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In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

Published on: March 26, 2012

Production of Fully Capped mRNA for Transfection into Mammalian Cells: A Protocol for Enzymatic Degradation of

Aneta Jurkiewicz¹, Damian Graczyk¹, Roman J Szczesny²

¹Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

Methods in Molecular Biology (Clifton, N.J.)

|November 13, 2024

Abstract:

The functionality of messenger RNA, such as stability and translation, is determined by several elements. In Eukaryotes, the 5' end of the mRNA is modified to contain a 5' cap structure, the presence of which protects the mRNA from degradation by 5' to 3' exoribonucleases and promotes mRNA translation. The in vitro synthesis of RNA has recently attracted ample attention for its application as a source of therapeutic agents or research tools. Although in vitro mRNA synthesis (IVT) methodology is well established and is still being improved, not all synthesized RNA molecules have the expected properties. For example, the co-transcriptional addition of a 5' cap is frequently incomplete. Yet, the uncapped mRNA molecules are undesirable and must be removed before further processing. Here, we present a protocol for the enzymatic removal of uncapped RNA molecules. This approach offers an excellent opportunity to enrich IVT products with fully competent, 5' cap-containing mRNA molecules.

Keywords:

Cap IVT In vitro transcription N1-methyl-pseudouridine mRNA

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