SYNTHETIC RNA-CLEAVING MOLECULES MIMICKING RIBONUCLEASE A ACTIVE-CENTER - DESIGN AND CLEAVAGE OF TRANSFER-RNA TRANSCRIPTS

RNA cleaving molecules were synthesized by conjugating imidazole resid ues imitating the essential imidazoles in the active center of pancrea tic ribonuclease to an intercalating compound, derivative of phenazine capable of binding to the double stranded regions of polynucleotides. action of the molecules on tRNA was investigated. It was found, that some of the compounds bearing two imidazole residues cleave tRNA under physiological conditions. The cleavage reaction shows a bell-shaped p H dependence with a maximum at pH 7.0 indicating participation of prot onated and non-protonated imidazole residues in the process. Under the conditions stabilizing the tRNA structure, a tRNA(ASp) transcript was cleaved preferentially at the junctions of the stem and loop regions of the cloverleaf tRNA fold, at the five positions C-56, C-43, C-20.1, U-13, and U-8, with a marked preference for C-56. This cleavage patte rn is consistent with a hydrolysis mechanism involving non-covalent bi nding of the compounds to the double-stranded regions of tRNA followed by an attack of the imidazole residues at the juxtaposed flexible sin gle-stranded regions of the molecule. The compounds provide new probes for the investigation of RNA structure in solution and potential reac tive groups for antisense oligonucleotide derivatives.