Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo
| Autor: | Jeff S. Nelson, Claude Hélène, Michael R. H. White, David G. Spiller, Daniel Scherman, Carine Giovannangeli, Catherine Dubertret, Marcella Faria |
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| Rok vydání: | 2001 |
| Předmět: |
Untranslated region
Transcription Genetic Genetic Vectors Biomedical Engineering Cytomegalovirus Bioengineering Biology Transfection Applied Microbiology and Biotechnology Oligodeoxyribonucleotides Antisense Eukaryotic translation Genes Reporter Gene expression Animals Humans Simplexvirus Phosphoric Acids RNA Messenger Luciferases Reporter gene Messenger RNA Oligonucleotide RNA Phosphoramidate Thionucleotides Molecular biology Amides Coleoptera Biochemistry Gene Expression Regulation Molecular Medicine 5' Untranslated Regions Biotechnology HeLa Cells |
| Zdroj: | Nature biotechnology. 19(1) |
| ISSN: | 1087-0156 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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