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Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics

Nature Nanotechnology volume 6, pages 658–667 (2011)Cite this article

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Abstract

RNA nanoparticles have applications in the treatment of cancers and viral infection; however, the instability of RNA nanoparticles has hindered their development for therapeutic applications. The lack of covalent linkage or crosslinking in nanoparticles causes dissociation in vivo. Here we show that the packaging RNA of bacteriophage phi29 DNA packaging motor can be assembled from 3–6 pieces of RNA oligomers without the use of metal salts. Each RNA oligomer contains a functional module that can be a receptor-binding ligand, aptamer, short interfering RNA or ribozyme. When mixed together, they self-assemble into thermodynamically stable tri-star nanoparticles with a three-way junction core. These nanoparticles are resistant to 8 M urea denaturation, are stable in serum and remain intact at extremely low concentrations. The modules remain functional in vitro and in vivo, suggesting that the three-way junction core can be used as a platform for building a variety of multifunctional nanoparticles. We studied 25 different three-way junction motifs in biological RNA and found only one other motif that shares characteristics similar to the three-way junction of phi29 pRNA.

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Figure 1: Sequence and secondary structure of phi29 DNA-packaging RNA.
Figure 2: Assembly and stability studies of 3WJ-pRNA.
Figure 3: Competition and dissociation assays of 3WJ-pRNA.
Figure 4: Construction of multi-module RNA nanoparticles harbouring siRNA, ribozyme and aptamer.
Figure 5: In vitro and in vivo binding and entry of 3WJ-pRNA nanoparticles into targeted cells.
Figure 6: Comparison of different 3WJ-RNA cores.

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Acknowledgements

This research was mainly supported by the National Institutes of Health (NIH; grants EB003730, GM059944 and CA151648 to P.G.). P.G. is also a co-founder of Kylin Therapeutics Inc. The authors thank L. Shlyakhtenko and Y. Lyubchenko for AFM images via the Nanoimaging Core Facility supported by the NIH SIG Program and the UNMC Program of ENRI, as well as N. Abdeltawab and Z. Zhu from M. Kotb's laboratory at the University of Cincinnati for help with qRT–PCR assays.

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  1. Dan Shu, Yi Shu and Farzin Haque: These authors contributed equally to this work

Authors and Affiliations

  1. Nanobiomedical Center, University of Cincinnati, Cincinnati, 45267, Ohio, USA

    Dan Shu, Yi Shu, Farzin Haque & Peixuan Guo

  2. Kylin Therapeutics, West Lafayette, Indiana, 47906, USA

    Sherine Abdelmawla

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  1. Dan Shu
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Contributions

P.G. conceived, designed and led the project. D.S., Y.S. and F.H. designed and conducted the in vitro experiments. S.A. performed animal imaging experiments. P.G., D.S., Y.S. and F.H. analysed the data and co-wrote the manuscript.

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Correspondence to Peixuan Guo.

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Shu, D., Shu, Y., Haque, F. et al. Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotech 6, 658–667 (2011). https://doi.org/10.1038/nnano.2011.105

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