EXAMINATION OF THE CATALYTIC FITNESS OF THE HAMMERHEAD RIBOZYME BY IN-VITRO SELECTION

We have designed a self-cleaving ribozyme construct that is rendered i nactive during preparative in vitro transcription by allosteric intera ctions with ATP. This allosteric ribozyme was constructed by joining a hammerhead domain to an ATP-binding RNA aptamer, thereby creating a r ibozyme whose catalytic rate can be controlled by ATP. Upon purificati on by PAGE, the engineered ribozyme undergoes rapid self-cleavage when incubated in the absence of ATP. This strategy of ''allosteric delay' ' was used to prepare intact hammerhead ribozymes that would otherwise self-destruct during transcription. Using a similar strategy, we have prepared a combinatorial pool of RNA in order to assess the catalytic fitness of ribozymes that carry the natural consensus sequence for th e hammerhead. Using in vitro selection, this comprehensive RNA pool wa s screened for sequence variants of the hammerhead ribozyme that also display catalytic activity. We find that sequences that comprise the c ore of naturally occurring hammerhead dominate the population of selec ted RNAs, indicating that the natural consensus sequence of this riboz yme Is optimal for catalytic function.