title: 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: Department of Biochemistry & Molecular Medicine, School of Medicine, UC Davis, Sacramento, CA 95817, USA Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors: Qiu-Xia
authors: QX
authors: Chen
authors: Department of Biochemistry & Molecular Medicine, School of Medicine, UC Davis, Sacramento, CA 95817, USA.
authors: Wei-Peng
authors: WP
authors: Wang
authors: Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors: Su
authors: S
authors: Zeng
authors: Department of Internal Medicine, School of Medicine, UC Davis, Sacramento, CA 95817, USA.
authors: Shiro
authors: S
authors: Urayama
authors: Department of Biochemistry & Molecular Medicine, School of Medicine, UC Davis, Sacramento, CA 95817, USA aimyu@ucdavis.edu.
authors: Ai-Ming
authors: AM
authors: Yu
mesh_terms: D000818:Animals; D001483:Base Sequence; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D009690:Nucleic Acid Conformation; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D011995:Recombination, Genetic
chemical_list: D049452:Green Fluorescent Proteins; D012313:RNA