RIBOsensor for FRET-based, real-time ribose measurements in live cells

d-Ribose is a building block of many essential biomolecules, including all nucleic acids, and its supplementation can enhance energy production, particularly under stress conditions such as ischemia and heart failure. The distribution, biosynthesis, and regulation of ribose in mammalian systems remain poorly understood. To explore intracellular ribose dynamics, we developed a genetically encoded fluorescence resonance energy transfer (FRET) sensor using ribose binding protein (RBP) and enhanced cyan and yellow fluorescent proteins (FPs). The RIBOsensor, which positions one FP near the active site of RBP, achieves the necessary sensitivity for cellular imaging by increasing the FRET signal upon ribose binding, compared to traditional N- and C-terminal FP orientations. This sensor rapidly, reversibly, and selectively detects labile ribose in live cells-enabling longitudinal studies-and can be employed for intracellular ribose quantitation, which provides a valuable tool for investigating ribose transport and metabolism in normal and disease states.