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

作者：Chen, Qiu-Xia; Wang, Wei-Peng; Zeng, Su; Urayama, Shiro; Yu, Ai-Ming^*

来源：Nucleic Acids Research, 2015, 43(7): 3857-3869.

DOI：10.1093/nar/gkv228

• RECOMBINANT RNA
• PANCREATIC RIBONUCLEASE
• SERUM RIBONUCLEASE
• MICRORNAS
• THERAPEUTICS
• PURIFICATION
• TECHNOLOGY
• EXPRESSION
• APTAMERS
• MIRNA

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 I 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/miR124 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.

• 出版日期2015-4-20
• 单位浙江大学