Fabrication of pRNA nanoparticles to deliver therapeutic RNAs and bioactive compounds into tumor cells

核糖核酸 核酶 适体 纳米颗粒 生物物理学 基因沉默 纳米技术 小干扰RNA 体外 DNA 化学 生物 材料科学 细胞生物学 生物化学 基因 分子生物学
作者
Yi Shu,Dan Shu,Farzin Haque,Peixuan Guo
出处
期刊:Nature Protocols [Nature Portfolio]
卷期号:8 (9): 1635-1659 被引量:113
标识
DOI:10.1038/nprot.2013.097
摘要

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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