KINETIC CHARACTERIZATION OF INTRAMOLECULAR AND INTERMOLECULAR HAMMERHEAD RNAS WITH STEM-II DELETIONS

A method is described to obtain intramolecular cleavage rates for the hammerhead ribozyme during in vitro transcription. By avoiding RNA purification and renaturation, the potential for formation of inactive conformations of the RNA is minimized. By showing that an intramolecular hammerhead and a closely related intermolecular hammerhead cleave at the same rate under a given set of conditions, we confirm that both reactions probably have the same rate-limiting step. An in vitro selection strategy was used to isolate active hammerheads from a library of molecules where six randomized nucleotides replaced stem-loop II. The sequence and number of nucleotides which replace stem-loop II have large effects on hammerhead cleavage activity. The relative activities of three sequences selected from the intramolecular library are the same when the sequences are transferred into an intermolecular hammerhead background.