title: Functional RNAs: combined assembly and packaging in VLPs.
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 5΄ 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 3΄UTR 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 target-specific and efficient.
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA.
authors.forename: Po-Yu
authors.initials: PY
authors.lastname: Fang
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA.|School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
authors.forename: Lizzette M
authors.initials: LM
authors.lastname: Gómez Ramos
authors.affiliation: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
authors.forename: Stefany Y
authors.initials: SY
authors.lastname: Holguin
authors.affiliation: Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, Republic of China.
authors.forename: Chiaolong
authors.initials: C
authors.lastname: Hsiao
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA.
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Bowman
authors.affiliation: Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan, Republic of China.
authors.forename: Hung-Wei
authors.initials: HW
authors.lastname: Yang
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA.
authors.forename: Loren Dean
authors.initials: LD
authors.lastname: Williams
mesh_terms: D020413:3' Untranslated Regions; D017908:Allolevivirus / Q000235:genetics; D036022:Capsid Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006801:Humans; D009690:Nucleic Acid Conformation; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D036022:Capsid Proteins; D034741:RNA, Small Interfering; C043366:coat protein, Bacteriophage Qbeta; D016283:Proto-Oncogene Proteins p21(ras)