%0 Journal Article
%T Effect of small interfering RNA 3'-end overhangs on chemosensitivity to thymidylate synthase inhibitors
%A John C Schmitz
%A Edward Chu
%J Silence
%D 2011
%I BioMed Central
%R 10.1186/1758-907x-2-1
%X It was recently shown that extension of the 3' end to five or eight dT molecules allows siRNAs to be effectively complexed with linear polyethylenimine (PEI), leading to enhanced cellular uptake and intracellular release. Here, we provide further evidence that only extended or 'sticky' siRNAs complexed with PEI result in significant target knockdown. However, when investigating the potential effects of these extended siRNAs on growth of human colon cancer RKO cells, we observed a dose-dependent reversal of cytotoxicity of a thymidylate synthase-targeted siRNA. In contrast, siRNAs with uridine overhangs maintained their growth inhibitory effects. We further demonstrated that dT-containing siRNAs prevented the cytotoxic effects of thymidylate synthase (TS) inhibitor compounds, such as ZD1694 and 5'-fluoro-deoxyuridine, while having no deleterious effect on cisplatin toxicity. We show that this rescue effect results from the rapid degradation of the siRNA.Given that TS is an important enzyme for cell growth and proliferation and that its expression is controlled by multiple pathways, the rescue of its growth inhibitory effects may have unintended consequences. As siRNAs are being developed as therapeutic molecules, it will be important to avoid such off-target effects due to dT release. Hence, siRNAs should contain only uridine residues in their 3'-end overhangs.Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) [1]. Once synthesized, dTMP is subsequently metabolized intracellularly to deoxythymidine triphosphate (dTTP), a key nucleotide for DNA replication and repair. Although dTMP can be formed via the salvage pathway, a reaction catalyzed by thymidine kinase, the TS-catalyzed reaction provides the only intracellular de novo source of dTMP. As such, inhibition of this enzymatic step results in suppression of cellular growth and proliferation. Given
%U http://www.silencejournal.com/content/2/1/1