Abstract
Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3′–P5′ phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5′-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.
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Acknowledgements
We wish to thank R.V. Giles and C.J. Ruddell for helpful technical assistance and discussions, A. Winter for generation of stable cell lines, G. Juslin for technical expertise in in vivo experiments. We thank S. Gryaznov, D. Lloyd, and J.K. Chen for providing the first samples of oligophosphoramidates employing the oxidative phosphorylation chemistry, and L. DeDionisio and A. Raible for many of the phosphoramidate syntheses by the phosphoramidite amine-exchange methods. This work was supported by Agence Nationale de Recherche sur le Sida, and Centre International des Etudiants et Stagiaires. M.F. is supported by Coordenaçao de Aperfeicoamento de Pessoal de nivel Superior.
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Faria, M., Spiller, D., Dubertret, C. et al. Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo. Nat Biotechnol 19, 40–44 (2001). https://doi.org/10.1038/83489
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DOI: https://doi.org/10.1038/83489
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