Unusual Interactions Between Cleavage Products of a Cis-Cleaving Hammerhead Ribozyme

Abstract

We have synthesized and tested a cis-cleaving ribozyme designed to have thermodvnamically stable stem-loop structures. This cis-ribozyme cleaves very efficiently in vitro, with a cleavage rate of about 0.5/min. Surprisingly, during the course of in vitro transcription and cleavage of our ribozyme, a product of unusual mobility accumulates and coincides with a sharp decline in the rate of formation of cleavage products. Analyses of this electrophoretic variant demonstrated that it is formed by interactions of the cleavage products. Despite the fact that the products and ribozyme transcript are of identical sequence, the cleavage products interact only with one another and not with the uncleaved precursor. This suggests a significant structural difference between the cleaved and uncleaved ribozyme transcripts. Testing of this cis-ribozyme in both yeast and mammalian cells shows no significant cleavage activity in vivo. We conclude that the structure of the ribozyme flanking sequences is important for optimizing the rate of ribozyme cleavage, but this enhanced rate does not necessarily correlate with enhanced in vivo function.