Template-directed interference footprinting for RNA based on inosine-specific cleavage

We report here the development of a Template-directed Interference (TDI) footprinting assay for RNA. The TDI nucleotide analogue inosine (I) lacks the exocyclic amine of G and is a suitable probe for the role of this group in RNA structure and function. Using an I-specific cleavage protocol we identified three functionally significant G residues in the Tetrahymena ribozyme. These residues are proximal to the active site of the folded intron and likely contribute to the positioning of substrates at the catalytic core.