A Robust and Versatile Method for Production and Purification of Large-scale RNA Samples for Structural Biology

Overview

Journal RNA

Specialty Molecular Biology

Date 2020 May 2

PMID 32354720

Citations 10

Authors

Hampus Karlsson

Lorenzo Baronti

Katja Petzold

Affiliations

Soon will be listed here.

Abstract

Recent findings in genome-wide transcriptomics revealed that RNAs are involved in almost every biological process, across all domains of life. The characterization of native RNAs of unknown function and structure is particularly challenging due to their typical low abundance in the cell and the inherent sensitivity toward ubiquitous RNA degrading enzymes. Therefore, robust in vitro synthesis and extensive work-up methods are often needed to obtain samples amenable for biochemical, biophysical, and structural studies. Here, we present a protocol that combines the most recent advances in T7 in vitro transcription methodology with reverse phase ion pairing and ion exchange HPLC purification of RNAs for the production of yield-optimized large-scale samples. The method is easy to follow, robust and suitable for users with little or no experience within the field of biochemistry or chromatography. The complete execution of this method, for example, for production of isotopically labeled NMR samples, can be performed in less than a week.

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References

Kanwal F, Chen T, Zhang Y, Simair A, Lu C . A Modified In Vitro Transcription Approach to Improve RNA Synthesis and Ribozyme Cleavage Efficiency. Mol Biotechnol. 2019; 61(7):469-476. DOI: 10.1007/s12033-019-00167-5. View

Price S, Ito N, Oubridge C, Avis J, Nagai K . Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. J Mol Biol. 1995; 249(2):398-408. DOI: 10.1006/jmbi.1995.0305. View

Tuma R, Beaudet M, Jin X, Jones L, Cheung C, Yue S . Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators. Anal Biochem. 1999; 268(2):278-88. DOI: 10.1006/abio.1998.3067. View

Hagen F, Young E . Preparative polyacrylamide gel electrophoresis of ribonucleic acid. Identification of multiple molecular species of bacteriophage T7 lysozyme messenger ribonucleic acid. Biochemistry. 1974; 13(16):3394-400. DOI: 10.1021/bi00713a033. View

McKenna S, Kim I, Puglisi E, Lindhout D, Aitken C, Marshall R . Purification and characterization of transcribed RNAs using gel filtration chromatography. Nat Protoc. 2007; 2(12):3270-7. DOI: 10.1038/nprot.2007.480. View

. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012; 489(7414):57-74. PMC: 3439153. DOI: 10.1038/nature11247. View

Schlagnitweit J, Friebe Sandoz S, Jaworski A, Guzzetti I, Aussenac F, Carbajo R . Observing an Antisense Drug Complex in Intact Human Cells by in-Cell NMR Spectroscopy. Chembiochem. 2019; 20(19):2474-2478. DOI: 10.1002/cbic.201900297. View

Pereira P, Queiroz J, Figueiras A, Sousa F . Affinity approaches in RNAi-based therapeutics purification. J Chromatogr B Analyt Technol Biomed Life Sci. 2016; 1021:45-56. DOI: 10.1016/j.jchromb.2016.01.022. View

Parisien M, Major F . The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data. Nature. 2008; 452(7183):51-5. DOI: 10.1038/nature06684. View

10.

Ferre-DAmare A, Doudna J . Use of cis- and trans-ribozymes to remove 5' and 3' heterogeneities from milligrams of in vitro transcribed RNA. Nucleic Acids Res. 1996; 24(5):977-8. PMC: 145725. DOI: 10.1093/nar/24.5.977. View

11.

Akoopie A, Muller U . Cotranscriptional 3'-End Processing of T7 RNA Polymerase Transcripts by a Smaller HDV Ribozyme. J Mol Evol. 2018; 86(7):425-430. DOI: 10.1007/s00239-018-9861-9. View

12.

McGinnis A, Chen B, Bartlett M . Chromatographic methods for the determination of therapeutic oligonucleotides. J Chromatogr B Analyt Technol Biomed Life Sci. 2011; 883-884:76-94. DOI: 10.1016/j.jchromb.2011.09.007. View

13.

Lorenz R, Bernhart S, Honer Zu Siederdissen C, Tafer H, Flamm C, Stadler P . ViennaRNA Package 2.0. Algorithms Mol Biol. 2011; 6:26. PMC: 3319429. DOI: 10.1186/1748-7188-6-26. View

14.

Wichlacz A, Legiewicz M, Ciesiolka J . Generating in vitro transcripts with homogenous 3' ends using trans-acting antigenomic delta ribozyme. Nucleic Acids Res. 2004; 32(3):e39. PMC: 373431. DOI: 10.1093/nar/gnh037. View

15.

Steiner E, Schlagnitweit J, Lundstrom P, Petzold K . Capturing Excited States in the Fast-Intermediate Exchange Limit in Biological Systems Using H NMR Spectroscopy. Angew Chem Int Ed Engl. 2016; 55(51):15869-15872. DOI: 10.1002/anie.201609102. View

16.

Kim I, McKenna S, Puglisi E, Puglisi J . Rapid purification of RNAs using fast performance liquid chromatography (FPLC). RNA. 2006; 13(2):289-94. PMC: 1781364. DOI: 10.1261/rna.342607. View

17.

Beckert B, Masquida B . Synthesis of RNA by in vitro transcription. Methods Mol Biol. 2010; 703:29-41. DOI: 10.1007/978-1-59745-248-9_3. View

18.

Kimsey I, Petzold K, Sathyamoorthy B, Stein Z, Al-Hashimi H . Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexes. Nature. 2015; 519(7543):315-20. PMC: 4547696. DOI: 10.1038/nature14227. View

19.

Weissman D, Pardi N, Muramatsu H, Kariko K . HPLC purification of in vitro transcribed long RNA. Methods Mol Biol. 2013; 969:43-54. DOI: 10.1007/978-1-62703-260-5_3. View

20.

Cunningham L, Kittikamron K, Lu Y . Preparative-scale purification of RNA using an efficient method which combines gel electrophoresis and column chromatography. Nucleic Acids Res. 1996; 24(18):3647-8. PMC: 146136. DOI: 10.1093/nar/24.18.3647. View