The 2′-hydroxyl group plays fundamental roles in both the structure and the function of RNA, and is the major determinant of the conformational and thermodynamic differences between RNA and DNA. Here, we report a conformational analysis of 2′-OH groups of the HIV-2 TAR RNA by means of NMR scalar coupling measurements in solution.

Our analysis supports the existence of a network of water molecules spanning the minor groove of an RNA A-form helix, as has been suggested on the basis of a high-resolution X-ray study of an RNA duplex. The 2′-OH protons of the lower stem nucleotides of the TAR RNA project either towards the O3′ or towards the base, where the 2′-OH group can favorably participate in H-bonding interactions with a water molecule situated in the nucleotide base plane. We observe that the k_ex rate of the 2′-OH proton with the bulk solvent anti-correlates with the base-pair stability, confirming the involvement of the 2′-OH group in a collective network of H-bonds, which requires the presence of canonical helical secondary structure.

The methodology and conformational analysis presented here are broadly applicable and facilitate future studies aimed to correlate the conformation of the 2′-OH group with both the structure and the function of RNA and RNA–ligand complexes.