Fluorogenic Interacting Protein Stabilization for Orthogonal RNA Imaging

Live imaging of RNAs is crucial to interrogate their cellular functions, but genetically encodable RNA imaging system compatible to multiplexed and in vivo applications remains a persistent challenge. We propose a new concept of fluorogenic interacting protein stabilization (FLIPS) that enables engineering of RNA‐binding proteins (RBPs) such as MCP, L7Ae, Cse3 and LIN28A into orthogonal RNA‐stabilized fluorogenic proteins for multiplexed RNA imaging. The FLIPS system comprises elaborate engineering of the fluorescence protein‐fused RBPs through circular permutation and incorporation with a C‐terminal poly(arginine)‐appended degron. We show that the RNA motifs bind and stabilize the cognate engineered RBPs with a proximity‐mediated synergistic effect from the poly(arginine) region due to enhanced electrostatic interactions. The FLIPS design affords a generally applicable strategy for different RNA motifs and RBPs, enabling orthogonal and multi‐color fluorescence‐activated RNA imaging. The design is demonstrated for multicolor and orthogonal imaging of RNAs, single molecule RNA imaging and tracking, simultaneous imaging of two RNAs in nuclear condensates, and biplexed tracking of RNA translocation into cytosolic condensates. The versatility of our system highlights its potential for interrogating RNA biology and developing RNA‐based imaging tools.