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J Biol Chem, Vol. 273, Issue 36, 23524-23533, September 4, 1998
From the Markey Center for Molecular Genetics, Department of
Microbiology and Molecular Genetics, University of Vermont,
Burlington, Vermont 05405
Combinatorial libraries of hairpin ribozymes
representing all possible cleavage specificities
(>105) were used to evaluate all ribozyme cleavage
sites within a large (4.2-kilobase) and highly structured viral
mRNA, the 26 S subgenomic RNA of Sindbis virus. The combinatorial
approach simultaneously accounts for target site structure and
dynamics, together with ribozyme folding, and the sequences that result
in a ribozyme-substrate complex with maximal activity. Primer extension
was used to map and rank the relative activities of the ribozyme pool
against individual sites and revealed two striking findings. First,
only a small fraction of potential recognition sites are effectively cleaved (activity-selected sites). Second, nearly all of the most effectively cleaved sites deviated substantially from the established consensus selection rules for the hairpin ribozyme and were not predicted by examining the sequence, or through the use of
computer-assisted predictions of RNA secondary structure. In
vitro selection methods were used to isolate ribozymes with
increased activity against substrates that deviate from the GUC
consensus sequence. trans-Acting ribozymes targeting nine
of the activity-selected sites were synthesized, together with
ribozymes targeting four sites with a perfect match to the cleavage
site consensus (sequence-selected sites). Activity-selected ribozymes
have much higher cleavage activity against the long, structured RNA
molecules than do sequence-selected ribozymes, although the latter are
effective in cleaving oligoribonucleotides, as predicted. These results
imply that, for Sindbis virus 26 S RNA, designing ribozymes based on
matches to the consensus sequence may be an ineffective strategy.
Cleavage of Highly Structured Viral RNA Molecules by
Combinatorial Libraries of Hairpin Ribozymes
THE MOST EFFECTIVE RIBOZYMES ARE NOT PREDICTED BY SUBSTRATE
SELECTION RULES
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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