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.

• Authors: Qiu-Xia Chen, Wei-Peng Wang, Su Zeng, Shiro Urayama, Ai-Ming Yu
• Journal: Nucleic acids research (Nucleic Acids Res) Vol. 43 Issue 7 Pg. 3857-69 (Apr 20 2015) ISSN: 1362-4962 [Electronic] England
• PMID: 25800741 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Chemical References

• Green Fluorescent Proteins
• RNA

Topics

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