Dynamic RNA nanotechnology with small conditional RNAs (scRNAs) offers a promising conceptual approach to introducing synthetic regulatory links into endogenous biological circuits. Here, we use human cell lysate containing functional Dicer and RNases as a testbed for engineering scRNAs for conditional RNA interference (RNAi). scRNAs perform signal transduction via conditional shape change: detection of a subsequence of mRNA input X triggers formation of a Dicer substrate that is processed to yield small interfering RNA (siRNA) output anti-Y targeting independent mRNA Y for destruction. Automated sequence design is performed using the reaction pathway designer within NUPACK to encode this conditional hybridization cascade into the scRNA sequence subject to the sequence constraints imposed by X and Y. Because it is difficult for secondary structure models to predict which subsequences of mRNA input X will be accessible for detection, here we develop the RNAhyb method to experimentally determine accessible windows within the mRNA that are provided to the designer as sequence constraints. We demonstrate the programmability of scRNA regulators by engineering scRNAs for transducing in both directions between two full-length mRNAs X and Y, corresponding to either the forward molecular logic "if X then not Y" (X [Formula: see text] Y) or the reverse molecular logic "if Y then not X" (Y [Formula: see text] X). In human cell lysate, we observe a strong OFF/ON conditional response with low crosstalk, corresponding to a ≈20-fold increase in production of the siRNA output in response to the cognate versus noncognate full-length mRNA input. 2'OMe-RNA chemical modifications protect signal transduction reactants and intermediates against RNase degradation while enabling Dicer processing of signal transduction products. Because diverse biological pathways interact with RNA, scRNAs that transduce between detection of endogenous RNA inputs and production of biologically active RNA outputs hold great promise as a synthetic regulatory paradigm.

中文翻译：

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通过动态RNA纳米技术在人细胞裂解液中进行信号转导。

具有小条件RNA（scRNA）的动态RNA纳米技术为将合成的调控联系引入内源性生物回路提供了一种有前途的概念方法。在这里，我们使用包含功能性Dicer和RNase的人类细胞裂解液作为工程scRNA的条件RNA干扰（RNAi）的测试床。scRNA通过有条件的形状变化执行信号转导：检测mRNA输入X的子序列会触发Dicer底物的形成，该酶被加工以产生小的干扰RNA（siRNA）输出抗Y，靶向独立的mRNA Y进行破坏。使用NUPACK中的反应途径设计器执行自动序列设计，以将这种条件杂交级联编码到scRNA序列中，该序列受X和Y施加的序列限制。由于二级结构模型很难预测将检测到哪些mRNA输入X子序列，因此在此我们开发了RNAhyb方法，以实验确定mRNA中的可访问窗口，这些窗口作为序列约束条件提供给设计人员。我们证明了通过工程scRNA在两个全长mRNA X和Y之间双向转导的scRNA调节剂的可编程性，对应于正向分子逻辑“如果X则不是Y”（X [公式：参见文本] Y）或反向分子逻辑“如果是Y，则不是X”（Y [公式：请参见文字X]）。在人类细胞裂解物中，我们观察到强烈的OFF / ON条件响应，并且串扰低，对应于对应同源和非同源全长mRNA输入的siRNA输出产量增加≈20倍。2'OMe-RNA化学修饰保护信号转导反应物和中间体免于RNase降解，同时使Dicer能够处理信号转导产物。由于多种生物途径与RNA相互作用，因此在检测内源性RNA输入与产生生物活性RNA输出之间转换的scRNA具有广阔的前景，可作为一种合成的调控范式。