Engineering high-speed allosteric hammerhead ribozymes
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Abstract
Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg2+. The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (kobs) as high as 8 min-1 in the presence of theophylline and maximal kobs increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.
©2007 by Walter de Gruyter Berlin New York
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- Protein import into chloroplasts: new aspects of a well-known topic
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- The structure of CMS2MS2, a mitogenic protein isolated from Carica candamarcensis
- The proteome of the heterocyst cell wall in Anabaena sp. PCC 7120
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