Metabolic heterogeneity, the occurrence of different metabolic phenotypes among cells, represents a key challenge in health and biotechnology. To unravel its molecular basis, tools probing metabolism of single cells are needed. While RNA devices harbor huge potential for the development of such tools, until today, it is challenging to create in vivo-functional sensors for any given metabolite. Here, we developed from scratch an RNA-based sensor for fructose-1,6-bisphosphate (FBP), a doubly phosphorylated intermediate of glycolysis. Starting from in vitro selection of an RNA aptamer and its structural analyses, we developed libraries of RNA-based regulatory devices with this aptamer and the hammerhead ribozyme as an actuator. Through FACS-seq-based high-throughput screening in yeast, we identified in vivo-functional FBP-sensing devices that generate fluorescent readout dependent on intracellular FBP concentration. As FBP reports the flux through glycolysis, the developed RNA device can be used to sense the glycolytic rate in single cells, offering unprecedented possibilities to investigate the causes of metabolic heterogeneity.

中文翻译：

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合成的基于RNA的果糖1,6-双磷酸酯生物传感器，报告糖酵解通量

代谢异质性，即细胞间不同代谢表型的出现，代表了健康和生物技术领域的关键挑战。为了揭示其分子基础，需要用于探测单细胞代谢的工具。尽管RNA设备具有开发此类工具的巨大潜力，但直到今天，为任何给定的代谢物创建具有体内功能的传感器都是具有挑战性的。在这里，我们从头开发了一种基于RNA的1,6-双磷酸果糖（FBP）传感器，一种糖酵解的双磷酸化中间体。从RNA适体的体外选择及其结构分析开始，我们开发了以该适体和锤头状核酶作为促动器的基于RNA的调控装置文库。通过酵母中基于FACS-seq的高通量筛选，我们确定了体内功能性的FBP传感设备，该设备可产生依赖于细胞内FBP浓度的荧光读数。当FBP报告通过糖酵解的通量时，开发的RNA装置可用于检测单个细胞中的糖酵解速率，为研究代谢异质性的原因提供了前所未有的可能性。