DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

Abstract

BackgroundManufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis.ResultsSequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse.ConclusionsThe feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.

A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications

“…With a great interest in developing new strategies to bioengineer ready-to-use RNAi agents on a large scale, a successful example has been reported very recently for the generation of fully-processed siRNAs from p19-expressing bacteria ( 12 ). On the other hand, tRNA ( 13 – 15 ) and rRNA ( 16 ) have been employed as scaffolds to produce a number of chimeric RNAs in common strains of bacteria, given the fact that tRNAs and rRNAs are present as stable RNA molecules in the cells. The recombinant RNA chimeras are thus isolated, and the target RNAs may be released in demand by corresponding RNase ( 13 , 14 ), ribozyme ( 15 ) or DNAzyme ( 16 ) for structural and biophysical analyses.…”

Rapid Production of Novel Pre-MicroRNA Agent hsa-mir-27b in Escherichia coli Using Recombinant RNA Technology for Functional Studies in Mammalian Cells

“…In vitro transcription (Beckert and Masquida, 2011) may produce RNA agents in variable lengths, whereas a large-scale production requires more but inexpensive RNA polymerases. Recently, tRNA (Ponchon and Dardel, 2007;Ponchon et al, 2009;Nelissen et al, 2012) and ribosomal RNA (Liu et al, 2010) have been used as scaffolds for successful production of recombinant RNAs in Escherichia coli (E. coli) for structural analyses. This recombinant RNA technology may also offer a novel means to biosynthesize RNA agents for functional studies.…”

Bioengineering of noncoding RNAs for research agents and therapeutics

“…Due to the abundance and stability of ribosomal RNAs in E. coli , the 5S rRNA was shown to be strongly expressed under the control of E. coli rrnB promoter and rrnB T1 and T2 terminators . Thus the 5S rRNA was exploited to serve as a scaffold for the production of recombinant ncRNA species (Figure ) . Specifically, a 71 nt artificial 3x pen RNA was inserted into stem II and completely replaced the stem III loop B and C regions of 5S rRNA, which maintained the highly structured rRNA intact, and cleavable by DNAzymes.…”