RNAi silencing of exogenous and endogenous reporter genes using a macrocyclic octaamine as a "compact" siRNA carrier. Studies on the nonsilenced residual activity.
RNAi silencing of exogenous and endogenous reporter genes using a macrocyclic octaamine as a "compact" siRNA carrier. Studies on the nonsilenced residual activity.
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DOI：
10.1021/bc050112l
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发表时间：
2006
影响因子：
4.7
通讯作者：
K. Matsui;Shohei Horiuchi;S. Sando;T. Sera;Y. Aoyama
中科院分区：
文献类型：
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作者：
K. Matsui;Shohei Horiuchi;S. Sando;T. Sera;Y. Aoyama
介绍了一种具有共价连接脂束结构的大环辛胺-1作为一种新型的siRNA载体。凝胶电泳、DLS和SPR结果表明，它与荧光素酶靶向的21-聚(42P)siRNA强烈结合，其比例为1/P=0.3(1/N=2.4)，得到了非常致密的平均尺寸约为10 nm的1-siRNA复合物。1介导的外源荧光素酶基因的siRNA沉默效率为90-95%。无论细胞类型(HeLa、HepG2或HEK293)、siRNA和荧光素酶编码质粒的供应顺序或时机(逐步或同时)、转录的mRNA水平或载体的类型(1vs Lipofetamine2000)，通常都能观察到饱和区残留活性为5-10%的总体抑制[siRNA]谱。稳定整合在HeLa细胞基因组中的内源性DsRed2基因的沉默也具有类似的整体特征。这些结果表明：(1)细胞对质粒的摄取和对siRNA的摄取基本上是相互独立的；(2)不完全沉默并不是由于siRNA递送不足所致。简要讨论了项目2的含义。
A macrocyclic octaamine 1 having a covalently linked lipid-bundle structure was introduced as a new type of siRNA carrier. Gel electrophoresis, DLS, and SPR results indicate that it strongly binds to a luciferase-targeting 21-mer (42P) siRNA with a ratio of 1/P congruent with 0.3 (1/N congruent with 2.4) to give remarkably compact 1-siRNA complexes with an average size of approximately 10 nm. The 1-mediated siRNA silencing of the exogenous luciferase gene occurs with a 90-95% efficiency. The overall suppression-[siRNA] profile with a 5-10% residual activity in the saturation region is commonly observed irrespective of the cell type (HeLa, HepG2, or HEK293), the order, or timing (stepwise or simultaneous) of supply of the siRNA and that of the luciferase-encoding plasmid, the level of mRNA transcribed, or the type of carriers (1 vs lipofectamine 2000). The silencing of the endogenous DsRed2 gene stably incorporated in the genome of HeLa cells also has a similar overall profile. These results suggest that (1) the cellular uptake of the plasmid and that of the siRNA are basically independent of each other and (2) the incomplete silencing is not due to insufficient siRNA delivery. Implication of item 2 is briefly discussed.