Imaging intracellular metabolite and protein changes in live mammalian cells with bright fluorescent RNA-based genetically encoded sensors

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 Sadenosylmethionine (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.

Automated summary

Engineered fluorescent RNA (FR)-based sensors have been used to detect essential metabolites in living systems, but their unfavorable characteristics limit their applications. Here, we demonstrate a strategy for converting Pepper fluorescent RNA into sensors with improved characteristics. These Pepper-based sensors showed expanded emission and improved brightness, allowing robust monitoring of intracellular changes and protein translocation in live cells. Furthermore, the use of Pepper-based sensors in the CRISPR-display strategy achieved signal amplification in fluorescence imaging. These results highlight the potential of Pepper as a high-performance FR-based sensor for detecting cellular targets.