FULL H-1-NMR ASSIGNMENT OF A 24-NUCLEOTIDE RNA HAIRPIN - APPLICATION OF THE H-1 3D-NOE 2QC EXPERIMENT/

The subject RNA models the binding site for the coat protein of the R1 7 virus, as well as the ribosome recognition sequence for the R17 repl icase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the H-1 NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axi s. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeati ng NOE-2Q pattern. In this experiment substantial J-coupling confers s pecial advantages. This always occurs for geminal pairs (H5'/H5 '' for RNA plus H2'/H2 '' for DNA), as well as for H5/H6, for H3'/H4' in sug ars with substantial populations of the N-pucker, for H1'/H2' for S-pu ckered sugars, and usually for H2'/H3'. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignmen t of all of the non-exchangeable protons, eliminating the need for sta ble-isotope labeling.