Direct identification of NH⋯N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy

It is shown that the recently developed quantitative JNN HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar 2hJNN couplings across NH⋯N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor 15N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding 2hJNN coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced confor-mational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements.