Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri, Kansas City, 2464 Charlotte Street, Kansas City, Missouri 64108-2718, United States.
J Am Chem Soc. 2011 Jan 26;133(3):440-6. doi: 10.1021/ja107226e. Epub 2010 Dec 16.
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.
基因表达的时空和量对于包括发育在内的多种生物过程至关重要。出于这个原因，人们已经尝试了许多方法来控制基因表达。我们之前已经证明，通过使用末端磷酸基团被二甲氧基硝基苯乙基（DMNPE）基团修饰的 siRNA 和 dsRNA，可以用光来控制 RNA 干扰（RNAi）。照射后，这些基团会光解并释放出天然 RNA。迄今为止，光激活 RNA 干扰（LARI）的主要问题是，所使用的基团在照射前只能部分阻断 RNA 干扰，从而限制了该方法的应用。在这里，我们描述了一种新的光可裂解基团，环十二烷基 DMNPE（CD-DMNPE），旨在在照射前完全阻断与负责 RNA 干扰的细胞机制相互作用的双链体。这使我们能够用光从正常切换到接近完全降低基因表达，并在细胞单层中构建定义明确的基因表达模式。由于这种方法是基于 RNA 干扰途径，它受益于能够快速识别在低浓度或亚纳摩尔浓度下有效的双链体的能力。此外，它允许靶向内源性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结合将允许在多种生物系统中轻松控制基因表达的时空和程度。
