Abstract
The MS2 system, with an MS2 binding site (MBS) and an MS2 coat protein fused to a fluorescent protein (MCP-FP), has been widely used to fluorescently label mRNA in live cells. However, one of its limitations is the constant background fluorescence signal generated from free MCP-FPs. To overcome this obstacle, we used a superfolder GFP (sfGFP) split into two or three nonfluorescent fragments that reassemble and emit fluorescence only when bound to the target mRNA. Using the high-affinity interactions of bacteriophage coat proteins with their corresponding RNA binding motifs, we showed that the nonfluorescent sfGFP fragments were successfully brought close to each other to reconstitute a complete sfGFP. Furthermore, real-time mRNA dynamics inside the nucleus as well as the cytoplasm were observed by using the split sfGFPs with the MS2-PP7 hybrid system. Our results demonstrate that the split sfGFP systems are useful tools for background-free imaging of mRNA with high spatiotemporal resolution.