408
Antisense Oligonucleotides as Potential Drugs
stable to degradation by nucleases. As in
the phosphorothioates, the methylphos-
phonates
exist
as
two
diastereomers
of
the
Rp
or
the
Sp
conFguration.
Uniformly
methylphosphonate-modiFed
oligonucleotides do not form duplexes
with RNA to induce RNase H cleavage.
However, chimeric oligonucleotides con-
taining both methylphosphonate and a
window of at least Fve to seven phospho-
diester linkages retain RNase H activity.
This class of ODNs has not been very
useful for the inhibition of gene ex-
pression. Besides the lack of RNase H
activity, it also turned out that, contrary
to expectation, cellular uptake is more
sluggish than that of ODNs with a neg-
ative charge.
2.4
2
0
-
O
-Modifcation
In order to limit the number of phos-
phorothioates in an AS-ODN, alternative
modiFcations were sought to enhance en-
zymatic stability without the inherent side
effects of the phosphorothioates. It was
recognized early that alkylation of the 2
0
position of the ribose indeed increases sta-
bility. Thus, 2
0
-
O
-methyl modiFcation was
used, followed by the 2
0
-
O
-methoxyethyl
derivatives. These analogues adopt an
RNA-like (A-form) conformation, assuring
high binding afFnities for the RNA target
and thus being superior in this respect to
the phosphorothioates. As such, 2
0
modi-
Fcation does not support RNase H activity
(see below). The general policy is to locate
them at the flanks of an AS-ODN, leaving
a gap of approximately six to eight phos-
phorothioate nucleotides in the center to
take advantage of RNase H activity. Thus,
the second-generation oligos of the ISIS
company have 2
0
-methoxy-ethoxy moiety
termed
MOE-Oligos
. Often, particularly
for animal model application, even the
flanks contain the phosphorothioates to
further improve stability. Apparently, in
conjunction with a 2
0
alkyl modiFcation,
the cytotoxic side effects of phosphoroth-
ioates are not severe.
2.5
Sterically Locked Nucleic Acids
Nucleic acid analogues with conformation-
ally restricted sugar–phosphate backbones
have been attractive as it was envisaged
that they might form more-stable du-
plexes for entropy reasons. Thus, the
DNA analogue bicyclo-[3.2.1]-DNA has a
rigid phosphodiester backbone, emulat-
ing a B-DNA-type conformation, to which
the nucleobases are attached via a flexi-
ble open-chain linker. Although bicyclo-
[3.2.1]-DNA forms less-stable duplexes
with complementary DNA than natural
DNA, base-mismatch discrimination is
slightly enhanced compared to pure DNA
duplexes. Importantly, bicyclo-[3.2.1]-DNA
oligomers are resistant to 3
0
-exonuclease
degradation. None of these analogues has
been tested for inhibition of gene expres-
sion yet.
A
further
development
along
these
lines are the locked nucleic acids (LNA),
which represent a totally new and very
promising class of compounds. In these
analogues, the 2
0
oxygen is linked by a
methylene bridge to the 4
0
carbon, forming
a methylene-linked bicyclic ribofuranosyl
nucleoside. These analogues, like the 2
0
-
O
-alkylated oligoribonucleotides, adopt an
RNA-like (A-form) conformation and thus
bind very well to target RNA. Probably
not surprisingly, they are not substrates
for nucleases. Oligonucleotides consisting
purely of LNA units do not activate RNase
H, but LNA/DNA chimeras do. Thus,
these analogues combine many favorable
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