The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.

DNA minicircles exist in biological contexts, such as kinetoplast DNA, and are promising components for creating functional nanodevices. They have been used to mimic the topological features of nucleosomal DNA and to probe DNA‐protein interactions such as HIV‐1 and PFV integrases, and DNA gyrase. Here, we synthesized the topologically‐interlocked minicircle rotaxane and catenane inside a frame‐shaped DNA origami. These minicircles are 183 bp in length, constitute six individual single‐stranded DNAs that are ligated to realize duplex interlocking, and adopt temporary base pairing of single strands for interlocking. To probe the DNA‐protein interactions, restriction reactions were carried out on DNAs with different topologies such as free linear duplex or duplex constrained inside origami and free or topologically‐interlocked minicircles. Except the free linear duplex, all tested structures were resistant to restriction digestion, indicating that the topological features of DNA, such as flexibility, curvature, and groove orientation, play a major role in DNA‐protein interactions.

In the frame: Topologically interlocked minicircle DNA rotaxanes and catenanes inside DNA origami frames have been designed and synthesized. With these assemblies, the DNA topology and its influence on DNA–protein interactions were examined by enzymatic restriction. Facile cleavage by restriction enzymes was observed for flexible linear duplexes, whereas free or interlocked minicircles were reluctant to split, thus indicating the role of the topology. More information can be found in the Research Article by T. Morii et al. (DOI: 10.1002/chem.202200108).

Invited for the cover of this issue are Prof. Takashi Morii and co‐workers at Kyoto University and Ewha Womans University. The cover image depicts the graphical design and atomic force microscopic (AFM) images of the synthesized topologically‐interlocked DNA catenane and rotaxanes inside a frame‐shaped DNA origami. Read the full text of the article at 10.1002/chem.202200108.