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Self-assembled DNA nanostructures can be used as scaffolds to organize small functional nanocomponents. In order to build working devices—electronic circuits, biochips, optical/photonics devices—controlled placement of DNA nanostructures on substrates must be achieved. Here we present a nanoimprint lithography-based process to create chemically patterned templates, rendering them capable of selectively binding DNA origami. Hexamethyldisilazane (HMDS) is used as a passivating layer on silicon dioxide substrates, which prevents DNA attachment. Hydrophilic areas, patterned by nanoimprint lithography with the same size and shape of the origami, are formed by selective removal of the HMDS, enabling the assembly of the origami scaffolds in the patterned areas. The use of nanoimprint lithography, a low cost, high throughput patterning technique, enables high precision positioning and orientation of DNA nanostructures on a surface over large areas.