Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments.

The advent of methods for preparing 15N- and 13C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studied by NMR spectroscopy. A practical limitation is the expense of the 13C label. It may therefore sometimes be desirable to prepare a relatively inexpensive 15N-labeled sample only. Here we show that the two-bond 1H-15N HSQC experiment can be used on 15N-labeled RNA to correlate the intranucleotide H1' and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected 1H-15N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RN oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1' and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.