Kinetic characterization of two I/II format hammerhead ribozymes

Five new hammerhead ribozymes were designed that assemble through the formation of helices I and II (I/II format) instead of the more standard assembly through helices I and III (I/III format). The substrate binding and cleavage properties of such hammerheads could potentially be different due to the absence of loop II and the requirement for the entire catalytic core to assemble. Two I/II format hammerheads, HHalpha1 and HHalpha5, which show structural homogeneity on native gels, were characterized kinetically. The association rate constants of both I/II hammerheads are unusually slow compared to the rate of RNA duplex formation. The dissociation rate constants indicate that the hammerhead core destabilizes an uninterrupted RNA helix somewhat less than was observed for I/III hammerheads. Whereas the cleavage rate constant of HHalpha5 is similar to that observed for I/III hammerheads, HHalpha1 cleaves 10-fold faster than any hammerhead previously reported. The temperature and pH dependence of the cleavage rate constant of HHalpha1 are similar to those reported for I/III hammerheads, suggesting a similar mechanism of cleavage.