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Bioengineering of noncoding RNAs for research agents and therapeutics

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The discovery of functional small noncoding RNAs (ncRNAs), such as microRNAs and small interfering RNAs, in the control of human cellular processes has opened new avenues to develop RNA‐based therapies for various diseases including viral infections and cancers. However, studying ncRNA functions and developing RNA‐based therapeutics relies on access to large quantities of affordable ncRNA agents. Currently, synthetic RNAs account for the major source of agents for RNA research and development, yet carry artificial modifications on the ribose ring and phosphate backbone in sharp contrast to posttranscriptional modifications present on the nucleobases or unmodified natural RNA molecules produced within cells. Therefore, large efforts have been made in recent years to develop recombinant RNA techniques to cost‐effectively produce biological RNA agents that may better capture the structure, function, and safety properties of natural RNAs. In this article, we summarize and compare current in vitro and in vivo methods for the production of RNA agents including chemical synthesis, in vitro transcription, and bioengineering approaches. We highlight the latest recombinant RNA approaches using transfer RNA (tRNA), ribosomal RNA (rRNA), and optimal ncRNA scaffold (OnRS), and discuss the applications of bioengineered ncRNA agents (BERAs) that should facilitate RNA research and development. WIREs RNA 2016, 7:186–197. doi: 10.1002/wrna.1324 This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Methods > RNA Analyses In Vitro and In Silico RNA Methods > RNA Analyses in Cells
RNA‐based drugs act mechanistically on genome derived target transcript, which are different from small‐molecule and protein therapeutics that interact with target proteins. In addition, RNA aptamers may directly bind to target protein and exert pharmacological effects.
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The robust optimal noncoding RNA scaffold (OnRS) approach may be employed to construct OnRS/sRNA library for high‐throughput screening of more effective OnRS/sRNA agents and examination of desired phenotype.
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Bioengineering ncRNA agents for research and development. The ncRNA coding sequence is cloned into a target vector and transformed in E. coli. Total RNAs are extracted from bacterial cultures and verified for the overexpression of target ncRNA (red arrow). BERAs are then purified by anion exchange FPLC method. Pure BERAs are subjected to structural characterization including posttranscriptional modifications, as well as preclinical and clinical investigation of biological fate, activity, effectiveness, and safety properties.
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Recombinant RNA approaches using tRNA, 5S rRNA, and chimeric tRNA/pre‐miRNA as a scaffold. The tRNA scaffold allows the replacement of anticodon sequence by RNA species of interest, and the 5S rRNA scaffold permits the insertion of desired RNAs into Stem II or substitution of Stems II and III. The optimal noncoding RNA scaffold (OnRS) approach encompassing a stable tRNA/pre‐miR‐34a structure offers the flexibility to accommodate various types of small RNAs (sRNAs) and most importantly, retain a high‐yield, large‐scale production of biological ncRNA chimeras.
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Comparison of some common posttranscriptional modifications of natural RNAs and artificial modifications of synthetic RNAs. It is noteworthy that natural RNAs are usually modified on the nucleobases (e.g., pseudouridine, 1‐methyladenosine, 7‐methylguanosine, 5‐methylcytidine, etc.), whereas synthetic RNAs display modifications on the phosphate linkage (e.g., phosphorothioate) or sugar ring (e.g., 2′‐fluoro, locked nucleic acid, 2′‐O‐methoxy‐ethyl RNA, etc.).
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RNA Methods > RNA Analyses in Cells
Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
RNA Methods > RNA Analyses In Vitro and In Silico

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