Light controllable siRNAs regulate gene suppression and phenotypes in cells
Light controllable siRNAs regulate gene suppression and phenotypes in cells
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DOI：
10.1016/j.bbamem.2006.01.003
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发表时间：
2006-03-01
影响因子：
3.4
通讯作者：
McMaster, Gary K.
中科院分区：
文献类型：
--
作者：
Nguyen, Quan N.;Chavli, Rajesh V.;McMaster, Gary K.
小干扰RNA（siRNA）是一种有效的靶向基因沉默工具。然而，siRNA基因沉默发生在转染过程中，限制了其在动力学研究中的使用，解释了毒性和脱靶效应以及需要时间和/或空间调节的表型测定。我们开发了一种新型的可控siRNA（csiRNA），它是由光激活的。在寡核苷酸合成期间，单个光可去除基团偶联至siRNA的反义链的5'末端，其阻断siRNA的活性。低剂量的光激活siRNA，不依赖于转染，导致特异性靶mRNA和蛋白质（GAPDH、p53、存活素、hNuf 2）的敲低，而不刺激非特异性作用，如调节的蛋白激酶PKR和干扰素应答的诱导。我们证明了生存素和hNuf 2 csiRNA暂时敲低它们的mRNA，分别通过有丝分裂停滞引起多核化和细胞死亡。此外，我们证明了hNuf 2 csiRNA活性和由此产生的表型的剂量依赖性光调节。使用包括基于MPG肽的递送系统的市售试剂将光可控siRNA引入细胞中。csiRNA在其转染效率和基因沉默效力方面与标准siRNA相当。这种技术应该是感兴趣的表型测定，如细胞存活，细胞周期调控和细胞发育。(c)2006 Elsevier B. V.保留所有权利。
Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5' end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development. (c) 2006 Elsevier B.V. All rights reserved.