Fluorogenic Interacting Protein Stabilization for Orthogonal RNA Imaging

Live imaging of RNAs is crucial to interrogate their cellular functions, and approaches allowing fluorogenic RNA imaging with high orthogonality and contrast remains underexplored. We propose a fluorogenic interacting protein stabilization (FLIPS) strategy that enables orthogonal RNA imaging in live cells with fluorescence-labeled RNA interacting proteins (RIPs) stabilized by cognate RNA motifs. We show that the RNA motif stabilizes the otherwise degraded cognate RIP fused to a poly(arginine)- extended C-terminal degron, and the stabilizing effect is enhanced with circular permutation of the RIP. This design affords a generally applicable strategy for different RNA motifs and RIPs, enabling orthogonal and multi-color fluorescence-activated RNA imaging. Our strategy is broadly demonstrated for multiplexed, high-contrast RNA detection and imaging, single molecule RNA imaging and RNA dynamic translocation tracking in live cells. The versatility of our system highlights its potential for interrogating RNA biology and developing RNA-based imaging tools.