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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 Mg 2+ . The population of allosteric ribozymes resulting from 15 rounds of  in vitro  selection yielded variants with observed rate constants ( k  obs ) as high as 8 min -1  in the presence of theophylline and maximal  k  obs  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.

Engineering high-speed allosteric hammerhead ribozymes