Abstract
During in vitro run-off transcription with T7 RNA polymerase, transcripts with heterogenous 3′ ends are commonly synthesized. Here, we describe an efficient procedure for correct processing of transcript 3′ ends with the use of antigenomic HDV ribozyme. The procedure involves the extension of nascent transcripts with seven nucleotides complementary to the ribozyme’s recognition site and, subsequently, the removal of those nucleotides with the HDV ribozyme acting in trans. Sufficient reaction rates and final cleavage extents of approx. 90% can be obtained with just twofold excess of the ribozyme. The highest concentration of RNA substrate suggested for practical applications turns out to be 3 μM. The procedure is an alternative to the use of ribozymes as cis-cleaving autocatalytic cassettes attached to transcript 3′ ends.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Milligan JF, Groebe JF, Witherell DR, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15:8783–8798
Cazenave C, Uhlenbeck OC (1994) RNA template-directed RNA synthesis by T7 RNA polymerase. Biochemistry 91:6972–6976
Price SR, Ito N, Outbridge C, Avis JM, Nagai K (1995) Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. J Mol Biol 249:398–408
Sherlin LD, Bullock TL, Nissan TA, Perona JJ, Lariviere FJ, Uhlenbeck OC, Scaringe SA (2001) Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization. RNA 7:1671–1678
Grosshans CA, Cech TR (1991) A hammerhead ribozyme allows synthesis of a new form of the Tetrahymena ribozyme homogenous in length with a 3′ end blocked for transestrification. Nucleic Acids Res 19:3875–3880
Ferre-D’Amare AR, Doudna JA (1996) Use of cis- and trans-ribozymes to remove 5′ and 3′ heterogeneities from milligrams of in vitro transcribed RNA. Nucleic Acids Res 24:977–978
Schürer H, Lang K, Schuster J, Mörl M (2002) A universal method to produce in vitro transcripts with homogenous 3′ ends. Nucleic Acids Res 30:e56
Walker SC, Avis JM, Conn GL (2003) General plasmids for producing RNA in vitro transcripts with homogenous ends. Nucleic Acids Res 31:e82
Mörl M, Lizano E, Willkomm DK, Hartmann RK (2005) Production of RNAs with homogenous 5′ and 3′ ends. In: Hartmann RK, Bindereif A, Schon A, Westhof E (eds) Handbook of RNA biochemistry. Wiley-VCH GmbH & Co, KGaA, Weinheim, pp 22–35
Wrzesinski J, Łęgiewicz M, Smolska B, Ciesiołka J (2001) Catalytic cleavage of cis-and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions. Nucleic Acids Res 29:4482–4492
Wichłacz A, Łęgiewicz M, Ciesiołka J (2004) Generating in vitro transcripts with homogenous 3′ ends using trans-acting antigenomic delta ribozyme. Nucleic Acids Res 32:e39
Świątkowska A, Dutkiewicz M, Ciesiołka J (2007) Structural features of target RNA molecules greatly modulate the cleavage efficiency of trans-acting delta ribozymes. Biochemistry 46:5523–5533
Acknowledgements
This work was supported by Wroclaw Research Center EIT+ under the project “Biotechnologies and advanced medical technologies—BioMed” (POIG 01.01.02-02-003/08-00) financed from the “European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2)” and earlier by grants from the Ministry of Science and Higher Education.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Szafraniec, M., Blaszczyk, L., Wrzesinski, J., Ciesiolka, J. (2013). Trans-Acting Antigenomic HDV Ribozyme for Production of In Vitro Transcripts with Homogenous 3′ Ends. 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_8
Download citation
DOI: https://doi.org/10.1007/978-1-62703-113-4_8
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-112-7
Online ISBN: 978-1-62703-113-4
eBook Packages: Springer Protocols