Correlation of Activity with Stability of Chemically Modified Ribozymes in Nuclei Suspension

Abstract

To examine hammerhead ribozyme activity in the nuclear environment, we have used nuclei isolated from HTLV-I tax transformed fibroblasts to evaluate ribozymes targeted against HTLV-I tax RNA. The ribozyme activity in nuclei suspension was strongly dependent on the resistance of the particular ribozyme to endogenous nucleases. A ribozyme containing exclusively 2′-deoxynucleotides in its stems cleaved target RNA by its catalytic activity in the absence of proteins and caused degradation in their presence by induction of nuclear RNase H activity. A ribozyme containing 2′-amino- and 2′-fluoropyrimidine nucleosides in combination with terminal phosphorothioate linkages was significantly more stable in nuclei suspension and also exhibited a more than threefold higher cleavage efficacy than its unmodified counterpart. The increased resistance against nuclease degradation is mainly due to terminal phosphorothioate linkages, suggesting that both 5′ and 3′-exonucleases are primarily responsible for the nuclear degradation of oligonucleotides.