Elucidation of the Mechanism for the Self-Cleavage Rection of RNA Using Chemically-Synthesized Oligonucleotides

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Self-cleaving RNA found in viruses and viroids is the smallest catalytic RNA molecule named "ribozyme", which requires a "hammerhead" structure for its activity. We have investigated this self-cleavage reaction using chemicallysynthesized oligonucleotides in order to elucidate its mechanism.First we have constructed a two-stranded ribozyme complex and its mutants with a sequence derived from a satellite DNA transcript from the newt, in order to analyze the self-cleavage reaction, and found the consensus sequence required for the reaction. Then we have measured CD spectra of a three-stranded complex containing 2'-O-methylcytidine at the cleavage site, which prevented the cleavage reaction, to show that formation of the tertiary structure is essential to this reaction. The kinetic parameters of the three-stranded complex were as follows : K_m = 0.53 uM, V_<max> = 0.015 uM^.min^<-1>, and k_<cat> = 0.03 min ^<-1>.As a dimer structure is reported to be preferential in the case of the hammerhead-type ribozyme containing a unstable stem-loop structure, we have analyzed the monomer/dimer structure of the two-stranded ribozyme complex derived from the newt sequence. The following results demonstrate that this complex exists as a monomer : 1) The free energy calculated from its T_m values was almost the same as that obtained for a monomer model. 2) Its electrophoretic mobility on a gel coincided with the monomer-type three-stranded complex. 3) Its half life in the cleavage reaction was very long, compared with that of a dimer complex. 4) In the case of the complex containing two inosines instead of guanosines in the stem region, only one signal assigned to the I^.C pair was observed by ^1H-NMR.