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Hetero-assembling of spherical building blocks with well-defined spatial distribution holds great significance in developing chiral nanostructures. Herein, a strategy for hetero-assembling of gold nanoparticles (AuNPs) was demonstrated using rigid bifacial DNA origami as templates. By tuning the sizes and the fixed location of AuNPs on DNA origami, right-handed and left-handed AuNPs nanostructures were respectively constructed. Gel electrophoresis indicated the formation of the DNA origami- AuNPs complex and transmission electron microscopy (TEM) visually displayed the arrangement of AuNPs in these two chiral structures. The spatial configuration and 3D geometry of AuNPs were further illustrated by the stereographic TEM with tilting angles from -30° to 30°. This strategy provides a universal approach to construct the asymmetrical 3D geometries, which may have potential applications in …