Abstract
RNA nanotechnology is a term that refers to the design, fabrication and use of nanoparticles that are mainly composed of RNAs via bottom-up self-assembly. The packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor has been developed into a nanodelivery platform. This protocol describes the synthesis, assembly and functionalization of pRNA nanoparticles on the basis of three 'toolkits' derived from pRNA structural features: interlocking loops for hand-in-hand interactions, palindrome sequences for foot-to-foot interactions and an RNA three-way junction for branch extension. siRNAs, ribozymes, aptamers, chemical ligands, fluorophores and other functionalities can also be fused to the pRNA before the assembly of the nanoparticles, so as to ensure the production of homogeneous nanoparticles and the retention of appropriate folding and function of the incorporated modules. The resulting self-assembled multivalent pRNA nanoparticles are thermodynamically and chemically stable, and they remain intact at ultralow concentrations. Gene-silencing effects are progressively enhanced with increasing numbers of siRNAs in each pRNA nanoparticle. Systemic injection of the pRNA nanoparticles into xenograft-bearing mice has revealed strong binding to tumors without accumulation in vital organs or tissues. The pRNA-based nanodelivery scaffold paves a new way for nanotechnological application of pRNA-based nanoparticles for disease detection and treatment. The time required for completing one round of this protocol is 3–4 weeks when including in vitro functional assays, or 2–3 months when including in vivo studies.
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Acknowledgements
The research was supported by US National Institutes of Health (NIH) grants EB003730 and CA151648, and by the Arnold and Mabel Beckman Initiative for Macular Research External Grant 1108 to P. Guo. AFM images were obtained by L. Shlyakhtenko and Y. Lyubchenko at the Nanoimaging Core Facility, University of Nebraska Medical Center, supported by the NIH Shared Instrumentations Grant Program and the University of Nebraska Medical Center Program Excellence (POE) and the Nebraska Research Initiative (NRI). We thank D. Rodger and M. Chow at University of Kentucky for the help with dynamic light scattering; F. Huang at The University of Southern Mississippi for providing Cy3- and Cy5-labeled AMP and GMP; and J. Haak, E. Khisamutdinov, E. Beabout, and F. Pi for help with the manuscript preparation.
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P.G. conceived and led the project. Y.S., D.S. and F.H. designed and conducted the experiments and co-wrote the manuscript with P.G.
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P.G. is a co-founder of Kylin Therapeutics and Biomotor and Nucleic Acid Nanotechnology Development Corp.
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Shu, Y., Shu, D., Haque, F. et al. Fabrication of pRNA nanoparticles to deliver therapeutic RNAs and bioactive compounds into tumor cells. Nat Protoc 8, 1635–1659 (2013). https://doi.org/10.1038/nprot.2013.097
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DOI: https://doi.org/10.1038/nprot.2013.097
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