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In Vitro Transcription of Modified RNAs

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Recombinant and In Vitro RNA Synthesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 941))

Abstract

RNAs containing a variety of terminal and internal modifications can be produced using bacteriophage polymerases often with a few simple adjustments to standard transcription protocols. RNAs containing a single phosphate or a cap structure at their 5′ ends can readily be generated either co-transcriptionally or through enzymatic treatments of transcription products. Likewise, a variety of modified bases, including fluorescent or biotinylated species, can be effectively incorporated co-transcriptionally. The key to effective co-transcriptional incorporation lies in determining the efficiency of incorporation of modified base relative to its standard counterpart. Finally, an approach to place a poly(A) tail at the exact 3′ end of a desired transcription product is presented. Collectively, these protocols allow one to synthesize RNAs with a variety of modifications to serve as versatile molecules to analyze biological questions.

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Acknowledgments

We wish to thank members of the Wilusz Laboratories for their input and helpful comments. RNA research in the laboratory is supported by grants from the NIH (R01-GM072481 and U54-AI-065357) to J.W. S.L.M. is supported by a training grant from the USDA (2010-38420-20367).

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Correspondence to Jeffrey Wilusz .

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Moon, S.L., Wilusz, J. (2013). In Vitro Transcription of Modified RNAs. In: Conn, G. (eds) Recombinant and In Vitro RNA Synthesis. Methods in Molecular Biology, vol 941. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-113-4_13

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  • DOI: https://doi.org/10.1007/978-1-62703-113-4_13

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-112-7

  • Online ISBN: 978-1-62703-113-4

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