Increasing the Potency of RNA Interference Using RNA Mimics

In 2002 the journal Science described RNA interference (RNAi) as the biggest scientific breakthrough of the year and since then it has remained one of the hottest topics under investigation, culminating in the award of the 2006 Nobel Prize to its discoverers. Excitement over this technique derives from the ease with which it can be used to switch off a specific gene in almost any organism, thereby allowing the role of that gene to be identified. More importantly, it offers the potential to treat certain diseases by switching off the causative genes. RNAi appears to be an ancient defence mechanism against double-stranded RNA (dsRNA) in which dsRNA is chopped into the smaller siRNAs by the enzyme called Dicer. The so-called antisense strand of the siRNA is used by a RNA-induced silencing complex (RISC) to direct mRNA cleavage. However, as an alternative to the production of the siRNA by Dicer, chemically synthesised siRNA of any sequence can be prepared and introduced into the cells. It is the use of synthetic RNAs that provide the flexibility such that any mRNA can be targeted. Whilst siRNAs derived from natural RNA show relatively good activity in gene silencing it is clear that synthetic RNA mimics that retain the natural conformation of RNA, but have increased nuclease resistance will improve the efficacy of RNAi. We propose to use 3'-S-phosphorothiolate analogues of the phosphodiester backbone, which are established mimics of RNA, to improve the efficacy of RNAi. The programme of research will involve the synthesis of siRNA duplexes, partially and fully substituted with the 3'-S-phosphorothiolate linkage. To enhance our understanding of the RNAi process and the design of siRNA constructs, we will correlate thermal duplex stability with the potency of gene knockout. We will also study the toxicity and cellular uptake of the modified siRNAs as compared to the unmodified systems.