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The catalytic activity of the trans cleaving hammerhead ribozyme 2as-Rz12, with long antisense flanks of 128 and 278 nt, was tested under a wide range of different reaction conditions for in vitro cleavage of a 422 nt RNA transcript derived from human immunodeficiency virus type 1 (HIV-1). Depending on the reaction conditions, in vitro cleavage rates varied by a factor of approximately 100. Increasing concentrations of magnesium up to 1 M were found to enhance the reaction. Sodium when added simultaneously with magnesium showed an inhibitory effect on the cleavage reaction. Addition of sodium during pre-annealing, however, produced a stimulating effect. It was found that the additional inclusion of spermidine during pre-annealing further increased the reaction rate markedly. In accordance with accelerated cleavage, it was possible to identify a distinct, spermidine-induced conformer of the ribozyme-substrate complex. Under the most favourable conditions cleavage rates of 1/min were obtained, which are in the range of rates obtained for conventional hammerhead ribozymes with short antisense flanks. A comparison of thermodynamic data for short- and long-armed hammerhead ribozymes suggested that the activation entropy became unfavourable when helices I and III formed a long chain ribozyme-substrate complex. We conclude that in the absence of spermidine folding into the active conformation is impaired by increased friction of long helices, resulting in relatively low cleavage rates in vitro.

A spermidine-induced conformational change of long-armed hammerhead ribozymes: ionic requirements for fast cleavage kinetics