Programmable orthogonal and rapid sequential DNA strand displacement for fluidic-exchange-free highly multiplexed fluorescent imaging

ABSTRACT Multiplexed fluorescent imaging methods are essential for studying cellular function by visualization of biomolecules in cells and tissues with high-resolution spatial information, but most suffer from limitations such as low multiplexity because of spectral overlap between the used fluorophores. Fluidic exchange based sequential multiplexed imaging overcomes this limitation but requires time-consuming incubation and washing steps. We introduce a novel multiplexed fluorescent imaging method that uses rapid, orthogonal DNA strand displacement reactions to enable unlimited multiplexed fluorescent imaging without fluidic exchange. The signal switching between targets is achieved by strand displacement with added non-fluorescent DNA displacer strands, no fluidic washing step is required for sequential multiplexed imaging. We experimentally screened a set of rapid and orthogonal DNA displacement sequences for probe design and applied it for RNA imaging, which takes less than 30 seconds per round to complete in fixed cells. Because of the vast sequence design space of DNA probes, theoretically unlimited multiplexity can be achieved. Using 25 developed rapid orthogonal probes, we achieved 25-plex RNA imaging in a single fluorophore channel in fixed cells within 20 minutes. To further demonstrate robustness and practical usage of DIRSE-based imaging, we showed 24-plex RNA imaging with the method in retinal tissues and resolved different cell types. This DIRSE mechanism significantly simplifies the high-plex fluorescent imaging process with pre-programmed DNA probes and has broad biotechnical applications for future medicine and diagnostics.