Studies on expression of RNA sequences embedded into a stable 5S rRNA-based scaffold

The feasibility of a fermentation-based system for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. A small RNA of interest is incorporated into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene. After transformation into Escherichia coli, the chimeric gene is expressed constitutively from a strong promoter to produce a chimeric RNA. Primary transcripts of the chimeric gene are processed and modified by the same set of maturation enzymes as endogenous 5S rRNA precursors to produce a single artificial RNA (aRNA) species of defined sequence and length. The 160 nucleotide 3xpen aRNA accumulated to levels that equaled or exceeded those of 5S rRNA. The insert of interest was quantitatively excised from the chimera at 23°C using a pair of 8-17 DNAzymes. It was found that increasing time of exposure to these enzymes was more important than increasing their amounts. An alternative method for purifying total RNA from bacterial cells was developed that uses non-toxic agents and separates a liquid from a precipitant. This protocol is more suitable for large-scale total RNA purification than traditional methods. It is anticipated that the 5S rRNA scaffold approach combined with fermentation will provide a valuable economic method for large-scale production of small functional RNAs such as shRNA, siRNAs and aptamers for use in clinical trials and biomedical research in general.;5S rRNA chimeras were also examined for use in in vivo studies of molecular evolution. An artificial operon was designed to express chimeric RNA molecules with 50 nucleotide randomized sequences inserted into the 5S rRNA scaffold. This plasmid-born gene library was transformed into E. coli cells and used as an initial source of genetic variance during stress-driven selection using subinhibitory concentrations of NiCl2. At the end of the selection, the minimal inhibitory concentration increased from 4 to 12 mM NiCl2 and each individual culture expressed an essentially unique chimeric RNA. However, the nickel resistance of the strain with the highest resistance was minimally enhanced when the plasmid borne RNA was induced using IPTG. Additional studies confirmed that the presence of the selected RNA was likely not responsible for the major portion of the enhanced resistance. The possibility that minimal expression of the RNA was increasing resistance through a regulatory role could not be ruled out. It is concluded that the enhanced resistance of the final cultures was likely largely the result of adaptive mutations in the host genomic enhanced with modest additional selective advantage imparted by the RNA. Thus, a minimally helpful, version of the RNA may have been largely carried along by genetic hitch-hiking. Improvements in the experimental design are proposed and other applications of the approach discussed.