## 2 Production of Recombinant dsRNA in Microorganisms
### Escherichia coli
The GST-p19-His protein/siRNA complex can be easily purified from _E. coli_ cell lysate by Ni-NTA affinity chromatography. The target siRNA species are eluted using 0.1% sodium dodecyl sulfate (SDS) solution. approach is to produce RNAs that have a stable and compact three-dimensional structure that is resistant to ribonucleases in vivo, and to carry the target RNA segment on this scaffold. Such scaffolds for the production of recombinant RNA molecules of interest include tRNA [46; 47; 48], 5S rRNA [49; 50], tRNA/pre-miRNA structures [51; 52], and viroid-derived RNA structures [53; 54]. The concept of using the structures of tRNA and 5S rRNA as scaffolds is based on the rationale that these structures will be recognized as endogenous molecules by the RNA metabolizing machinery in the producing bacteria, resulting in suppression of rapid degradation of the additional RNA segment of interest. [PERSON] et al. [46; 47] designed a method that uses tRNA as a protective scaffold for the production of desired RNA molecules in _E. coli_ and for convenient isolation of the target chimeric RNA. Specifically, the RNA segment of interest was inserted into the anticodon stem of the tRNA-scaffold, and an aptamer-tag (e.g., a Sephadex aptamer-based affinity tag) was also incorporated into the scaffold for efficient purification of the chimeric RNA molecules. The authors succeeded in obtaining a chimeric RNA molecule containing the target RNA segment, which could be easily purified. They produced several RNA fragments of pharmacological interest by this method, and tens of milligram quantities of the RNA (as purified chimeras) were obtained from 1 L scale culture. To finally obtain the target RNA from the chimera with the scaffold, an in vitro RNase H-cleavage method directed by two DNA oligonucleotides complementary to the invariable part of the tRNA-scaffold was employed. Subsequently, [PERSON] et al. [48] improved this tRNA-scaffold approach. First, to further enhance RNA expression, the promoter was changed from the constitutive promoter of the _lpp_ gene (encoding the major outer membrane lipoprotein in _E. coli_) to the inducible T7 promoter. Then, to enable efficient and simple excision of the target RNA segment from the produced tRNA-scaffold chimera, a _cis_-acting hammerhead (HH) ribozyme was incorporated into the chimeric structure, and a method of isolating the target RNA molecule by HH ribozyme-mediated cleavage was devised. In this system, several of the chimeric RNAs showed a yield of approximately 10-30 mg per liter of culture medium. These RNA expression systems are suitable for the production of relatively short RNAs, such as therapeutic siRNAs, for applications that require structurally homogeneous and highly pure RNAs. For agricultural applications, high structural homogeneity of the target RNA is not necessarily required, but such technology is also expected to be applied for the higher accumulation of RNAs of interest for agricultural uses in bacteria. Subsequently, based on tRNA-scaffold technology, [PERSON] et al. [51] further investigated the expression of chimeric RNAs by fusing several kinds of pre-miRNAs to the tRNA-scaffold with _E. coli_ as the host. They developed an RNA expression system that uses the tRNA/pre-miR-34a scaffold as a new, optimal non-coding RNA scaffold (named OnRS) for production of functional small RNAs; the fusion construct with pre-miR-34a was particularly highly expressed in _E. coli_. This scaffold was also resistant to nucleolytic digestion in _E. coli_, and multi-milligrams of the chimeric RNAs were readily obtained per liter of bacterial culture. They further developed this system and designed a new scaffold, tRNA/pre-miR-34a/pre-miR-34a, by adding an additional human pre-miR-34a to the OnRS structure, and used it to produce multiple small RNAs of interest [52]. The chimeras containing the target RNAs were successfully expressed in _E. coli_ at high levels (>40% of the total bacterial RNA).