Patterning of Gene Expression Using New Photolabile Groups Applied to Light Activated RNAi
Patterning of Gene Expression Using New Photolabile Groups Applied to Light Activated RNAi
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DOI：
10.1021/ja107226e
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发表时间：
2011-01-26
影响因子：
15
通讯作者：
Friedman, Simon H.
中科院分区：
文献类型：
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作者：
Jain, Piyush K.;Shah, Samit;Friedman, Simon H.
基因表达的间隔、时间和数量对于包括发育在内的一系列生物过程至关重要。出于这个原因，已经有许多尝试将基因表达置于光的控制之下。我们之前已经表明，RNA干扰（RNAi）可以通过使用siRNA和dsRNA来控制，这些siRNA和dsRNA的末端磷酸盐被二甲氧基硝基苯乙基（DMNPE）基团修饰。在辐射下，这些基团光解并释放天然RNA。迄今为止，光激活RNA干扰（LARI）的主要问题是，在照射之前，所使用的基团仅部分阻断RNA干扰，从而限制了该方法的实用性。在这里，我们描述了一个新的光裂解组，环十二烷基DMNPE（CD-DMNPE），旨在完全阻断双链体与负责RNA干扰的细胞机器的相互作用照射前。这使我们能够从正常切换到使用光的基因表达几乎完全减少，并在细胞单层中构建明确的基因表达模式。因为这种方法是建立在RNA干扰途径上的，所以它受益于快速鉴定在低或亚纳摩尔浓度下有效的双链体的能力。此外，它允许靶向内源基因而无需额外的遗传操作。最后，由于CD-DMNPE的区域特异性，它允许在单个步骤中快速修饰标准双链体。其功效和易于应用的组合将允许在一系列生物系统中容易地控制基因表达的间隔、时间和程度。
The spacing, timing, and amount of gene expression are crucial for a range of biological processes, including development. For this reason, there have been many attempts to bring gene expression under the control of light. We have previously shown that RNA interference (RNAi) can be controlled with light through the use of siRNA and dsRNA that have their terminal phosphates modified with the dimethoxy nitro phenyl ethyl (DMNPE) group. Upon irradiation, these groups photolyze and release native RNA. The main problem with light activated RNA interference (LARI) to date is that the groups used only partially block RNA interference prior to irradiation, thus limiting the utility of the approach. Here, we describe a new photocleavable group, cyclo-dodecyl DMNPE (CD-DMNPE), designed to completely block the interaction of duplexes with the cellular machinery responsible for RNA interference prior to irradiation. This allowed us to switch from normal to a near complete reduction in gene expression using light, and to construct well-defined patterns of gene expression in cell monolayers. Because this approach is built on the RNA interference pathway, it benefits from the ability to quickly identify duplexes that are effective at low or subnanomolar concentrations. In addition, it allows for the targeting of endogenous genes without additional genetic manipulation. Finally, because of the regiospecificity of CD-DMNPE, it allows a standard duplex to be quickly modified in a single step. The combination of its efficacy and ease of application will allow for the facile control of the spacing, timing, and degree of gene expression in a range of biological systems.