UC Irvine

A complete understanding of RNA dynamics requires methods to continuously image transcripts in living systems. We recently developed a panel of bioluminescent tools for multiplexed RNA detection, comprising luciferase fragments fused to RNA-binding proteins (RNA lanterns). The RNA lanterns assembled a light-emitting luciferase upon binding unique, structured RNA tags. Different colors of emission were produced via bioluminescence resonance energy transfer to fluorescent protein fusions. In this work, we systematically optimized the RNA lantern platform for enhanced performance in live cells. A luciferase-fluorescent protein linkage was identified that enabled improved energy transfer and enhanced photon outputs. The RNA lantern was further optimized for selective recognition of structured RNA tags. The new RNA lantern-tag combination is orthogonal to existing probes and enabled the first demonstration of three-color bioluminescent RNA visualization in live cells. Overall, this work expands the suite of probes for real-time, multiplexed imaging of RNA.