Factors altering ribozyme-mediated cleavage of tumor necrosis factor-alpha mRNA in vitro

Hammerhead ribozymes are capable of cleaving RNA in a sequence specific manner in vitro. However, the complex environment of the cell differs dramatically from the conditions in vitro. Therefore, we explored cleavage of full-length target RNA with two ribozymes targeted against the murine tumor necrosis factor-alpha (TNF-alpha) mRNA. These ribozymes cleaved TNF-alpha mRNA within a pool of total cellular RNA in vitro, but less efficiently than previously reported for similar ribozymes. Although there may be several factors that could affect ribozyme activity, two of these factors were tested. The first factor was whether non-target polynucleotides inhibited ribozyme-mediated cleavage. Total cellular RNA and to a lesser degree DNA inhibited ribozyme activity. This inhibition was a combination of competitive and non-competitive inhibition. Non-target RNA with minimal complementarity to the ribozyme or target showed no effect on cleavage rates. The second factor was whether denaturing conditions improved ribozyme cleavage efficiency. Hammerhead ribozymes with 24 complementary bases had increased cleavage efficiency in formamide. Thus, the ribozymes may have had too long of an antisense flanking sequence which hybridized with the target RNA and resulted in a high melting temperature. These studies demonstrate that ribozyme cleavage was influenced by the amount of non-target polynucleotide and the strength of the ribozyme-substrate interaction.