Antisense Oligonucleotides as Potential Drugs
that, in addition, harbor a hydroxyl func-
tion from which the oligonucleotide chain
is extended during solid-phase synthesis.
After oligonucleotide synthesis is com-
plete, the oligonucleotide conjugate is
cleaved from the solid support and depro-
tected by ammonia treatment. Similarly to
the 5
-conjugation, appropriate 3
modiFer solid supports are commercially
available, which allow coupling of suitably
activated derivatives of the molecule to be
conjugated with the 3
-amino alkyl group
in a postsynthetic solution-phase step.
Mechanism of Action
±or most of the AS-ODNs, activation of
RNase H is the mechanism of action to
interfere with translation. This enzyme
recognizes a duplex between DNA and
RNA, resulting in the cleavage of the RNA
strand of this duplex. The essential role of
this enzyme in inhibiting gene expression
is based on the observation that the target
RNA is degraded, which would not be ex-
pected if a simple blockage of translation,
as originally conceived, was the mech-
anism of inhibition of translation. The
most convincing evidence that the enzyme
RNase H is responsible for this degrada-
tion relies on the fact that AS-ODNs that
are incompatible with the action of this
enzyme do not result in degradation of the
mRNA. This enzymatic step, of course,
makes AS-ODN turnover a key step in the
process requiring, in principle, substoi-
chiometric amounts of the oligonucleotide
for complete destruction of the RNA.
Activation of RNase H demands a DNA-
like or B-conformation of the ODN. This
requirement is obviously best met by
oligodeoxynucleotides as they exist in this
conformation. However, DNA–RNA com-
plexes are not as stable as RNA–RNA
complexes, which makes ODN with an
RNA-like or A-conformation more desir-
able for hybridization with RNA. This
criterion is met by the 2
-alkylated ODNs
and the LNAs, which indeed show high
thermal stability. Thus, there is a dilemma
in that one conformation is most suitable
for one of the desired properties that is, un-
fortunately, not optimal for the other. The
phosphorothioates apparently represent a
good compromise for these two qualities.
As mentioned above, the alkylated ana-
logues do not activate RNase as the 2
group interferes with recognition by this
enzyme or prevents substrate cleavage.
Neither the 2
alkylated analogues, the
alkylphosphonates, the morpholino, the
-phosphoramidates, nor the PNAs
activate RNase H, but they do show in-
hibition of gene expression. The simplest
explanation is that they follow the original
concept in physically blocking translation
by preventing the ribosome either to bind
at the entry site or to move along the
mRNA. Alternatively, the oligo may re-
place one strand in a duplex. Whether this
is a sufFcient explanation or whether other
factors are involved remains to be seen.
An important factor in obtaining efFcient
inhibition of gene expression is the po-
sitioning of the AS-ODN on the mRNA.
The secondary structure of the latter will
have regions that are more suitable than
others for the annealing process. Although
a number of computational methods exist
to predict such regions, many researchers
prefer experimental approaches to identify
such positions. The method based on the
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