Synthetic gene circuits allow the behavior of living cells to be reprogrammed, and non-coding small RNAs (sRNAs) are increasingly being used as programmable regulators of gene expression. However, sRNAs (natural or synthetic) are generally used to regulate single target genes, while complex dynamic behaviors would require networks of sRNAs regulating each other. Here, we report a strategy for implementing such networks that exploits hybridization reactions carried out exclusively by multifaceted sRNAs that are both targets of and triggers for other sRNAs. These networks are ultimately coupled to the control of gene expression. We relied on a thermodynamic model of the different stable conformational states underlying this system at the nucleotide level. To test our model, we designed five different RNA hybridization networks with a linear architecture, and we implemented them in Escherichia coli. We validated the network architecture at the molecular level by native polyacrylamide gel electrophoresis, as well as the network function at the bacterial population and single-cell levels with a fluorescent reporter. Our results suggest that it is possible to engineer complex cellular programs based on RNA from first principles. Because these networks are mainly based on physical interactions, our designs could be expanded to other organisms as portable regulatory resources or to implement biological computations.

中文翻译：

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在活细胞中实现的RNA杂交网络的基于模型的设计

合成基因电路允许对活细胞的行为进行重新编程，并且非编码小RNA（sRNA）越来越多地用作基因表达的可编程调节物。然而，sRNA（天然或合成的）通常用于调节单个靶基因，而复杂的动态行为则要求sRNA的网络相互调节。在这里，我们报告了一种实施这种网络的策略，该网络利用了专门由多面sRNA进行的杂交反应，而sRNA既是其他sRNA的目标，又是其他sRNA的触发因素。这些网络最终与基因表达的控制耦合。我们依赖于该系统在核苷酸水平上的不同稳定构象状态的热力学模型。为了测试我们的模型，大肠杆菌。我们通过天然聚丙烯酰胺凝胶电泳在分子水平上验证了网络架构，并通过荧光报告基因在细菌群体和单细胞水平上验证了网络功能。我们的研究结果表明，有可能根据第一原理设计出基于RNA的复杂细胞程序。由于这些网络主要基于物理交互，因此我们的设计可以扩展为其他有机体，作为可移植的监管资源或实现生物计算。