Effect of small interfering RNA 3'-end overhangs on chemosensitivity to thymidylate synthase inhibitors

Abstract

BackgroundSmall interfering RNAs (siRNAs) are double-stranded RNAs that effectively inhibit expression of its complimentary target mRNA. Standard siRNAs contain two nucleotide overhangs on their 3' end. While these overhangs are usually comprised of deoxythymidines (dT), it has been shown that any nucleotide can be used on the 3' end without affecting RNAi silencing.ResultsIt 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.ConclusionsGiven 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.

Development of modified siRNA molecules incorporating 5-fluoro-2'-deoxyuridine residues to enhance cytotoxicity

“…After transfection of TS6-dU/dA, antisense strand targeting was maintained, whereas sense strand targeting was significantly reduced. This finding suggests that the 3′-end dU modification interferes with incorporation of the sense strand into RISC and provides an explanation for the slightly greater knockdown of TS previously observed with this ssiRNA (17). Transfection of the modified ssiRNA TS6-FdU/dA resulted in similar strand selection as with TS6-dU/dA.…”

“…We had previously shown that the activity of siRNAs with extended complementary 3′-end overhangs [termed ‘sticky’ siRNAs or ssiRNAs (20)] was at least equivalent or perhaps slightly more effective than standard siRNAs with respect to target mRNA suppression (17). However, in these studies, we determined that the extended dT overhangs on the ssiRNA were degraded, resulting in rescue from the cytotoxic effects of a TS-targeted ssiRNA.…”

“…In earlier studies, we had shown that traditional siRNAs and ssiRNAs with 3′-end dT overhangs are rapidly degraded after transfection with intracellular release of dT and other nucleosides (17). The release of these nucleosides was sufficient to rescue from the growth inhibitory effects of a TS-targeted siRNA.…”

“…RNAs were resuspended in RNase-free water and allowed to anneal for 30 min at room temperature before aliquots were stored at −80°C. Synthesis of TS6 ssiRNA has been previously described (17). All TS6 siRNA/ssiRNA sequences are listed in Table 1.…”

“…We have previously demonstrated that the growth inhibitory effects of a specific TS-targeted siRNA was completely reversed by thymidine, suggesting that the siRNA specifically targets TS with minimal off-target effects on other genes that might impact cell growth and proliferation (11). Recent studies from our laboratory have shown that the intracellular degradation of siRNA released dT nucleosides from the 3′-end overhang, which, in turn, rescued against the cytotoxicity resulting from TS inhibition (17). This dT release was able to reverse the growth inhibitory effects of TS siRNA as well as the cytotoxic effects of small molecule inhibitors of TS, such as raltitrexed and FdU.…”