Antisense Oligonucleotides as Potential Drugs
409
features for application. Although some
cell culture data have been published,
further studies should demonstrate the
advantages of this class of compounds in
a wider setting, in particular, in animal
model studies.
2.6
N3
0
-P5
0
-Phosphoramidates
Oligonucleotides based on this structural
element also show remarkable resistance
to nucleases but do not activate RNase H.
Extensive studies for the inhibition of gene
expression are still awaited.
2.7
Morpholino Oligonucleotides
This class of compounds again is RNase
H–irresponsive but, of course, is very
stable to nucleases. It has found acceptance
mainly in developmental studies in the
zebra Fsh and Xenopus, both systems in
which the compounds can be administered
(injected) to the embryo. ±or other
in vivo
studies, uptake of the compound seems to
be limited, as discussed below.
2.8
Peptide Nucleic Acids (PNA)
Peptide nucleic acids (PNA) deviate from
all other analogues in that the linkage
between the monomeric units is a peptide
bond and not a phosphodiester bond.
They show
excellent afFnity
to
RNA
targets and thus should be ideally suited
for interference with gene expression,
even though they cannot rely on RNase
H activity to destruct the target. Some
cell culture data have been published,
but further studies are desired to fully
appreciate
the
potential
of
this
class
of compounds.
2.9
Oligonucleotide Conjugates
Certain properties of ODNs can be in-
fluenced by the covalent attachment of
nonnucleosidic moieties to either the 3
0
or
5
0
ends. This type of derivatization allows
modulation of nuclease stability, cellular
uptake, and organ distribution of ODNs.
In addition, the internucleoside linkages,
the C5 position of pyrimidine bases, and
the 2
0
position of ribose are also positions
for derivatisation, but these will not be
considered here.
2.10
5
0
-end Conjugates
Conjugation of molecules to the 5
0
end
of ODNs is straightforward by coupling
a
phosphoramidite
or
H-phosphonate
derivative of the desired molecule to the 5
0
-
hydroxy group of the oligomer, following
chain elongation by solid-phase synthesis.
A broad range of phosphoramidite deriva-
tives of ligands, such as fluorescein, biotin,
cholesterol, cholic acids, dinitrophenyl,
acridine, and psoralen derivatives, are
commercially available. Alternatively, the
5
0
-terminal hydroxy group of the oligonu-
cleotide is reacted with an aminoalkyl
linker phosphoramidite, which, after de-
protection, results in a free aminoalkyl
function. This amino function can then
be reacted in solution with suitably acti-
vated conjugate molecule derivatives, such
as active esters, isothiocyanates, or iodoac-
etamides.
2.11
3
0
-end Conjugates
The conjugation of molecules to the 3
0
end
of oligonucleotides is achieved by using ap-
propriately functionalized solid supports
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