Abstract:

A comprehensive understanding of RNA biology requires methods to visualize transcripts and their interactions within physiological environments. However, few technologies can report on multiple RNAs continuously and in living systems. To address this gap, I have developed new bioluminescent RNA imaging probes and platforms for multiplexed analysis. Bioluminescence does not require excitation light and can enable sensitive, noninvasive readouts in a variety of settings. In this work, a panel of structured RNA tags and luciferase fragments (RNA lanterns) was engineered for orthogonal RNA detection. The lanterns emit few photons on their own, but assemble and emit light when brought into proximity by the RNA tags. The tools were extensively optimized for sensitive targeting of a variety of transcripts in living systems. Bioluminescent phasor was further harnessed for tandem RNA imaging in live mammalian cells. Finally, a smartphone-based spectral phasor platform was developed for multiplexed analysis of bioluminescent reporters. Collectively, these advancements set the stage for real-time RNA tracking and more accessible bioluminescence multiplexing at all scales.