Genetically encoded fluorogenic RNA-based bioluminescence resonance energy transfer (BRET) sensors for cellular imaging and target detection

Fluorescence- and bioluminescence-based probes are valuable tools for understanding cell functions in health and disease. Bioluminescence offers an ideal complementary readout to fluorescence due to its minimal background interference and self-illuminating nature. We previously introduced the first type of genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors. These RNA-based probes are highly programmable and can be modularly engineered to detect various cellular targets. While this system was successfully validated in vitro and from the entire cell population within a microplate, the BRET signals were quite dim and difficult to visualize at the single-cell level under a microscope. The ability of single-cell bioluminescence imaging is critical for studying cell-to-cell variations and spatiotemporal changes of cellular targets in different signaling pathways or upon drug treatment. In this study, we will introduce strategies that can enhance the functionality and capability of RNA-based BRET sensors for real-time cellular imaging and sensing. Using commonly used widefield microscopes, single-cell bioluminescent detection of various metabolites and other small molecules can be achieved in both bacterial and mammalian cells. This advancement represents a significant step toward the future development of genetically encoded RNA-based bioluminescent tools for studying disease mechanisms, high-throughput drug screening, and in vivo imaging.