Nucleic Acids Research, Vol 25, Issue 24 4985-4993, Copyright © 1997 by Oxford University Press
TB Campbell, CK McDonald and M Hagen
Inhibition of gene expression by catalytic RNA (ribozymes) requires that
ribozymes efficiently cleave specific sites within large target RNAs.
However, the cleavage of long target RNAs by ribozymes is much less
efficient than cleavage of short oligonucleotide substrates because of
higher order structure in the long target RNA. To further study the effects
of long target RNA structure on ribozyme cleavage efficiency, we determined
the accessibility of seven hammerhead ribozyme cleavage sites in a target
RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr .
The base pairing- availability of individual nucleotides at each cleavage
site was then assessed by chemical modification mapping. The ability of
hammerhead ribozymes to cleave the long target RNA was most strongly
correlated with the availability of nucleotides near the cleavage site for
base pairing with the ribozyme. Moreover, the accessibility of the seven
hammerhead ribozyme cleavage sites in the long target RNA varied by up to
400-fold but was directly determined by the availability of cleavage sites
for base pairing with the ribozyme. It is therefore unlikely that steric
interference affected hammerhead ribozyme cleavage. Chemical modification
mapping of cleavage site structure may therefore provide a means to
identify efficient hammerhead ribozyme cleavage sites in long target RNAs.
ARTICLES
The effect of structure in a long target RNA on ribozyme cleavage efficiency
Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA. thomas.campbell@uchsc.edu
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