Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Improved tools for providing specific intraoperative diagnoses could improve patient care. In neurosurgery, intraoperatively differentiating non-operative lesions such as CNS B-cell lymphoma from operative lesions can be challenging, often necessitating immunohistochemical (IHC) procedures which require up to 24-48 hours. Here, we evaluate the feasibility of generating rapid ex vivo specific labeling using a novel lymphoma-specific fluorescent switchable aptamer. Our B-cell lymphoma-specific switchable aptamer produced only low-level fluorescence in its unbound conformation and generated an 8-fold increase in fluorescence once bound to its target on CD20-positive lymphoma cells. The aptamer demonstrated strong binding to B-cell lymphoma cells within 15 minutes of incubation as observed by flow cytometry. We applied the switchable aptamer to ex vivo xenograft tissue harboring B-cell lymphoma and astrocytoma, and within one hour specific visual identification of lymphoma was routinely possible. In this proof-of-concept study in human cell culture and orthotopic xenografts, we conclude that a fluorescent switchable aptamer can provide rapid and specific labeling of B-cell lymphoma, and that developing aptamer-based labeling approaches could simplify tissue staining and drastically reduce time to histopathological diagnoses compared with IHC-based methods. We propose that switchable aptamers could enhance expeditious, accurate intraoperative decision-making.

DNA can be programmed to self-assemble into high molecular weight 3D assemblies with precise nanometer-scale structural features. Although numerous sequence design strategies exist to realize these assemblies in solution, there is currently no computational framework to predict their 3D structures on the basis of programmed underlying multi-way junction topologies constrained by DNA duplexes. Here, we introduce such an approach and apply it to assemblies designed using the canonical immobile four-way junction. The procedure is used to predict the 3D structure of high molecular weight planar and spherical ring-like origami objects, a tile-based sheet-like ribbon, and a 3D crystalline tensegrity motif, in quantitative agreement with experiments. Our framework provides a new approach to predict programmed nucleic acid 3D structure on the basis of prescribed secondary structure motifs, with possible application to the design of such assemblies for use in biomolecular and materials science.

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein–DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.