Non-Invasive Visualization of Cells Expressing Unmodified Target mRNA Using a Programmable RNA-Mediated BiFC Platform

Abstract

Real-time visualization of specific mRNA expression in living cells is essential for understanding diverse biological processes; however, many existing approaches require modifications to target genes. In this study, we developed a programmable RNA-mediated bimolecular fluorescence complementation (RmBiFC) system by fusing split Venus fluorescent protein fragments with the Pumilio homology domain as a modular RNA-binding domain. Using murine leukemia virus mRNA as a model target, we systematically optimized the orientation of the probes and demonstrated robust detection of cells expressing target mRNA. This system enables live-cell imaging of target mRNA-expressing cells without modifying the target gene. This provides a valuable means of tracking cells expressing wild-type genes, which is challenging to achieve using conventional reporter-based approaches. The RmBiFC system represents a versatile and non-invasive platform with broad potential for identifying specific cell populations based on their transcriptomic profiles at the cellular level.