Fabrication of pRNA nanoparticles to deliver therapeutic RNAs and bioactive compounds into tumor cells.
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.
Nanobiotechnology Center, Markey Cancer Center, Lexington, Kentucky, USA.
Yi
Y
Shu
Dan
D
Shu
Farzin
F
Haque
Peixuan
P
Guo
D000818:Animals; D017103:Bacillus Phages / Q000235:genetics; D001483:Base Sequence; D045744:Cell Line, Tumor; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods*; D064593:Heterografts; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry; D034741:RNA, Small Interfering / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry*; D013816:Thermodynamics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; D016337:RNA, Catalytic; D034741:RNA, Small Interfering; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases