文摘

The aim of this work was to shed some more light on factors influencing the effectiveness ofdelta ribozyme cleavage of structured RNA molecules. An oligoribonucleotide that corresponds to the3'-terminal region X of HCV RNA and yeast tRNA^Phe were used as representative RNA targets. Only afew sites susceptible to ribozyme cleavage were identified in these targets using a combinatorial libraryof ribozyme variants, in which the region responsible for ribozyme-target interaction was randomized.On the other hand, the targets were fairly accessible for binding of complementary oligonucleotides, aswas shown by 6-mer DNA libraries and RNase H approach. Moreover, the specifically acting ribozymescleaved the targets precisely but with unexpectedly modest efficacy. To explain these observations, sixmodel RNA molecules were designed, in which the same seven nucleotide long sequence recognized bythe delta ribozyme was always single stranded but was embedded into different RNA structural context.These molecules were cleaved with differentiated rates, and the corresponding k₂ values were in the rangeof 0.91-0.021 min^-1; thus they differed almost 50-fold. This clearly shows that cleavage of structuredRNAs might be much slower than cleavage of a short unstructured oligoribonucleotide, despite fullaccessibility of the targeted regions for hybridization. Restricted possibilities of conformational transitions,which are necessary to occur on the cleavage reaction trajectory, seem to be responsible for these differences.Their magnitude, which was evaluated in this work, should be taken into account while considering theuse of delta ribozymes for practical applications.