The unique structure-directing properties of DNA origami nanostructures (DONs) show great potential to specifically manipulate intracellular processes. We report an innovative concept to selectively activate the transcription of a single gene in the developing zebrafish embryo. We reason that engineering a designer transcription factor in which a rigid DON imposes a fixed distance between the DNA-binding domain (DBD) and the transactivation domain (TAD) would allow the selective activation of a gene harboring the same distance between the corresponding transcription factor binding site and the core promoter. As a test case, a rigid tubular DON was designed to separate the DBD of the GAL4 transcription factor and the VP16 viral protein as a TAD. This construct was microinjected in the yolk of one-cell-stage zebrafish embryos, together with a reporter plasmid to assess its functionality. The large DON was efficiently distributed to cells of the developing embryo and showed no signs of toxicity. However, because the DON showed only a cytosolic localization, it did not activate transcription of the reporter gene. Although this work clearly demonstrates that DON microinjection enables the intracellular distribution of multi-protein architectures in most of the cells of the developing zebrafish embryo, further refinements are necessary to enable selective gene activation in vivo.