Alam Khalid K, Tawiah Kwaku D, Lichte Matthew F, Porciani David, Burke Donald H
Department of Biochemistry, University of Missouri , Columbia, Missouri 65211, United States.
Bond Life Sciences Center, University of Missouri , Columbia, Missouri 65211, United States.
ACS Synth Biol. 2017 Sep 15;6(9):1710-1721. doi: 10.1021/acssynbio.7b00059. Epub 2017 May 26.
RNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second-order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.
RNA-RNA组装调控着关键的生物学过程，并且是用于构建合成遗传回路的强大工具。在活细胞中表征RNA组装通常涉及监测荧光报告蛋白，而荧光报告蛋白充其量只是对潜在RNA-RNA杂交事件的间接测量，并且会受到与报告蛋白翻译和激活相关的额外时间和负载限制。相比之下，能够螯合小分子染料并激活其荧光的RNA适体越来越多地被用于基因编码策略中以报告RNA水平的事件。分裂适体系统已被合理设计，以便在两个或更多个离散RNA转录本杂交时产生信号，但没有一个在体内表达时能直接发挥作用。我们推测，西兰花适体改善的生理学特性能够构建出一个可在活细胞中发挥作用的分裂适体系统。在此，我们展示了分裂西兰花系统，其中自组装由一个热稳定的三向接头RNA结构成核，并且荧光激活需要两条链。该系统的功能组装在体外大致遵循二级动力学，并且在共转录时而非从纯化组分组装时有所改善。分裂西兰花荧光在体内是数字化的，并且当与通过RNA-RNA杂交调节回路功能的RNA融合时保留功能模块性，如用一个RNA引发开关所证明的那样。分裂西兰花代表了首个在体内运行的功能性分裂适体系统。它提供了一个基因编码且非破坏性的平台，用于监测和利用RNA-RNA杂交，无论是作为全RNA、独立的与门还是作为监测RNA-RNA杂交体组装的工具。
