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

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.