First evidence of geometrical patterns and defined distances of biomolecules as fundamental parameters to regulate receptor binding and cell signaling have emerged recently. Here, we demonstrate the importance of controlled nanospacing of immunostimulatory agents for the activation of immune cells by exploiting DNA-based nanomaterials and pre-existing crystallography data. We created DNA origami nanoparticles that present CpG-motifs in rationally designed spatial patterns to activate Toll-like Receptor 9 in RAW 264.7 macrophages. We demonstrated that stronger immune activation is achieved when active molecules are positioned at the distance of 7 nm, matching the active dimer structure of the receptor. Moreover, we show how the introduction of linkers between particle and ligand can influence the spatial tolerance of binding. These findings are fundamental for a fine-tuned manipulation of the immune system, considering the importance of spatially controlled presentation of therapeutics to increase efficacy and specificity of immune-modulating nanomaterials where multivalent binding is involved.