Characterization of a native hammerhead ribozyme derived from schistosomes

A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop–loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop–loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop–loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of R_p-phosphorothioate substitutions at the cleavage site and 5′ to A9, both of which could be rescued with Cd^{. Here, phosphorothioate modifications at the cleavage site and 5′ to A9 were made in the schistosome-derived sequence. In Mg, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence.}