An Activatable siRNA Probe: Trigger-RNA-Dependent Activation of RNAi Function
An Activatable siRNA Probe: Trigger-RNA-Dependent Activation of RNAi Function
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DOI：
10.1002/anie.200903925
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发表时间：
2009-01-01
影响因子：
16.6
通讯作者：
Sando, Shinsuke
中科院分区：
文献类型：
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作者：
Masu, Hiroki;Narita, Atsushi;Sando, Shinsuke
随着RNA在复杂细胞系统中的作用不断被揭示，人们的兴趣集中在扩大RNA工具的范围，以人工控制细胞功能其中最有前途的工具是利用细胞RNA干扰（RNAi）的强大催化活性的基于小干扰RNA的调节剂。几个研究小组已经报道了RNAi[2,3]或dicer加工活性的触发依赖性控制方法。[4,5]然而，只有小分子被确定为成功的触发器，并且所有报道的程序都在关闭控制下运行，即RNAi活性被触发器与RNA探针的结合所抑制。更积极地使用这种基于RNAi的调节剂的一个挑战是建立一种策略来生产一种新的RNA工具，其RNAi活性可以通过触发依赖性的开启方式增强。在此，我们报道了一种新设计的合成发夹状（Hp）义链/反义链对作为可激活的小干扰RNA （siRNA）探针，并证明该系统能够通过RNA触发器首次开启RNAi活性控制。细胞质中的细胞RNAi过程是由双链RNA （dsRNA）的存在通过两个激活步骤启动的：1)dsRNA通过Dicer加工成具有两个核苷酸（nt）悬空的19-21碱基对（bp）义链（SS)/反义链（AS）， 2）加载到RNA诱导的沉默复合物（RISC）中以切割与AS具有互补序列的信使RNA (mRNA)（图1a）。我们专注于dicer处理步骤，并设计了一个可激活的siRNA系统，该系统包括Hp-SS和AS探针，用于以开启方式对siRNA功能的触发rna依赖控制（图1b）。Hp-SS探针是siRNA的SS结构域（红色）、调控茎结构域（粉色）和与触发RNA互补的识别环结构域（绿色）的合成融合体（黑色；图1b）。在缺乏触发RNA的情况下，调控系统被设计为抑制SS/AS杂交（OFF状态），而触发RNA与伴随RNA的结合
As the roles of RNA in complex cellular systems continue to be revealed, interest has focused on expansion of the repertoire of RNA tools for the artificial control of cellular functions.[1] Among the most promising tools are smallinterfering-RNA-based modulators that use the powerful catalytic activity of cellular RNA interference (RNAi). Several research groups have reported approaches to the trigger-dependent control of RNAi [2, 3] or Dicer-processing activity.[4, 5] However, only small molecules have been identified as successful triggers, and all of the reported procedures operate under turn-OFF control, whereby RNAi activities are suppressed by the binding of triggers to RNA probes. One challenge to the more active use of such RNAi-based modulators is the establishment of a strategy to produce a new RNA tool whose RNAi activity can be enhanced in a triggerdependent turn-ON manner. Herein, we report a newly designed synthetic hairpin-shaped (Hp) sense strand/antisense strand pair as an activatable small-interfering-RNA (siRNA) probe and demonstrate that this system enables the first turn-ON control of RNAi activity with an RNA trigger.The cellular RNAi process in the cytoplasm is initiated by the presence of double-stranded RNA (dsRNA) through two activation steps: 1) Dicer processing of dsRNA to a 19–21 base pair (bp) sense strand (SS)/antisense strand (AS) with a two-nucleotide (nt) overhang, and 2) loading into an RNA-induced silencing complex (RISC) to cleave messenger RNA (mRNA) that has a complementary sequence to the AS (Figure 1a). We focused on the Dicer-processing step and designed an activatable siRNA system comprising Hp-SS and AS probes for the trigger-RNA-dependent control of siRNA function in a turn-ON manner (Figure 1b). The Hp-SS probe is a synthetic fusion of an SS domain of siRNA (red), a regulatory stem domain (pink), and a recognition loop domain (green) complementary to the trigger RNA (black; Figure 1b). In the absence of a trigger RNA, the regulatory stem is designed to inhibit SS/AS hybridization (OFF state), whereas the binding of the trigger RNA with concomitant