Hammerhead ribozyme activity in the presence of low molecular weight cellular extract

Hammerhead ribozymes cleave RNA in vitro at magnesium concentrations that are not thought to be available in vivo. To search for cellular factors that could substitute for the role of magnesium, we investigated the influence of size-fractionated cell extracts on the rate of association with its target and the cleavage rate of the HIV-1-directed long-chain hammerhead ribozyme alphaYRz60. When using a fraction containing cellular compounds smaller than a molecular weight cutoff of 5000 (MWCO5000), we observed no influence on the annealing of the ribozyme with its target but strongly increased cleavage activity under single turnover conditions. The cleavage rate constant in the presence of the MWCO5000 extract was similar to the effect of approximately 25 mM magnesium in vitro, indicating that hammerhead ribozyme cleavage could occur at considerable rates in vivo.