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Assembly of multienzyme complexes on DNA nanostructures

Nature Protocols volume 11, pages 2243–2273 (2016)Cite this article

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Abstract

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.

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Figure 1: The assembly of enzymes on spatially addressable DNA scaffolds.
Figure 2: Overview of DNA nanostructure-scaffolded assembly of multienzyme complexes.
Figure 3: Chemical conjugation of oligonucleotides with enzymes.
Figure 4: Chemical conjugation of oligonucleotides with cofactors.
Figure 5: Characterization of enzyme-assembled DNA nanostructures.
Figure 6: Purification and structural characterization of enzyme-assembled DNA nanostructures.
Figure 7: Activity characterization of the enzyme-assembled DNA nanostructures.
Figure 8: Enzyme activities vs labeled number of SPDP molecules.
Figure 9: Single-enzyme activity data collection and analysis.
Figure 10: Schematic of prism-based TIRF setup.
Figure 11: The assembly of multienzyme complexes on DNA nanostructures.

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Acknowledgements

This work was supported by an Army Research Office YIP award (W911NF-14-1-0434) and the Cottrell College Science Award to J.F., and an Army Research Office MURI award (W911NF-12-1-0420) to H.Y. and N.G.W. J.F. is supported by startup funds from Rutgers University. H.Y. is also supported by the NIH Transformative award R01GM104960 and the Presidential Strategic Initiative Fund from Arizona State University. We thank K. Taylor for proofreading the manuscript.

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Authors and Affiliations

  1. Department of Chemistry, Rutgers University–Camden, Camden, New Jersey, USA

    Jinglin Fu & Ting Zhang

  2. Center for Computational and Integrative Biology, Rutgers University–Camden, Camden, New Jersey, USA

    Jinglin Fu

  3. Center for Molecular Design and Biomimetics, Arizona State University, Tempe, Arizona, USA

    Yuhe Renee Yang, Minghui Liu & Hao Yan

  4. School of Molecular Sciences, Arizona State University, Tempe, Arizona, USA

    Yuhe Renee Yang, Minghui Liu & Hao Yan

  5. Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA

    Soma Dhakal & Nils G Walter

  6. Department of Biological Chemistry and Molecular Pharmacology, Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts, USA

    Zhao Zhao

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  1. Jinglin Fu
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Contributions

J.F., Y.R.Y., M.L. and T.Z. developed the DNA–enzyme conjugation and purification procedure; J.F., Y.R.Y. and M.L. developed the process for enzyme assembly on 2D DNA nanostructures, and characterized the activity; J.F. and Z.Z. designed DNA nanocages for enzyme encapsulation and performed activity assay in bulk solution; S.D. developed single-molecule fluorescence experiments and analyzed data; and J.F., N.G.W. and H.Y. conceived the concepts and supervised the projects. All authors contributed to the writing of the manuscript.

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Correspondence to Jinglin Fu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Assembly of a DNA origami tile.

A M13 mp 18 ssDNA scaffold is incubated with a set of hundreds of staples strands, which are thermally annealed for assembling DNA origami nanostructures.

Supplementary Figure 2 Designed DNA nanostructures for enzyme assembly.

(a) A DNA DX tile. (b) A four-way junction tile. (c) A rectangular DNA origami tile. (d) A 3D DNA nanocage based on honeycomb folding pattern.

Supplementary Figure 3 AFM quantification of the co-assembly of GOx and HRP on a rectangular DNA origami tile.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Methods (PDF 373 kb)

Supplementary Data

DNA structures. (ZIP 550 kb)

Supplementary Software

Custom-written IDL (Interactive Data Language) and MATLAB scripts. (ZIP 78 kb)

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Fu, J., Yang, Y., Dhakal, S. et al. Assembly of multienzyme complexes on DNA nanostructures. Nat Protoc 11, 2243–2273 (2016). https://doi.org/10.1038/nprot.2016.139

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