Design strategy for DNA rotaxanes with a mechanically reinforced PX100 axle.

Rotaxanes are interlocked molecular architectures that can be perceived as simple mechanical devices. A macrocycle that is threaded onto an axle and is deterred from dethreading by bulky stoppers can move translationally along the vector of the axle as well as rotate around it. To ensure that these molecular assemblies can carry out directional mechanical motion, the respective components require sufficient dimensional stability, or stiffness, over the entire working space. In case of rotaxanes, it is primarily the axle that needs to exist as a non-deformable unit to efficiently convert the microscopic movement of the macrocycle into mechanical energy and to employ it for power transmission, otherwise the momentum of the moving macrocycle simply leads to a deformation of the axle, and thus cannot be further employed. We have recently described a DNA rotaxane that has a translational amplitude of about 100 base pairs (bp). In a double-stranded DNA, however, the length of persistence of approximately 130 bp is too short to meet the required mechanical stability along the dumbbell axle. Many systematic studies have devised methods in structural DNA nanotechnology that not only allow for the construction of topologically defined architectures by selfassembly of DNA sequences, but also lead to robust twoand three-dimensional objects. Seminal work in this field was established by Seeman, who demonstrated that two DNA double strands that are interwoven by multiple reciprocal strand exchange can lead to molecular assemblies that exhibit increased stiffness. 7] Among them, particularly the so-called paranemic crossover structures PX and JX were often applied for mechanical switching in DNA nanotechnology. PX elements are characterized by a strand exchange that occurs at each contact point of two antiparallel DNA double strands. In a JX element, however, the strand exchange is abrogated at two consecutive positions. What makes the PX and JX elements so special is that two independent DNA double strands can be held together by reciprocal base pairing. Consequently, a paranemic crossover structure always exists in equilibrium with the respective DNA double strands. In presence of Mg ions, the equilibrium is strongly shifted towards the crossover product. For the assembly of dsDNA rotaxanes, we devised a threading strategy that relies on the formation of eight bp between the DNA axle and the macrocycle. The hybridization of these two components occurs highly efficiently, leading to quantitative rotaxane formation. Owing to the highly flexible single-stranded region, the DNA axle is able to easily accommodate its conformation to the geometry inherent to the macrocycle, thus leading to quantitative threading of the axle. However, higher-order DNA architectures like paranemic crossover DNA or even DNA origami 12] do not permit this flexibility anymore. On the contrary: it is precisely their mechanical robustness that accounts for their importance in DNA nanotechnology. Conversely, however, this lack of flexibility constitutes a major challenge for the threading of a rotaxane axle into a macrocycle that necessitates novel design strategies, which we describe herein. To expand the range of application of mechanically interlocked DNA architectures to these higher-order DNA structures, we sought to apply our threading strategy to a robust paranemic crossover system and to assemble the PX100 rotaxane (Figure 1a). The reinforcement of the dumbbell axle is achieved by an extended PX-JX2 crossover system in which two parallel DNA double strands are interwoven by six double crossovers (Figure 1b). A pivotal