Single-Stranded Silencing RNAs: Hit Rate and Chemical Modification.

Single-stranded silencing RNAs (ss-siRNAs) are chemically modified single-stranded oligomers that engage the RNA interference machinery normally used by duplex RNAs to silence gene expression. ss-siRNAs have the potential to combine advantages of antisense oligonucleotides and siRNAs. Previous work has explored the chemistry of the phosphate and the oligonucleotide body. We now describe the process of attempting to develop and optimize ss-siRNAs based on five active siRNA duplexes. Three of the sequences failed to show any activity as ss-siRNAs, and in two of those cases the ss-siRNAs showed significantly increased toxicity relative to the parent duplexes. For the two sequences that did work well as ss-siRNAs, we show that the chemistry of the 3'-terminal dinucleotide also has a significant effect on the potency of ss-siRNAs. Previously published work on ss-siRNAs has been based on a 2'-O-methoxyethyl-RNA (MOE) dinucleotide at the 3'-terminus. To our surprise, oligomers containing 2'-O-Me-RNA modifications at the 3'-terminus showed significantly improved potency and activity relative to those modified with MOE at the same sites. Oligonucleotides with two locked nucleic acid units at the 3'-terminus showed improved activity over the MOE-modified analog for one sequence. Importantly, the fact that 2'-O-Me-RNA works so well makes the ss-siRNA approach accessible to a wider range of researchers since it can be achieved with inexpensive commercially available modifications.