Conditional Dicer substrate formation via shape and sequence transduction with small conditional RNAs.
Conditional Dicer substrate formation via shape and sequence transduction with small conditional RNAs.
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DOI：
10.1021/ja404676x
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发表时间：
2013-11-20
影响因子：
15
通讯作者：
Pierce NA
中科院分区：
文献类型：
--
作者：
Hochrein LM;Schwarzkopf M;Shahgholi M;Yin P;Pierce NA
由小干扰RNA(SiRNAs)介导的RNA干扰(RNAi)使选择的基因能够被击倒，执行逻辑操作：沉默基因Y。siRNA在结构上是活跃的事实是一个重要的限制，使得很难将击倒限制在特定的位置和时间。为了实现对沉默的时空控制，我们试图设计小的条件RNAs(ScRNAs)来介导与逻辑操作相对应的条件RNAi：如果基因X被转录，沉默独立的基因Y。通过适当地选择基因X，基因Y的敲除可以以组织和时间特异性的方式受到限制。为了实现条件RNAi的逻辑，我们的方法是设计scRNAs，当它与mRNAs‘检测靶’X结合时，进行形状和序列转导，形成靶向独立的mRNAs‘沉默靶’Y的DICER底物，随后的DICER加工产生靶向mRNAY的siRNA用于破坏。为此，我们在这里设计并实验验证了用于条件性底物形成的各种scRNA机制。试管研究显示出强烈的OFF/ON条件性反应，在存在同源mRNA检测目标的情况下，底物产量至少增加了一个数量级。通过适当地确定scRNA的尺寸和/或对scRNA进行化学修饰，只有信号转导的产物，而不是反应物或中间体，才能被Dier有效地处理，从而产生siRNAs。这些机制研究探索了工程单链RNA信号转导级联的不同设计原则，包括反应物稳定性与亚稳性、催化转导与非催化转导、转录前与转录后转导、反应物与产物的分子性以及分子自组装和拆解模式。
RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) enables knockdown of a gene of choice, executing the logical operation: silence gene Y. The fact that the siRNA is constitutively active is a significant limitation, making it difficult to confine knockdown to a specific locus and time. To achieve spatiotemporal control over silencing, we seek to engineer small conditional RNAs (scRNAs) that mediate ‘conditional RNAi’ corresponding to the logical operation: if gene X is transcribed, silence independent gene Y. By appropriately selecting gene X, knockdown of gene Y could then be restricted in a tissue- and time-specific manner. To implement the logic of conditional RNAi, our approach is to engineer scRNAs that, upon binding to mRNA ‘detection target’ X, perform shape and sequence transduction to form a Dicer substrate targeting independent mRNA ‘silencing target’ Y, with subsequent Dicer processing yielding an siRNA targeting mRNA Y for destruction. Toward this end, here we design and experimentally validate diverse scRNA mechanisms for conditional Dicer substrate formation. Test tube studies demonstrate strong OFF/ON conditional response, with at least an order of magnitude increase in Dicer substrate production in the presence of the cognate mRNA detection target. By appropriately dimensioning and/or chemically modifying the scRNAs, only the product of signal transduction, and not the reactants or intermediates, is efficiently processed by Dicer, yielding siRNAs. These mechanism studies explore diverse design principles for engineering scRNA signal transduction cascades including reactant stability vs metastability, catalytic vs noncatalytic transduction, pre- vs post-transcriptional transduction, reactant and product molecularity, and modes of molecular self-assembly and disassembly.
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影响因子：
14.9
作者：
Xie Z;Liu SJ;Bleris L;Benenson Y
通讯作者：
Benenson Y
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Masu, Hiroki;Narita, Atsushi;Sando, Shinsuke
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Sando, Shinsuke
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作者：
Kumar, Deepak;Kim, Sang Hoon;Yokobayashi, Yohei
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作者：
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通讯作者：
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影响因子：
38.3
作者：
Venkataraman, Suvir;Dirks, Robert M.;Pierce, Niles A.
通讯作者：
Pierce, Niles A.