In Vivo Production of Small Recombinant RNAs Embedded in a 5S rRNA-Derived Protective Scaffold



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Methods in Molecular Biology


Preparative synthesis of RNA is a challenging task that is usually accomplished using either chemical or enzymatic polymerization of ribonucleotides in vitro. Herein, we describe an alternative approach in which RNAs of interest are expressed as a fusion with a 5S rRNA-derived scaffold. The scaffold provides protection against cellular ribonucleases resulting in cellular accumulations comparable to those of regular ribosomal RNAs. After isolation of the chimeric RNA from the cells, the scaffold can be removed if necessary by deoxyribozyme-catalyzed cleavage followed by preparative electrophoretic separation of the cleavage reaction products. The protocol is designed for sustained production of high quality RNA on the milligram scale.



Recombinant RNA, In vivo RNA production, 5S rRNA-derived scaffold


Copyright 2015 Methods in Molecular Biology. Recommended citation: Stepanov, Victor G., and George E. Fox. "In vivo production of small recombinant RNAs embedded in a 5S rRNA-derived protective scaffold." In Rna Scaffolds, pp. 45-65. Humana Press, New York, NY, 2015. DOI: 10.1007/978-1-4939-2730-2_5. URL: Reproduced in accordance with the original publisher's licensing terms and with permission from the author(s),