Efficient and specific gene knockdown by small interfering RNAs produced in bacteria

Abstract

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells1,2 and may be used for therapeutic purposes to knockdown genes implicated in disease3. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in E. coli. This method relies on ectopic expression of p19, a siRNA-binding protein found in a plant RNA virus4, 5. When expressed in E. coli, p19 stabilizes ~21 nt siRNA-like species produced by bacterial RNase III. Transfection of mammalian cells with siRNAs, generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene, at low nanomolar concentrations reproducibly knocks down gene expression by ~90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.

Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization

“…The better expression of recombinant ncRNA in HST08 strain may be related to the lack of gene clusters in HST08 cells for digesting methylated DNA or a lower capacity to polyadenylate ncRNA for degradation. Notably, our strategy is different from a newly reported approach of generating fully-processed small interfering RNAs using p19-expressing bacteria (Huang et al, 2013). A high-yield accumulation of recombinant tRNA/mir-34a in bacteria (∼15% of total RNAs) also facilitated the purification by anion-exchange FPLC method to a high degree of homogeneity (.98%).…”

Bioengineered non-coding RNA agent (BERA) in action

“…One reported approach is to use siRNA-binding protein to stabilize and enrich target siRNA molecules in cells 22 (Table 1). A 19 kD siRNA-binding protein, p19, [23][24][25] was employed to bind to recombinant siRNA to form a siRNA-p19 complex and then purified by nickel affinity chromatography and followed by anion exchange high performance liquid chromatography (HPLC).…”

“…A 19 kD siRNA-binding protein, p19, [23][24][25] was employed to bind to recombinant siRNA to form a siRNA-p19 complex and then purified by nickel affinity chromatography and followed by anion exchange high performance liquid chromatography (HPLC). 22 While this method allows the production of fully-processed, ready-to-use siRNAs or miRNAs, the overall yield is very low and it unlikely provides a much desired large quantity of target siRNA agents. Figure 2.…”

“…37,38 In addition, recombinant ncRNAs from, such as the siRNAs isolated from p19 complex and miRNAs/ siRNAs from OnRS platform (Table 1), are biologically active in the regulation of target gene expression in mammalian cells, which have been demonstrated in many studies. 22,34,35,40,43,44 Specifically, bioengineered tRNA/ mir-27b was found to be processed to mature miR-27b in human carcinoma cells, which consequently reduced CYP3A4 protein expression and led to a lower midazolam 1'-hydroxylase activity. 35 The tRNA/mir-1291 was readily processed to mature miR-1291 in breast cancer MCF-7 cells and pancreatic cancer PANC-1 cells.…”

Production of highly potent recombinant siRNAs in Escherichia coli

“…Large quantities of long dsRNAs can be made in RNase III-deficient E. coli overexpressing T7 RNA polymerase 18 or ϕ6 RNA-dependent RNA polymerase 19 . Recently, we developed a protocol for producing highly potent and pure siRNAs directly from E. coli cells 20 . Those siRNAs, which induce ~90% gene knockdown when used at 2 nM concentrations 20 , are generated from long dsRNAs (>100 nt in length) and contain multiple siRNA sequences.…”