Synthesis of nuclease-resistant siRNAs possessing benzene-phosphate backbones in their 3′-overhang regions

We describe the synthesis and silencing activities of siRNA possessing N1-[3,5-bis(hydroxymethyl)phenyl]thymine (bt) in their 3′-overhang regions. We found that an siRNA possessing bt in the 3′-overhang region was more effective than an siRNA with natural nucleosides and the siRNA possessing 1,3-bis(hydroxymethyl)benzene (b) without a nucleobase at the 3′-overhang region in in vitro experiment using HeLa cells system. Furthermore, the RNA possessing bt at its 3′-end was more resistant to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3′-exonuclease) than the RNA possessing the natural nucleoside 2′-deoxythymidine at the 3′-end. Thus, the compound bt will be a novel 3′-overhang moiety that can enhance the silencing activity and nuclease-resistant property of siRNAs.