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In vitro assembly of cubic RNA-based scaffolds designed in silico

Nature Nanotechnology volume 5, pages 676–682 (2010)Cite this article

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Abstract

The organization of biological materials into versatile three-dimensional assemblies could be used to build multifunctional therapeutic scaffolds for use in nanomedicine. Here, we report a strategy to design three-dimensional nanoscale scaffolds that can be self-assembled from RNA with precise control over their shape, size and composition. These cubic nanoscaffolds are only ∼13 nm in diameter and are composed of short oligonucleotides, making them amenable to chemical synthesis, point modifications and further functionalization. Nanocube assembly is verified by gel assays, dynamic light scattering and cryogenic electron microscopy. Formation of functional RNA nanocubes is also demonstrated by incorporation of a light-up fluorescent RNA aptamer that is optimally active only upon full RNA assembly. Moreover, we show that the RNA nanoscaffolds can self-assemble in isothermal conditions (37 °C) during in vitro transcription, which opens a route towards the construction of sensors, programmable packaging and cargo delivery systems for biomedical applications.

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Figure 1: Three-dimensional models for 6- and 10-stranded cubes with corresponding two-dimensional schematics of sequence interactions.
Figure 2: Characterization of 6-stranded cube assemblies (without dangling ends).
Figure 3: Structural characterization of RNA cubes by cryo-EM with single-particle image reconstruction.
Figure 4: Functionalization of the RNA nanocube scaffold with malachite green (MG) aptamer.

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Acknowledgements

The authors thank V. A. Piunova for assistance with DLS, K. Kahn for helping with Kd curve analysis, and C. Potter and B. Carragher for their invaluable scientific input regarding cryo-EM and single-particle reconstruction. Cryo-EM imaging was performed at National Resources for Automated Molecular Microscopy, which is supported by the National Institutes of Health (NIH) through the National Center for Research Resources P41 program (RR17573). This research was supported (in part) by the Intramural Research Program of the NIH, the National Cancer Institute, the Center for Cancer Research (B.A.S.) and by NIH R01 GM079604 (L.J.). This project has been funded in whole or in part with federal funds from the National Cancer Institute, NIH, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does any mention of trade names, commercial products or organizations imply endorsement by the US government. K.A. and L.J. wish to dedicate this work to Sts. Cyril and Methodius.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Biomolecular Science and Engineering Program, University of California, Santa Barbara, 93106-9510, California, USA

    Kirill A. Afonin, Alan J. Yaghoubian & Luc Jaeger

  2. Basic Science Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, 21702, Maryland, USA

    Eckart Bindewald

  3. Department of Cell Biology, Automated Molecular Imaging Group, The Scripps Research Institute, MC CB129, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    Neil Voss & Erica Jacovetty

  4. Center for Cancer Research Nanobiology Program, NCI-Frederick, Frederick, 21702, Maryland, USA

    Bruce A. Shapiro

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Contributions

K.A.A. and L.J. conceived and designed the experiments. E.B., B.A.S., K.A.A. and L.J. contributed to the sequence and 3D model design. K.A.A. and A.J.Y. performed self-assembly PAGE and fluorescence experiments. K.A.A. performed DLS experiments. K.A.A. and L.J. analysed the data. N.V. and E.J. performed cryo-EM characterization and reconstruction. K.A.A., B.A.S. and L.J. co-wrote the paper.

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Correspondence to Bruce A. Shapiro or Luc Jaeger.

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Afonin, K., Bindewald, E., Yaghoubian, A. et al. In vitro assembly of cubic RNA-based scaffolds designed in silico. Nature Nanotech 5, 676–682 (2010). https://doi.org/10.1038/nnano.2010.160

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