Laboratory of Molecular Cell Biology, Department of Life Science, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, Japan.
Mol Pharm. 2011 Dec 5;8(6):2193-203. doi: 10.1021/mp200250f. Epub 2011 Oct 24.
Short interfering RNA (siRNA) technology is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with palmitic acid at the 5'-end of the sense strand (C16-siRNAs) using our novel synthesis strategy in order to improve the potency of siRNA. The C16-siRNAs exhibited enhanced nuclease stability. In addition, they showed potent gene-silencing efficacy against exogenous Renilla luciferase in HeLa cells compared with a nonmodified siRNA in the presence of Lipofectamine 2000. The C16-siRNAs also had a more potent inhibitory effect on Renilla luciferase activity than the other siRNA conjugated with lipids at the 5'-end and the 3'-end by palmitoyl conjugation. For further improvement, the gene silencing potency of the C16-siRNAs against the endogenous vascular endothelial growth factor (VEGF) gene in HeLa cells was investigated. In this investigation, the siRNAs were prepared not only with the normal RNA sequence but also coupled with an inverted thymidine (idT) at the 3'-ends of both the sense and antisense strands (siRNA-idT), including palmitic acid conjugations at the 5'-end of the sense strand, to improve stability. The C16-siRNA including idT modifications exhibited a significantly greater inhibitory effect on the VEGF gene in the presence of Lipofectamine 2000. It is noteworthy that C16-siRNA-idT demonstrated long-term gene-silencing efficacy of up to 5 days. Interestingly, the C16-siRNAs, including that with idT modifications, exhibited strong RNAi potency in the absence of any transfection reagents, although only at high concentrations. Both the C16-siRNAs and C16-siRNA-idT induced a high level of membrane permeability in HeLa cells. Our developed C16-siRNAs, particularly C16-siRNA-idT, are thus among the promising candidates for a new generation of modified siRNAs that can solve the many problems associated with siRNA technology.
短干扰 RNA（siRNA）技术是一种在哺乳动物细胞中抑制基因表达的强大工具。在这项研究中，我们专注于使用我们的新型合成策略开发在正义链 5'-端连接棕榈酸的 siRNA（C16-siRNA），以提高 siRNA 的效力。C16-siRNA 表现出增强的核酸酶稳定性。此外，与存在 Lipofectamine 2000 时未修饰的 siRNA 相比，它们在 HeLa 细胞中外源 Renilla 荧光素酶的基因沉默效果更强。C16-siRNA 对 Renilla 荧光素酶活性的抑制作用也比通过棕榈酰化连接在 5'-端和 3'-端连接脂质的其他 siRNA 更强。为了进一步提高效率，研究了 C16-siRNA 对 HeLa 细胞内源性血管内皮生长因子（VEGF）基因的基因沉默效力。在这项研究中，不仅制备了具有正常 RNA 序列的 siRNA，还在正义链和反义链的 3'-末端连接了反向胸腺嘧啶（idT）（包括在正义链 5'-端连接棕榈酸），以提高稳定性。包含 idT 修饰的 C16-siRNA 在存在 Lipofectamine 2000 时对 VEGF 基因的抑制作用显著增强。值得注意的是，C16-siRNA-idT 表现出长达 5 天的长期基因沉默效果。有趣的是，包括带有 idT 修饰的 C16-siRNA 在内，在没有任何转染试剂的情况下表现出很强的 RNAi 效力，尽管仅在高浓度下。C16-siRNA 和 C16-siRNA-idT 都在 HeLa 细胞中诱导了高水平的膜通透性。因此，我们开发的 C16-siRNA，特别是 C16-siRNA-idT，是一种很有前途的新一代修饰 siRNA 候选物，可以解决与 siRNA 技术相关的许多问题。
