Synthesis of nuclease-resistant siRNAs possessing universal overhangs

RNA interference (RNAi) induced by small interfering RNA (siRNA) has emerged as a powerful technique for the silencing of gene expression at the post-transcriptional level. It has been shown that in the RNAi machinery, the 3'-overhang region of a guide strand (an antisense strand) of siRNA is recognized by the PAZ domain in the Argonaute protein, and the 2-nucleotide (nt) 3'-overhang is accommodated into a binding pocket composed of hydrophobic amino acids in the PAZ domain. Based on this background information, we designed and synthesized siRNAs possessing aromatic compounds at their 3'-overhang regions. It was found that the modified siRNAs possessing aromatic compounds are more potent than the siRNAs without the 3'-overhang regions. Further, the silencing activities of the modified siRNAs are almost equal to those of normal siRNAs with natural nucleosides at their 3'-overhang regions. We also found that the siRNAs possessing the aromatic compounds at their 3'-overhang region could be used to inhibit hepatitis C virus (HCV) replication. Moreover, the RNAs with aromatic groups at their 3'-ends were more resistant to nucleolytic degradation by snake venom phosphodiesterase (SVPD) (a 3'-exonuclease) than natural RNAs. The aromatic compounds described in this report do not have functional groups capable of forming hydrogen bonds with nucleobases. Therefore, we expect that they can serve as the universal overhang units that can improve the nuclease resistance of siRNAs.