Nucleic Acids Research, Vol 27, Issue 4 942-948, Copyright © 1999 by Oxford University Press
G Roy, S Ananvoranich and JP Perreault
We report here the first demonstration of the cleavage of an mRNA in trans
by delta ribozyme derived from the antigenomic version of the human
hepatitis delta virus (HDV). We characterized potential delta ribozyme
cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using
oligonucleotide binding shift assays and ribonuclease H hydrolysis.
Ribozymes were synthesized based on the structural data and then tested for
their ability to cleave the mRNA. Of the nine ribozymes examined, three
specifically cleaved a derivative HDV mRNA. All three active ribozymes gave
consistent indications that they cleaved single-stranded regions. Kinetic
characterization of the ability of ribozymes to cleave both the full-length
mRNA and either wild-type or mutant small model substrate suggests: (i)
delta ribozyme has turnovers, that is to say, several mRNA molecules can be
successively cleaved by one ribozyme molecule; and (ii) the substrate
specificity of delta ribozyme cleavage is not restricted to C/UGN6.
Specifically, substrates with a higher guanosine residue content upstream
of the cleavage site (i.e. positions -4 to -2) were always cleaved more
efficiently than wild-type substrate. This work shows that delta ribozyme
constitutes a potential catalytic RNA for further gene-inactivation
therapy.
ARTICLES
Delta ribozyme has the ability to cleave in transan mRNA
Departement de Biochimie, Faculte de Medecine, Universite de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.
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