DNA origami, the folding of a long single-stranded DNA sequence (scaffold strand) by hundreds of short synthetic oligonucleotides (staple strands) into parallel aligned helices, is a highly efficient method to form advanced self-assembled DNA-architectures. Since molecules and various materials can be conjugated to each of the short staple strands, the origami method offers a unique possibility of arranging molecules and materials in well-defined positions on a structured surface. Here we combine the action of light with AFM and DNA nanostructures to study the production of singlet oxygen from a single photosensitizer molecule conjugated to a selected DNA origami staple strand on an origami structure. We demonstrate a distance-dependent oxidation of organic moieties incorporated in specific positions on DNA origami by singlet oxygen produced from a single photosensitizer located at the center of each origami.

CONSPECTUS: Singlet oxygen ((1)O2), the first excited electronic state of molecular oxygen, is a significant molecule, despite its minute size. For more than half a century, the molecule has been widely used and studied in organic synthesis, due to its characteristic oxygenation reactions. Furthermore, (1)O2 plays a key role in mechanisms of cell death, which has led to its use in therapies for several types of cancer and other diseases. The high abundance of oxygen in air provides a wonderful source of molecules that can be excited to the reactive singlet state, for example, by UV/vis irradiation of a photosensitizer molecule. Although convenient, this oxygen abundance also presents some challenges for purposes that require (1)O2 to be generated in a controlled manner. In the past decade, we and others have employed DNA nanostructures to selectively control and investigate the generation, lifetime, and reactions of (1)O2. DNA-based structures are one of the most powerful tools for controlling distances between molecules on the nanometer length scale, in particular for systems that closely resemble biological settings, due to their inherent ability to specifically form duplex structures with well-defined and predictable geometries. Here, we present some examples of how simple DNA structures can be employed to regulate (1)O2 production by controlling the behavior of (1)O2-producing photosensitizers through their interactions with independent quencher molecules. We have developed different DNA-based systems in which (1)O2 production can be switched ON or OFF in the presence of specific DNA sequences or by changing the pH of the solution. To further illustrate the interplay between DNA structures and (1)O2, we present three pieces of research, in which (1)O2 is used to activate or deactivate DNA-based systems based on the reaction between (1)O2 and cleavable linkers. In one example, it is demonstrated how a blocked oligonucleotide can be released upon irradiation with light of a specific wavelength. In more complex systems, DNA origami structures composed of more than 200 individual oligonucleotides were employed to study (1)O2 reactions in spatially resolved experiments on the nanoscale.

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of ~90 nm diameter and ~250 nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of ~8 nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology.

Immobilized antibodies are extensively employed for medical diagnostics, such as in enzyme-linked immunosorbent assays. Despite their widespread use, the ability to control the orientation of immobilized antibodies on surfaces is very limited. Herein, we report a method for the covalent and orientation-selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA)-modified strands are inserted into the cavity to form NTA-metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysine residues. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) confirmed the efficient immobilization of the antibodies in the origami structures. This increased control over the orientation of antibodies in nanostructures and on surfaces has the potential to direct the interactions between antibodies and targets and to provide more regular surface assemblies of antibodies.