For the functionalization with the enzyme, we first selected urease, because this is often used for the development of enzyme-driven micro-and nanoswimmers thanks to the propulsion generated by the conversion of urea (CO(NH2)2) into ammonium (NH4 + ) and bicarbonate (CO3 2-). More specifically, this movement is thought to be the result of a gradient of ionic products (ionic self-diffusiophoresis) around the swimmer triggered by the enzymatic reaction. To better visualize the enzyme, we first labeled urease with a Cy5 fluorophore using a Cy5-NHS ester that is reactive toward amine groups in the protein (see Supporting Information for more details). The Cy5labeled urease was then functionalized with an N3-containing reagent that adds an azide handle to urease by reacting with the amine groups. The resulting azide-Cy5 urease was then conjugated to a dibenzocyclooctyne (DBCO)-modified ssDNA strand using strain-promoted azide-alkyne cycloaddition (SPAAC). The DNA-enzyme conjugate was then purified by anion-exchange chromatography (Figure To find the best conditions for enzyme functionalization, we assembled DNA structures with different percentage distribution of the anchor strand by using different proportions of tiles with and without anchor strand during self-assembly. The efficiency of the self-assembly process was not affected by the presence of the anchor strand, as the size and number of nanostructures formed did not show significant differences in the absence and presence of different proportions of anchor strands (Figures ). To all assembled structures, we then added the DNA strand conjugated with urease at a fixed excess concentration (i.e., 200 nM) and quantified the mean fluorescence intensity of Cy5 urease on the surface of the DNA structures (Cy3). The binding of the enzyme to the structure increases with the percentage of tiles with the anchor strand until it levels off at 50%. By further increasing the relative proportion of anchor strands in the structure, less enzyme decoration is observed, likely due to enzyme aggregation effects (Figures ). For future experiments, we then selected DNA structures containing 30% of the tiles with the anchor strand (Figure ). Under these conditions, we find a homogeneous distribution of urease across the DNA structures. For example, the values for the average length (Cy3-DNA structure: 5.0 ± 0.3; Cy5-urease: 4.4 ± 0.1 µm) and count (Cy3-DNA structure: 3.3 ± 0.6; Cy5-urease: 3.6 ± 0.4 x 10 3 count/mm 2 ) obtained by measuring either the Cy3 (DNA tiles) or Cy5 (enzyme) signals are comparable (Figure ). The homogenous enzyme distribution is also confirmed by analysing the pixel intensity of DNA tiles and urease along the entire length of a single DNA structure (Figure ). From these analyses we derived a homogeneity factor (defined here as the average ratio of urease (Cy5) signal to DNA structure signal (Cy3) along the length of the DNA structure) of 0.7 ± 0.1. Finally, to better quantify the distribution of urease on the DNA structure, we calculated a co-localization factor that we used to estimate the overlap of the urease signal (Cy5) with the DNA structure signal (Cy3). A value of this factor around 1 would indicate a high co-localization of the enzyme with the DNA structure, whereas no overlap would result in a value around 0. In our case, a co-localization factor of 0.9 ± 0.1 suggests that the enzyme is highly co-localised with the DNA tiles (Figure ).