SELECTIVE ISOTOPIC ENRICHMENT OF SYNTHETIC RNA - APPLICATION TO THE HIV-1 TAR ELEMENT

The introduction of isotopically enriched nucleotides into NMR quantities of a synthetic 29-mer RNA derived from the HIV-1 TAR element is described. RNA enriched in C-13 and/or N-15 is produced by a procedure which involves isolation of whole cellular RNA from Escherichia coli, nucleolysis, separation of mononucleotides, chemical or enzymatic pyrophosphorylation, and in vitro transcription by T7 RNA polymerase. Spectral characteristics of each residue type are examined in isolation. C-13 chemical shifts provide an alternative method to determine ribose puckers for larger RNAs. Nonprotonated sites such as purine N7 groups can now be monitored through the use of multiple-bond H-1-N-15 coupling. When applied conservatively, coordinate analysis of chemical shift values should prove valuable for NMR studies of RNA structure and recognition. H-1, C-13, and N-15 chemical shift data suggest that TAR residue A35 has an unusual local environment, consistent with extrusion of its base from the terminal loop.