Assembly of multienzyme complexes on DNA nanostructures

Jinglin Fu, Yuhe Renee Yang, Soma Dhakal, Zhao Zhao, Minghui Liu, Ting Zhang, Nils G. Walter, Hao Yan

Research output: Contribution to journalArticlepeer-review

88 Scopus citations


In nature, the catalytic efficiency of multienzyme complexes highly depends on their spatial organization. The positions and orientations of the composite enzymes are often precisely controlled to facilitate substrate transport between them. Self-Assembled DNA nanostructures hold great promise for organizing biomolecules at the nanoscale. Here, we present detailed protocols for exploiting DNA nanostructures as assembly scaffolds that organize the spatial arrangements of multienzyme cascades with control over their relative distance, compartmentalization and substrate diffusion paths. The protocol describes the preparation and purification of DNA-conjugated enzymes and cofactors, along with the assembly of these prepared complexes on DNA nanostructures. The architecture of assembled enzyme complexes is then readily characterized using a broad selection of techniques from routine gel electrophoresis to advanced single-molecule imaging. We also describe methods of purifying these nano-Assemblies and testing them with functional assays based on either bulk or single-molecule fluorescence measurements. The entire assembly and characterization of a multienzyme complex can be completed within 1-2 weeks.

Original languageEnglish (US)
Pages (from-to)2243-2273
Number of pages31
JournalNature protocols
Issue number11
StatePublished - Nov 1 2016

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)


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