Page 84 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

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Encyclopedia of Physical Science and Technology EN014F-661 July 28, 2001 20:35

256 Ribozymes

B. Hairpin Ribozyme to overcome the lack of stability of introduced RNAs by
using modified nucleotides, 2 -fluoro- and 2 -amino-, or

A second small catalytic domain is the hairpin structure,
2 -O-allyl- and 2 -O-methyl-, mixed DNA/RNA mole-

which has four helical domains and five loops. Two he-
cules, or by the addition of terminal sequences (such as
lices of the hairpin domain form between the substrate
the bacteriophage T7 transcriptional terminator) at the Y-
and ribozyme, and this allows the design and specificity
end of the RNA to protect against cellular nucleases. En-
of binding for trans-acting hairpin ribozymes. The hairpin
dogenous expression has been achieved by inserting ri-
ribozyme has a more complicated substrate requirement
bozyme sequences into the untranslated regions of genes
than the hammerhead ribozyme, but generally can cleave
transcribed by RNA polymerase II (pol II), which have
to the 5 -side of any GUC.

strong promoters, such as the CMV promoter. Ribozymes
have also been inserted into the anticodon loop of tRNA,
C. Hepatitis Delta Virus transcribed by RNA polymerase III (pol III), or have been
HDV genomic and antigenomic RNAs contain a self- inserted into nonessential loop structures of small nuclear
cleavage site hypothesized to function during rolling circle RNAs and a small nucleolar RNA. They have been shown
replication of this satellite virus. Like the plant pathogens, to be functional in both plant and animal cells and in trans-
the sites in HDV are postulated to have a related secondary genic plants and animals.
structure, three models of which have been proposed: Successful use of ribozymes to knockdown target gene
cloverleaf, pseudoknot, and axehead, none of which is expression is dependent on a number of factors, including
similar to the catalytic domains previously described. target site selection as well as ribozyme gene delivery,
Like the other ribozyme motifs, the HDV ribozymes re- expression, stability, and intracellular localization. The
quire a divalent cation, and cleavage results in products two types of ribozymes that have been used most exten-

with 2 ,3 -cyclic phosphate and 5 -OH termini. Investi- sively for knockdown studies are the hairpin and hammer-
head ribozymes. Not all target sites for these ribozymes
gations of trans-cleavage with the HDV ribozyme have
are accessible for cleavage: secondary structures, bind-
not advanced to those of the hammerhead or hairpin
ing of proteins and nucleic acids, and other factors influ-
ribozymes.
ence intracellular ribozyme efficacy. Computer-assisted
RNA folding predictions and in vitro cleavage analyses
D. Neurospora Mitochondrial VS RNA are not necessarily predictive of intracellular or vivo ac-
tivity, and the best ribozyme target sites often must be
The Neurospora mitochondrial VS RNA, a single-
determined empirically in vivo. Alternative strategies uti-
stranded circular RNA of 881 nt, shares some features of
lizing cell extracts with native mRNAs have also proven
the self-catalytic RNAs of HDV, group I introns, and some
useful for determining accessible ribozyme binding
plant viral satellite RNAs. Although VS RNA can be de-
sites.
picted as having a secondary structure like group I introns,
Ribozymes can be introduced into cells as genes by
it is missing essential base-pairing regions, the cleavage
transfection or viral vector transduction, or as chemically
site is in a different position, and the termini produced
synthesized molecules stabilized with various base sub-
are 2 ,3 -cyclic phosphate and 5 -OH. Like the hammer-

stitutions and 3 and 5 modifications. Intracellular deliv-

head ribozymes, the VS RNA requires divalent cations
ery, in this case, is typically achieved by some form of
for cleavage in vitro. The catalytic core of Neurospora VS
carrier molecule, such as cationic lipids. Depending on
RNA has been shown to consist of 154 nt.
the application, either delivery method has its advantages
and disadvantages. Delivery of the ribozyme genes can
provide stable intracellular expression and the promoter
V. RIBOZYMES AS TOOLS AND GENE
THERAPY AGENTS choice can allow cell- or tissue-specific expression. Deliv-
ery of synthetic ribozymes allows short-term, high-level
availability, making it easier to use as a “drug;” however,
A. Ribozyme Delivery
ribozyme stability and pharmacokinetics may present sig-
Whatever type of ribozyme is chosen, it must be intro- nificant challenges.
duced into its target cell. Two general mechanisms exist Stable intracellular expression of transcriptionally ac-
for introducing catalytic RNA molecules into cells: ex- tive ribozymes can be achieved by viral vector-mediated
ogenous delivery of the preformed ribozyme and endoge- delivery. Currently, retroviral vectors are the most com-
nous expression from a transcriptional unit. Preformed monly used in cell culture, primary cells, and in transgenic
ribozymes can be delivered into cells using liposomes, animals. Retroviral vectors have the advantage of stable
electroporation, or microinjection. Efforts have been made integration into a dividing host cell genome, and the