Universal, label-free, single-molecule visualization of DNA origami nanodevices across biological samples using origamiFISH

Signal amplification through hybridization chain reaction by targeting conserved regions of the M13mp18 bacteriophage-based scaffold sequences is used for in situ imaging of unlabelled DNA origami nanostructures. Structural DNA nanotechnology enables the fabrication of user-defined DNA origami nanostructures (DNs) for biological applications. However, the role of DN design during cellular interactions and subsequent biodistribution remain poorly understood. Current methods for tracking DN fates in situ, including fluorescent-dye labelling, suffer from low sensitivity and dye-induced artifacts. Here we present origamiFISH, a label-free and universal method for the single-molecule fluorescence detection of DNA origami nanostructures in cells and tissues. origamiFISH targets pan-DN scaffold sequences with hybridization chain reaction probes to achieve 1,000-fold signal amplification. We identify cell-type- and DN shape-specific spatiotemporal distribution patterns within a minute of uptake and at picomolar DN concentrations, 10,000x lower than field standards. We additionally optimize compatibility with immunofluorescence and tissue clearing to visualize DN distribution within tissue cryo-/vibratome sections, slice cultures and whole-mount organoids. Together, origamiFISH enables the accurate mapping of DN distribution across subcellular and tissue barriers for guiding the development of DN-based therapeutics.

Automated summary

Signal amplification through hybridization chain reaction targets conserved regions of M13mp18 bacteriophage-based scaffold sequences for imaging of unlabelled DNA origami nanostructures. origamiFISH is a label-free and universal method for single-molecule fluorescence detection of DNA origami nanostructures in cells and tissues. It achieves 1,000-fold signal amplification by targeting pan-DN scaffold sequences with hybridization chain reaction probes.