Assessment of high-affinity hybridization, RNase H cleavage, and covalent linkage in translation arrest by antisense oligonucleotides

Gee, J. E.; Robbins, I.; van der Laan, A. C.; van Boom, J. H.; Colombier, C.; Leng, M.; Raible, A. M.; Nelson, J. S.; Lebleu, B.

Antisense Nucleic Acid Drug Dev

1998-04 / vol 8 / pages 103-11


Antisense oligonucleotides (ONs) are designed to hybridize target mRNA in a sequence-specific manner and inhibit gene expression by preventing translation, either by activation of RNase H or steric blockage of the ribosome complex. Second-generation ONs, which possess greater binding affinity for target RNA relative to the isosequential phosphodiester (PO) ONs, have been developed and include, among others, peptide nucleic acids (PNA) and N3′ P5′ phosphoramidate oligonucleotides (npONs). In the present study, PNA and npON derivatives were targeted to the coding portion of the complementary mRNA of the N protein of the vesicular stomatitis virus (VSV) in order to evaluate their ability to arrest translation in an in vitro rabbit reticulocyte lysate system. High-affinity hybridization of ONs lacking RNase H activity was not sufficient to block translation in this test system. Only antisense ONs acting via an RNase H mechanism or by steric hindrance through covalent attachment (via transplatin modification) to the target mRNA were found to definitively arrest translation in this study.

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Animals; Mice; Temperature; Structure-Activity Relationship; Rabbits; L Cells (Cell Line); Cell-Free System; Macromolecular Substances; Protein Biosynthesis/*drug effects; Nucleic Acid Denaturation; *Nucleic Acid Hybridization; *Nucleocapsid Proteins; Molecular Structure; Nucleocapsid/*genetics; Oligonucleotides, Antisense/*pharmacology; Reticulocytes/drug effects/metabolism; Ribonuclease H/*pharmacology; Ribosomes/physiology; RNA, Messenger/*antagonists & inhibitors/genetics

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