Kristian H. Link, Lixia Guo, Tyler D. Ames, Laising Yen, Richard C. Mulligan, Ronald R. Breaker
Engineering high-speed allosteric hammerhead ribozymes
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 the
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.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 2007
Pages: -
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