Antisense oligonucleotides (ON) allow the specific control of gene expression and phosphorothioate derivatives are currently being evaluated for possible clinical applications. Numerous second generation ON analogues with improved pharmacological properties have been described. Most of them, however, do not recruit RNase H, which is known to increase ON potency by eliciting the specific degradation of the target RNA. Silverman, Torrence and colleagues have conjugated 2,5A to natural antisense ON and demonstrated the preferential cleavage of a target RNA in cell-free and intact cell experiments. We have established for the first time that RNase H-incompetent ON, viz. alpha-anomeric ON analogues, can be converted into sequence-specific nucleases upon conjugation to 2,5A. The use of alpha-ON- and beta-ON-2,5A chimeras has allowed us to delineate the part played by RNase H and RNase L in target RNA degradation and translation arrest. Finally, the present studies have revealed limitations which are encountered in the choice of a suitable target for such ON-2,5A chimeras.

Animals↗

PubMedDate not supplied

Biological activity of oligonucleotide-poly(L-lysine) conjugates: mechanism of cell uptake.

We have previously shown that antisense oligomers linked to poly(L-lysine) (PLL) exhibit antiviral properties against vesicular stomatitis virus (VSV) at concentrations lower than 1 microM. The conjugation to PLL provides an interesting alternative to natural or neutral oligomers to increase the biological effects of antisense oligomers. The internalization pathway of oligomer-PLL conjugates as compared to unconjugated oligomers has been studied in L929 cells. In parallel to their enhanced antiviral activity, PLL increases greatly the uptake of fluorescently tagged oligomers. This internalization follows a classical endocytic pathway and the oligomer has to be cleaved from PLL in the cell to exhibit an antiviral effect.

Animals↗

PubMedDate not supplied

Oligonucleotide-poly(L-lysine)-heparin complexes: potent sequence-specific inhibitors of HIV-1 infection.

Poly(L-lysine)-conjugated oligonucleotides complementary to the translation initiation region of the tat protein were tested for their capacity to inhibit HIV-1 replication in de novo infected cells. Sequence-specific antiviral effects were observed with these conjugates at 0.5 microM; their activity was transient, and the viral production was only delayed for a few days. Interestingly, their efficiency was significantly increased by the addition of heparin, a sulfated polyanion that also presents antiviral properties against HIV-1. A single addition, at the time of virus exposure, of the ternary complex formed between oligonucleotide-poly(L-lysine) (75 nM) and heparin (50 micrograms/mL) totally protects cells from HIV-1 infection. Primary interference with virus adsorption is essential for the strong antiviral effect. However, this protection remains strictly sequence specific as demonstrated in experiments performed with different HIV-1 isolates. As comparison, treatments that combine AZT and heparin at the same concentrations did not promote such a complete protection.

Acquired Immunodeficiency Syndrome↗

PubMedDate not supplied

Uptake of oligonucleotides by keratinocytes.

Oligonucleotides (ODNs) conjugated to rhodamin (Rh) and 4-[(N-2-chloroethyl-N-methyl)amino] benzylamine were used to investigate ODNs transport into keratinocytes. Affinity labeling of two proteins, 63 and 35 kDa, and the inhibition of the affinity labeling and ODNs uptake by the cells in the presence of nucleic acids, polyanions and trypsin suggest, that the proteins are involved in transport of nucleic acids in keratinocytes.

Autoradiography↗