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

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.

References

  1. Seeman, N. C. An overview of structural DNA nanotechnology. Mol. Biotechnol. 37, 246–257 (2007).

    CAS  Article  Google Scholar 

  2. Aldaye, F. A., Palmer, A. L. & Sleiman, H. F. Assembling materials with DNA as the guide. Science 321, 1795–1799 (2008).

    CAS  Article  Google Scholar 

  3. Lin, C., Liu, Y. & Yan, H. Designer DNA nanoarchitectures. Biochemistry 48, 1663–1674 (2009).

    CAS  Article  Google Scholar 

  4. Chen, J. H. & Seeman, N. C. The electrophoretic properties of a DNA cube and its substructure catenanes. Electrophoresis 12, 607–611 (1991).

    CAS  Article  Google Scholar 

  5. Goodman, R. P. et al. Reconfigurable, braced, three-dimensional DNA nanostructures. Nature Nanotech. 3, 93–96 (2008).

    CAS  Article  Google Scholar 

  6. He, Y. et al. Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra. Nature 452, 198–201 (2008).

    CAS  Article  Google Scholar 

  7. Erben, C. M., Goodman, R. P. & Turberfield, A. J. A self-assembled DNA bipyramid. J. Am. Chem. Soc. 129, 6992–6993 (2007).

    CAS  Article  Google Scholar 

  8. Andersen, F. F. et al. Assembly and structural analysis of a covalently closed nano-scale DNA cage. Nucleic Acids Res. 36, 1113–1119 (2008).

    CAS  Article  Google Scholar 

  9. Shih, W. M., Quispe, J. D. & Joyce, G. F. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature 427, 618–621 (2004).

    CAS  Article  Google Scholar 

  10. Zimmermann, J., Cebulla, M. P., Monninghoff, S. & von Kiedrowski, G. Self-assembly of a DNA dodecahedron from 20 trisoligonucleotides with C(3h) linkers. Angew. Chem. Int. Ed. 47, 3626–3630 (2008).

    CAS  Article  Google Scholar 

  11. Erben, C. M., Goodman, R. P. & Turberfield, A. J. Single-molecule protein encapsulation in a rigid DNA cage. Angew. Chem. Int. Ed. 45, 7414–7417 (2006).

    CAS  Article  Google Scholar 

  12. Bhatia, D. et al. Icosahedral DNA nanocapsules by modular assembly. Angew. Chem. Int. Ed. 48, 4134–4137 (2009).

    CAS  Article  Google Scholar 

  13. Rothemund, P. W. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

    CAS  Article  Google Scholar 

  14. Andersen, E. S. et al. Self-assembly of a nanoscale DNA box with a controllable lid. Nature 459, 73–76 (2009).

    CAS  Article  Google Scholar 

  15. Ke, Y. et al. Scaffolded DNA origami of a DNA tetrahedron molecular container. Nano Lett. 9, 2445–2447 (2009).

    CAS  Article  Google Scholar 

  16. Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into twisted and curved nanoscale shapes. Science 325, 725–730 (2009).

    CAS  Article  Google Scholar 

  17. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

    CAS  Article  Google Scholar 

  18. Kim, D. H. & Rossi, J. J. Strategies for silencing human disease using RNA interference. Nature Rev. Genet. 8, 173–184 (2007).

    CAS  Article  Google Scholar 

  19. Famulok, M., Hartig, J. S. & Mayer, G. Functional aptamers and aptazymes in biotechnology, diagnostics and therapy. Chem. Rev. 107, 3715–3743 (2007).

    CAS  Article  Google Scholar 

  20. Joyce, G. F. Directed evolution of nucleic acid enzymes. Annu. Rev. Biochem. 73, 791–836 (2004).

    CAS  Article  Google Scholar 

  21. Davidson, E. A. & Ellington, A. D. Engineering regulatory RNAs. Trends Biotechnol. 23, 109–112 (2005).

    CAS  Article  Google Scholar 

  22. Wendell, D. et al. Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nature Nanotech. 4, 765–772 (2009).

    CAS  Article  Google Scholar 

  23. Khaled, A., Guo, S., Li, F. & Guo, P. Controllable self-assembly of nanoparticles for specific delivery of multiple therapeutic molecules to cancer cells using RNA nanotechnology. Nano Lett. 5, 1797–1808 (2005).

    CAS  Article  Google Scholar 

  24. Guo, P. RNA nanotechnology: engineering, assembly and applications in detection, gene delivery and therapy. J. Nanosci. Nanotechnol. 5, 1964–1982 (2005).

    CAS  Article  Google Scholar 

  25. Jaeger, L. & Leontis, N. B. Tecto-RNA: one-dimensional self-assembly through tertiary interactions. Angew. Chem. Int. Ed. 39, 2521–2524 (2000).

    CAS  Article  Google Scholar 

  26. Chworos, A. et al. Building programmable jigsaw puzzles with RNA. Science 306, 2068–2072 (2004).

    CAS  Article  Google Scholar 

  27. Shu, D., Moll, W.-D., Deng, Z., Mao, M. & Guo, P. Bottom-up assembly of RNA arrays and superstructures as potential parts in nanotechnology. Nano Lett. 4, 1717–1723 (2004).

    CAS  Article  Google Scholar 

  28. Jaeger, L. & Chworos, A. The architectonics of programmable RNA and DNA nanostructures. Curr. Opin. Struct. Biol. 16, 531–543 (2006).

    CAS  Article  Google Scholar 

  29. Afonin, K. A., Cieply, D. J. & Leontis, N. B. Specific RNA self-assembly with minimal paranemic motifs. J. Am. Chem. Soc. 130, 93–102 (2008).

    CAS  Article  Google Scholar 

  30. Severcan, I., Geary, C., Verzemnieks, E., Chworos, A. & Jaeger, L. Square-shaped RNA particles from different RNA folds. Nano Lett. 9, 1270–1277 (2009).

    CAS  Article  Google Scholar 

  31. Koyfman, A. Y. et al. Controlled spacing of cationic gold nanoparticles by nanocrown RNA. J. Am. Chem. Soc. 127, 11886–11887 (2005).

    CAS  Article  Google Scholar 

  32. Nasalean, L., Baudrey, S., Leontis, N. B. & Jaeger, L. Controlling RNA self-assembly to form filaments. Nucleic Acids Res. 34, 1381–1392 (2006).

    CAS  Article  Google Scholar 

  33. Severcan, I. et al. A polyhedron made of tRNAs. Nature Chem. 2, 772–779 (2010).

    CAS  Article  Google Scholar 

  34. Bindewald, E., Grunewald, C., Boyle, B., O'Connor, M. & Shapiro, B. A. Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler. J. Mol. Graph. Model 27, 299–308 (2008).

    CAS  Article  Google Scholar 

  35. Seiffert, J. & Huhle, A. A full-automatic sequence design algorithm for branched DNA structures. J. Biomol. Struct. Dyn. 25, 453–466 (2008).

    CAS  Article  Google Scholar 

  36. Seeman, N. C. Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247 (1982).

    CAS  Article  Google Scholar 

  37. Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940 (1999).

    CAS  Article  Google Scholar 

  38. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    CAS  Article  Google Scholar 

  39. Bernhart, S. H. et al. Partition function and base pairing probabilities of RNA heterodimers. Algorithms Mol. Biol. 1, 3 (2006).

    Article  Google Scholar 

  40. Hofacker, I. L. Fast folding and comparison of RNA secondary structures. Monatshefte f Chemie 125, 167–188 (1994).

    CAS  Article  Google Scholar 

  41. Freier, S. M. et al. Improved free-energy parameters for predictions of RNA duplex stability. Proc. Natl Acad. Sci. USA 83, 9373–9377 (1986).

    CAS  Article  Google Scholar 

  42. SantaLucia, J. Jr. A unified view of polymer, dumbbell and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl Acad. Sci. USA 95, 1460–1465 (1998).

    CAS  Article  Google Scholar 

  43. Sugimoto, N., Nakano, S., Yoneyama, M. & Honda, K. Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res. 24, 4501–4505 (1996).

    CAS  Article  Google Scholar 

  44. Kato, T., Goodman, R. P., Erben, C. M., Turberfield, A. J. & Namba, K. High-resolution structural analysis of a DNA nanostructure by cryoEM. Nano Lett. 9, 2747–2750 (2009).

    CAS  Article  Google Scholar 

  45. Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).

    CAS  Article  Google Scholar 

  46. Baugh, C., Grate, D. & Wilson, C. 2.8 Å crystal structure of the malachite green aptamer. J. Mol. Biol. 301, 117–128 (2000).

    CAS  Article  Google Scholar 

  47. Duxbury, D. F. The photochemistry and photophysics of triphenylmethane dyes in solid and liquid media. Chem. Rev. 93, 381–433 (1993).

    CAS  Article  Google Scholar 

  48. Afonin, K. A., Danilov, E. O., Novikova, I. V. & Leontis, N. B. TokenRNA: a new type of sequence-specific, label-free fluorescent biosensor for folded RNA molecules. Chembiochem 9, 1902–1905 (2008).

    CAS  Article  Google Scholar 

  49. Marky, L. A. & Breslauer, K. J. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26, 1601–1620 (1987).

    CAS  Article  Google Scholar 

  50. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    CAS  Article  Google Scholar 

  51. Lander, G. C. et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95–102 (2009).

    CAS  Article  Google Scholar 

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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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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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