RNA-Stabilized Coat Proteins for Sensitive and Simultaneous Imaging of Distinct Single mRNAs in Live Cells

RNA localization and regulation are critical for cellular function, yet many live RNA imaging tools suffer from limited sensitivity due to background emissions from unbound probes. Here, we introduce conditionally stable variants of MS2 and PP7 coat proteins (which we name dMCP and dPCP) designed to decrease background in live-cell RNA imaging. Using a protein engineering approach that combines circular permutation and degron masking, we generated dMCP and dPCP variants that rapidly degrade except when bound to cognate RNA ligands. These enhancements enabled the sensitive visualization of single mRNA molecules undergoing differential regulation within various sub-compartments of live cells. We further demonstrate dual-color imaging with orthogonal MS2 and PP7 motifs, allowing simultaneous low-background visualization of distinct RNA species within the same cell. Overall, this work provides versatile, low-background probes for RNA imaging, which should have broad utility in the imaging and biotechnological utilization of MS2- and PP7-containing RNAs.