RNA structure analysis using methidiumpropyl-EDTA.Fe(II): a base-pair-specific RNA structure probe.

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Abstract
Methidiumpropyl-EDTA.cntdot.Fe(II) [MPE.cntdot.Fe(II)], in the presence of dithiothreitol, cleaves [yeast] tRNAPhe in a structure-specific fashion. Molar ratios of MPE.cntdot.Fe(II) to tRNAPhe of < 1 preferentially cleave phosphodiester bonds known to occur in double-stranded regions of the tRNAPhe molecule. Microdensitometric analysis of autoradiograms of MPE.cntdot.Fe(II) cleavage products following gel electrophoresis reveals a correspondence between preferred sites of MPE.cntdot.Fe(II) cleavage and sites in tRNAPhe most sensitive to cobra venom RNase, a double-strand-specific endoribonuclease. Conversely, sites of cleavage by the single-strand-specific S1 nuclease correspond to those nucleotides that are least susceptible to MPE.cntdot.Fe(II) hydrolysis. Sensitive helical regions in tRNAPhe included the dihydrouracil and the T.PSI.C stems, which cannot be detected by cobra venom RNase because of steric constraints. Phosphodiester bonds within the T.PSI.C and dihydrouracil loop regions which are not detected by S1 nuclease under rigorously controlled digestion conditions, are revealed by inference from their relative insensitivity to MPE.cntdot.Fe(II). MPE.cntdot.Fe(II) should be useful as a general small MW probe of RNA structure, having a greater accessibility to base-paired regions than do the more bulky enzymic probes.