Detection of N-H center dot center dot center dot N hydrogen bonding in RNA via scalar couplings in the absence of observable imino proton resonances

Hydrogen bond networks stabilize RNA secondary and tertiary structure and a re thus essentially important for protein recognition. During structure ref inements using either NMR or X-ray techniques, hydrogen bonds were usually inferred indirectly from the proximity of donor and acceptor functional gro ups. Recently, quantitative heteronuclear J(N,N)-HNN COSY NMR experiments w ere introduced that allowed the direct identification of donor and acceptor nitrogen atoms involved in hydrogen bonds. However, protons involved in ba se pairing interactions in nucleic acids are often not observable due to ex change processes. The application of a modified quantitative J(N,N)-HNN COS Y pulse scheme permits observation of (2h)J(N,N) couplings via non-exchange able protons. This approach allowed the unambiguous identification of the A 27.U23 reverse Hoogsteen base pair involved in a U-A.U base triple in the H IV-2 transactivation response element-argininamide complex. Despite a wealt h of NOE information, direct evidence for this interaction was lacking due to the rapid exchange of the U23 imino proton. The ability to directly obse rve hydrogen bonds, even in D2O and in the presence of rapid exchange, shou ld facilitate structural studies of RNA.