Messenger RNA-encoded reporters for monitoring cellular stress and bioenergetics

Abstract The ability to monitor cellular processes in real-time is essential for understanding cell function, disease progression, and therapeutic responses. Engineered reporter proteins have been developed for monitoring cellular metabolism, stress responses, and bioenergetics. However, their use in primary cells is limited by inefficient plasmid transfection and the impracticality of generating and validating stable cell lines for each application. Here, we use in vitro transcription to generate mRNA-encoded metabolic trackers and achieve high transfection efficiencies in primary fibroblasts, cancer cells, and induced pluripotent stem cells. This approach provides a flexible platform for real-time monitoring of cellular processes in diverse cell types and overcomes the technical barriers of establishing stable cell lines by genetic modification. We confirm the activity of three ratiometric reporters that monitor pH, H 2 O 2 , and ATP levels in subcellular compartments. Our mRNA-based approach provides a versatile, efficient tool for real-time metabolic studies across basic and applied research, reducing reliance on commercially available reporters and broadening the applicability of metabolic reporters in patient-derived cell models.