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

Abstract

RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.

Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity

“…Consequently, mature miR-1291 levels were increased by 2 orders of magnitude, indicating a successful processing of tRNA/mir-1291 to mature miR-1291. It is noteworthy that all recombinant ncRNAs, such as tRNA/mir-27 , tRNA/ mir-1291 (this study), and tRNA/mir-34a (Chen et al, 2015) (unpublished data), showed favorable cellular stability and selective generation of target mature miRNAs, suggesting that tRNA carrier also offers a "stealth delivery" of target miRNAs into human cells beyond the production of chimeric ncRNAs in bacteria. Nevertheless, further studies are warranted to define the specific RNases involved in the processing of recombinant ncRNAs in human cells.…”

“…In addition, miR-1291 has been revealed to act as a tumor suppressor in various human carcinoma cell lines by our studies Bi et al, 2014) (unpublished data) as well as others (Hidaka et al, 2012;Yamasaki et al, 2013). Motivated by the idea of producing natural miRNA agents to perform miRNA actions, we made large efforts to biosynthesize pre-miR-1291 agents using the tRNA scaffold to examine miR-1291 biologic functions and therapeutic implications in vitro and in vivo Chen et al, 2015). The success and efficiency in producing recombinant RNAs relies on the structure and metabolic stability of target RNA in the organism.…”

“…The success and efficiency in producing recombinant RNAs relies on the structure and metabolic stability of target RNA in the organism. It is obvious that any target RNAs labile to bacterial RNase digestion are undoubtedly subjected to nucleolytic cleavage, and thus there will be limited or no accumulation of recombinant RNAs (e.g., pre-miR-27b) Chen et al, 2015). Recombinant pre-miR-1291 chimeras were revealed to be expressed successfully in a number of common E. coli strains, and a high-level accumulation was observed in HST08 and DH5a cells, similar to premiR-34a (Chen et al, 2015).…”

“…It is obvious that any target RNAs labile to bacterial RNase digestion are undoubtedly subjected to nucleolytic cleavage, and thus there will be limited or no accumulation of recombinant RNAs (e.g., pre-miR-27b) Chen et al, 2015). Recombinant pre-miR-1291 chimeras were revealed to be expressed successfully in a number of common E. coli strains, and a high-level accumulation was observed in HST08 and DH5a cells, similar to premiR-34a (Chen et al, 2015). Consistent with previous findings (Ponchon and Dardel, 2007;Ponchon et al, 2009), lower levels of recombinant RNAs were found for longer pre-miR-1291 species, which is presumably due to an increase of unstructured regions that are misfolded and/or cleaved by bacterial RNases.…”

“…Consistent with previous findings (Ponchon and Dardel, 2007;Ponchon et al, 2009), lower levels of recombinant RNAs were found for longer pre-miR-1291 species, which is presumably due to an increase of unstructured regions that are misfolded and/or cleaved by bacterial RNases. High-level expression also facilitated the purification of recombinant tRNA/ mir-1291-123nt, which was consistently produced in multimilligram quantity from 1-l fermentation that would allow us to assess miR-1291 activities in vivo, as we have done with other efficiently expressed ncRNAs (Chen et al, 2015) (unpublished data). Despite that the purification yields using Sephadex G-100 were relatively low (e.g., 2-3%), this method is simple, and the Sephadex-tagged recombinant ncRNAs that failed to bind to Sephadex in the first round (e.g., flow through and wash 1-3) may be combined for repurification to offer a much improved total yield (e.g., 5-10%) close to anion exchange fast protein liquid chromatography method Chen et al, 2015).…”