RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications |
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| Authors: | Margit Mutso Andrei Nikonov Arno Pihlak Eva ?usinaite Liane Viru Anastasia Selyutina T?nu Reintamm Merike Kelve Mart Saarma Mati Karelson Andres Merits |
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| Affiliation: | 1. Institute of Technology, University of Tartu, Tartu, Estonia.; 2. GeneCode, Ltd., Tallinn, Estonia.; 3. Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia.; 4. Institute of Biotechnology, University of Helsinki, Helsinki, Finland.; 5. Institute of Chemistry, University of Tartu, Tartu, Estonia.; International Centre for Genetic Engineering and Biotechnology, ITALY, |
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| Abstract: | The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2’-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum. |
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