Bacterial Expression of Gene‐Encoding DNA Origami Structures

Previous studies have shown that DNA origamis assembled from gene‐encoding scaffolds can be taken up by mammalian cells and ultimately lead to the expression of the encoded genes. While interactions of DNA origami structures with mammalian cells have been extensively investigated, their interactions with bacteria remain poorly understood. Here, we systematically study how structure, folding state, chemical functionalization, and polycationic coatings influence the uptake and expression of gene‐encoding DNA origami structures in E. coli . Using complementary positive‐ and negative selection assays, we show that DNA origami structures display markedly lower transformation efficiencies than their corresponding circular single‐stranded scaffolds. Folding geometry, compactness, and scaffold accessibility strongly modulate transformation, with partially folded origami structures showing considerably higher activity. Chemical functionalization did not improve uptake, whereas polyplex formation can improve stability and transformation. Fluorescence and electron microscopy reveal that DNA origami structures predominantly associate with the bacterial surface. Leveraging the intrinsically low transformability of intact DNA origamis, we engineered photocleavable nanostructures that enable UV‐triggered release of gene‐encoding scaffolds, providing switchable activation of transformation. Our results offer fundamental insights into the bacterial processing of DNA nanostructures and establish a framework for controlling DNA entry and expression using programmable nanoscale carriers.