Scaling Up Sequential Stepwise DNA Displacement-Based Signal Exchange for Rapid Ultraplex Fluorescent Imaging in Cells and Tissues

Multiplexed fluorescent imaging methods are essential for studying cellular function by visualization of biomolecules in cells and tissues with high-resolution spatial information but usually suffer from limitations of 3-5 multiplexity because of spectral overlap between the used fluorophores. We introduce a method using scaled-up sequential DNA displacement reactions to enable highly multiplexed fluorescent imaging. The fluorescent signals of targets are encoded in different DNA probes in a single step. Each DNA probe's fluorescent signal can be activated for imaging and removed to avoid signal overlap with targets in the next round by two sequential, independent DNA displacement reactions. Massive targets in situ can be imaged when a scaled-up DNA displacement reaction is applied sequentially. We experimentally screened a set of rapid and orthogonal DNA displacement sequences from 144 different in situ reactions and developed 25 DNA probes with 50 selected displacement sequences for multiplexed imaging. We demonstrated 25-plex RNA imaging in a single fluorophore channel in fixed cells within 20 min, using the 50 sequential displacement reactions that consisted of 100 DNA strands simultaneously. To further demonstrate the robustness and practical usage, we showed 24-plex RNA imaging with the method in retinal tissues and resolved different cell types. Because of the vast sequence design space of DNA probes, theoretically unlimited multiplexity can be achieved. This method significantly simplifies the high-plex fluorescent imaging process with preprogrammed complex dynamic DNA nanotechnology and has broad biotechnical applications for future medicine and diagnostics.