins and nucleic acids.