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.

Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiencies of these cascades depend on the precise spatial organization of the constituent enzymes, which is optimized to facilitate substrate transport and regulate activities. Mimicry of this organization in a non-living, artificial system would be very useful in a broad range of applications—with impacts on both the scientific community and society at large. Self-assembled DNA nanostructures are promising applications to organize biomolecular components into prescribed, multidimensional patterns. In this review, we focus on recent progress in the field of DNA-scaffolded assembly and confinement of multienzyme reactions. DNA self-assembly is exploited to build spatially organized multienzyme cascades with control over their relative distance, substrate diffusion paths, compartmentalization and activity actuation. The combination of addressable DNA assembly and multienzyme cascades can deliver breakthroughs toward the engineering of novel synthetic and biomimetic reactors.