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.
Highlights
The application of complementary DNA or RNA molecules or their derivatives for the modulation biological functions of specific RNA(s) is referred to as antisense technology
To analyze the effects of 8-oxo-dG residues on the binding of Antisense oligonucleotides (ASOs) to DNA and RNA molecules, a set of all-DNA oligonucleotides was prepared in which none, one, or two of the centrally located dG residues were substituted with 8-oxo-dG residues
ASOs with a very high Tm tend to bind to secondary targets, and their ability to trigger ribonuclease H (RNase H)-mediated target RNA degradation may be reduced or even completely abolished [25]
Summary
The application of complementary DNA or RNA molecules or their derivatives for the modulation biological functions of specific RNA(s) is referred to as antisense technology. This mechanism is not associated with the destruction of targeted molecules, and, it is most effective for coding RNAs if the ASO target site overlaps with or is located upstream of the initiation codon [2]. The second mechanism relies on the ability of ribonuclease H (RNase H), a ubiquitous group of cellular enzymes, to cleave the RNA part of the heteroduplexes formed between DNA ASOs and targeted RNA [3,4]. This mechanism results in the degradation of the targeted RNA and is effective regardless of the position of the ASO binding site [2]
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