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.