RNA interference (RNAi) is now widely used to knockdown gene expression in a sequence-specific manner, making it a powerful tool not only for studying gene function, but also for therapeutic purposes, including antiviral treatments (1
). Currently, the replication of a wide range of viruses can be inhibited successfully using RNAi, with both short interfering RNAs (siRNAs) and siRNA expression vectors (5
In mammalian RNAi, the efficacy of each siRNA varies widely depending on its sequence; only a limited fraction of randomly designed siRNAs is highly effective. Many experiments have been conducted to clarify possible sequence requirements of functional siRNAs. Of these, our work incorporates guidelines from three major studies (6
) of selecting functional siRNAs. However, designing functional siRNAs that target viral sequences is problematic because of their extraordinarily high genetic diversity. For example, about 500 entries of near full-length sequences of HIV-1 group M, which is largely responsible for global pandemic, are stored in the sequence databases, but it proved impossible to select a common 21mer from among all of them. Moreover, RNAi-resistant viral mutants achieved through point mutation or deletion emerge rapidly when targeting viruses in cell culture. These problems suggest a strong need to select highly conserved target sites for designing antiviral siRNAs. Furthermore, the off-target silencing effects of siRNA are also a serious problem that could affect host gene expression (9
). Off-target silencing effects arise when an siRNA has sequence similarities with unrelated genes. In antiviral RNAi, it is desirable to minimize off-target effects against human genes.
Consequently, only a limited fraction of 21mers is suitable for use as antiviral siRNAs. In this study, we developed a novel web-based online software system, siVirus, which provides functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences.