Molecular interlocked systems with mechanically trapped components can serve as versatile building blocks for dynamic nanostructures. Here we report the synthesis of unprecedented double-stranded (ds) DNA - and rotaxanes with two distinct stations for the hybridization of the macrocycles on the axle. In the rotaxane, the release and migration of the “shuttle ring” mobilizes a second macrocycle in a highly controlled fashion. Different oligodeoxynucleotides (ODNs) employed as inputs induce structural changes in the system that can be detected as diverse logically gated output signals. We also designed nonsymmetrical rotaxanes which allow unambiguous localization of the position of the macrocycle by use of atomic force microscopy (AFM). Either light irradiation or the use of fuel ODNs can drive the threaded macrocycle to the desired station in these shuttle systems. The DNA nanostructures introduced here constitute promising prototypes for logically gated cargo delivery and release shuttles.