Functional RNAs: Combined assembly and packaging in VLPs
Journal
Nucleic Acids Research
Journal Volume
45
Journal Issue
6
Pages
3519 - 3527
Date Issued
2017
Author(s)
Fang, Po-Yu
Ramos, Lizzette M. Gomez
Holguin, Stefany Y.
Bowman, Jessica C.
Yang, Hung-Wei
Williams, Loren Dean
Abstract
We describe here a one pot RNA production, packaging and delivery system based on bacteriophage Qβ. We demonstrate a method for production of a novel RNAi scaffold, packaged within Qβ virus-like particles (VLPs). The RNAi scaffold is a general utility chimera that contains a functional RNA duplex with paired silencing and carrier sequences stabilized by a miR-30 stem-loop. The Qβ hairpin on the 5β end confers affinity for the Qβ coat protein (CP). Silencing sequences can include mature miRNAs and siRNAs, and can target essentially any desired mRNA. The VLP-RNAi assembles upon co-expression of CP and the RNAi scaffold in E. coli. The annealing of the scaffold to form functional RNAs is intramolecular and is therefore robust and concentration independent. We demonstrate dose-and time-dependent inhibition of GFP expression in human cells with VLP-RNAi. In addition, we target the 3βUTR of oncogenic Ras mRNA and suppress Pan-Ras expression, which attenuates cell proliferation and promotes mortality of brain tumor cells. This combination of RNAi scaffold design with Qβ VLP packaging is demonstrated to be targetspecific and efficient. ? 2016 The Author(s).
SDGs
Other Subjects
coat protein; functional RNA; green fluorescent protein; messenger RNA; microRNA; microRNA 30; Ras protein; RNA; small interfering RNA; small interfering RNA let 7; unclassified drug; 3' untranslated region; capsid protein; coat protein, Bacteriophage Qbeta; protein p21; small interfering RNA; 3' untranslated region; Article; binding affinity; brain tumor cell line; cell killing; cell proliferation; concentration response; confocal microscopy; controlled study; Enterobacteria phage Qbeta; flow cytometry; fluorescence microscopy; gene expression; gene silencing; HeLa cell line; human; human cell; nonhuman; Northern blotting; priority journal; protein expression; RNA assembly; RNA interference; RNA packaging; RNA processing; RNA stability; RNA synthesis; viral gene delivery system; viral gene therapy; virus like agent; Allolevivirus; antagonists and inhibitors; chemistry; conformation; genetics; metabolism; RNA interference; tumor cell line; virion; 3' Untranslated Regions; Allolevivirus; Capsid Proteins; Cell Line, Tumor; Cell Proliferation; Humans; Nucleic Acid Conformation; Proto-Oncogene Proteins p21(ras); RNA Interference; RNA, Small Interfering; Virion
Type
journal article