Fluorescent RNA (FR)-based genetically encoded sensors have been engineered to detect various essential metabolites in living systems. However, the unfavorable characteristics of FR impede sensor applications. Here, we describe a strategy for converting Pepper fluorescent RNA into a series of fluorescent sensors to detect their cognate targets both in vitro and in live cells. Compared to previously developed FR-based sensors, Pepper-based sensors exhibited expanded emission of up to 620 nm and markedly improved cellular brightness, allowing robust and real-time monitoring of the pharmacologic-triggered dynamics changes in the intracellular level of S-adenosylmethionine (SAM) and the optogenetic manipulated protein translocation in live mammalian cells. Furthermore, signal amplification in fluorescence imaging of the target was achieved using the CRISPR-display strategy by incorporating a Pepper-based sensor into the sgRNA scaffold. Together, these results demonstrate that Pepper can be readily developed into high-performance FR-based sensors to detect various cellular targets.
Keywords: Fluorogenic RNA aptamer; Genetically encoded sensor; Metabolite and protein dynamics; Pepper.
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