DNA Nanoscaffolds for Multienzyme Systems Assembly

Multienzyme reactions play an important role in cellular metabolic functions. The assembly of a metabolon is often observed, in which the position and the orientation of composite enzymes are optimized to facilitate the substrate transport. The recent progress of DNA nanotechnology is promising to organize the assembly of bimolecular complexes with precise controlled geometric patterns at nanoscale, such as enzyme cascades assembly, biomimetic substrate channeling, and compartmentalization. Here, we present detailed protocols of using DNA nanoscaffolds to assemble a multienzyme system with control over spatial interactions and arrangements of individual components. The protocols include the preparation and purification of DNA nanostructures, the bioconjugation of DNA with proteins and cofactors, the chromatography purification of DNA-conjugated biomolecules, the characterization of assemblies by routine gel electrophoresis and advanced AFM imaging, as well as the activity evaluation of multienzyme assemblies.