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

被引:73
作者
Chen, Qiu-Xia [1 ,2 ]
Wang, Wei-Peng [1 ]
Zeng, Su [2 ]
Urayama, Shiro [3 ]
Yu, Ai-Ming [1 ]
机构
[1] Univ Calif Davis, Sch Med, Dept Biochem & Mol Med, Sacramento, CA 95817 USA
[2] Zhejiang Univ, Coll Pharmaceut Sci, Lab Pharmaceut Anal & Drug Metab, Zhejiang Prov Key Lab Anticanc Drug Res, Hangzhou 310058, Zhejiang, Peoples R China
[3] Univ Calif Davis, Sch Med, Dept Internal Med, Sacramento, CA 95817 USA
基金
美国国家卫生研究院;
关键词
RECOMBINANT RNA; PANCREATIC RIBONUCLEASE; SERUM RIBONUCLEASE; MICRORNAS; THERAPEUTICS; PURIFICATION; TECHNOLOGY; EXPRESSION; APTAMERS; MIRNA;
D O I
10.1093/nar/gkv228
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:3857 / 3869
页数:13
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