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Design, assembly, characterization, and operation of double-stranded interlocked DNA nanostructures

Nature Protocols volume 14, pages 2818–2855 (2019)Cite this article

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Abstract

Mechanically interlocked DNA nanostructures are useful as flexible entities for operating DNA-based nanomachines. Interlocked structures made of double-stranded (ds) DNA components can be constructed by irreversibly threading them through one another to mechanically link them. The interlocked components thus remain bound to one another while still permitting large-amplitude motion about the mechanical bond. The construction of interlocked dsDNA architectures is challenging because it usually involves the synthesis and modification of small dsDNA nanocircles of various sizes, dependent on intrinsically curved DNA. Here we describe the design, generation, purification, and characterization of interlocked dsDNA structures such as catenanes, rotaxanes, and daisy-chain rotaxanes (DCRs). Their construction requires precise control of threading and hybridization of the interlocking components at each step during the assembly process. The protocol details the characterization of these nanostructures with gel electrophoresis and atomic force microscopy (AFM), including acquisition of high-resolution AFM images obtained in intermittent contact mode in liquid. Additional functionality can be conferred on the DNA architectures by incorporating proteins, molecular switches such as photo-switchable azobenzene derivatives, or fluorophores for studying their mechanical behavior by fluorescence quenching or fluorescent resonance energy transfer experiments. These modified interlocked DNA architectures provide access to more complex mechanical devices and nanomachines that can perform a variety of desired functions and operations. The assembly of catenanes can be completed in 2 d, and that of rotaxanes in 3 d. Addition of azobenzene functionality, fluorophores, anchor groups, or the site-specific linkage of proteins to the nanostructure can extend the time line.

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Fig. 1: Overview of the interlocked dsDNA nanostructures presented in this protocol.
Fig. 2: Flowchart of the design and assembly process.
Fig. 3: dsDNA ring structures.
Fig. 4: Diagram showing the process for assembling a spherical stopper.
Fig. 5: PX100 axle (Step 1C).
Fig. 6: Catenane assembly.
Fig. 7: Rotaxane assembly.
Fig. 8: DCR assembly.
Fig. 9: Light-controlled DNA rotaxane shuttle.
Fig. 10: Catenane nanoengine assembly (Step 2A) and operation.
Fig. 11: Catenane walker movement.
Fig. 12: Expected results during [2]catenane walker assembly.
Fig. 13: Expected results during [3]catenane assembly.
Fig. 14: Expected results during [2]rotaxane assembly.
Fig. 15: AFM image and graphic representation of the purified [2]Rotmec with PX100 axle.
Fig. 16: Expected results during DCR assembly.

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Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Acknowledgements

This work was made possible through funding from the European Research Council (ERC; grant no. 267173), the Max-Planck-Society (Max-Planck fellowship to M.F.), the Alexander von Humboldt Foundation (postdoctoral grants to J.V. and Y.M.), the German Academic Exchange Service (doctoral grant to M.Š.), and the China Scholarship Council CSC (doctoral scholarship to Z.Y.). M.F. extends his most sincere thanks to all the co-workers and collaborators who have contributed to this project over the past 10 years. Their names appear throughout the references. We also thank V. Adam for the synthesis of azobenzene derivatives.

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Authors and Affiliations

  1. Chemical Biology and Medicinal Chemistry Unit, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany

    Julián Valero, Mathias Centola, Yinzhou Ma, Marko Škugor, Ze Yu, Michael W. Haydell, Daniel Keppner & Michael Famulok

  2. Max-Planck-Fellowship Group Chemical Biology, Center of Advanced European Studies and Research, Bonn, Germany

    Julián Valero, Mathias Centola & Michael Famulok

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Contributions

J.V., M.C., Y.M., M.Š., Z.Y., M.W.H., and M.F. conceived and designed the protocol and wrote the manuscript. J.V., M.C., Y.M., M.Š., Z.Y., M.W.H., and D.K. carried out the experiments.

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Correspondence to Michael Famulok.

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Peer review information: Nature Protocols thanks Yamuna Krishnan, Francesco Ricci and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Rasched, G. et al. Angew. Chem. Int. Ed. Engl. 47, 967–970 (2008): https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.200704004

Ackermann, D. et al. Nat. Nanotechnol. 5, 436–442 (2010): https://www.nature.com/articles/nnano.2010.65

Lohmann, F., Weigandt, J., Valero, J., & Famulok, M. Angew. Chem. Int. Ed. Engl. 53, 10372–10376 (2014): https://onlinelibrary.wiley.com/doi/pdf/10.1002/anie.201405447

Valero, J. Pal, N., Dhakal, S., Walter, N. G. & Famulok, M. Nat. Nanotechnol. 13, 496–503 (2018): https://www.nature.com/articles/s41565-018-0109-z

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Valero, J., Centola, M., Ma, Y. et al. Design, assembly, characterization, and operation of double-stranded interlocked DNA nanostructures. Nat Protoc 14, 2818–2855 (2019). https://doi.org/10.1038/s41596-019-0198-7

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