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

Nucleic Acids Res. 2015 Apr 20;43(7):3857-69. doi: 10.1093/nar/gkv228. Epub 2015 Mar 23.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence
  • Gene Knockdown Techniques
  • Green Fluorescent Proteins / genetics
  • Mice
  • Mice, Transgenic
  • Nucleic Acid Conformation
  • RNA / biosynthesis*
  • RNA / genetics
  • RNA / physiology
  • Recombination, Genetic


  • Green Fluorescent Proteins
  • RNA