Abstract
This chapter enables the reader to carry out the solid-phase synthesis of ribonucleic acid (RNA) using β-cyanoethyl phosphoramidite chemistry combined with tert-butyldimethylsilyl protection of the ribose 2′-hydroxyl group. Phosphoramidite monomers are activated with 5-benzylmercapto-1H-tetrazole enabling fast and highly efficient coupling to the 5′-hydroxyl group of the support-bound oligonucleotide. On completion of the synthesis, the stepwise deprotection of the nucleobase, phosphate, and ribose protecting groups is carried out using optimized protocols. Subsequently the various high-pressure (performance) liquid chromatography (HPLC) procedures are described enabling the purification and analysis of the RNA. For this purpose anion-exchange and reversed-phase HPLC are used singly or in combination according to the final purity requirement of the RNA.
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
Ramaswamy, G. and Slack, F. J. (2002) siRNA: a guide for RNA silencing. Chem. Biol. 9, 1053–1055.
Couzin, J. (2002) Small RNAs make big splash. Science 298, 2296–2297.
Sinha, N. D., Biernat, J., McManus, J., and Köter, H. (1984) Polymer support oligonucleotide synthesis XVIII: use of β-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 12, 4539–4557.
Usman, N., Ogilvie, K. K., Jiang, M.-Y., and Cedergren, R. J. (1987) Automated chemical synthesis of long oligoribonucleotides using 2′-O-silylated ribonucleoside 3′-O-phosphoramidites on a CPG support: synthesis of a 43-nucleotide sequence similar to the 3′-half molecule of an Escherichia coli formylmethionine tRNA. J. Am. Chem. Soc. 109, 7845–7854.
Sinha, N. D., Davis, P., Usman, N., et al. (1993) Labile exocyclic amine protection of nucleosides in DNA, RNA and oligonucleotide analog synthesis facilitating N-deacylation, minimizing depurination and chain degradation. Biochimie 75, 13–23.
Welz, R. and Müller, S. (2002) 5-(Benzylmercapto)-1H-tetrazole as activator for 2′-O-TBDMS phosphoramidite building blocks in RNA synthesis. Tetrahedron Lett. 43, 795–797.
Gasparutto, D., Livache, T., Bazin, H., et al. (1992) Chemical synthesis of a biologically active natural tRNA with its minor bases. Nucleic Acids Res. 20, 5159–5166.
Westman, E. and Ströberg, R. (1994) Removal of t-butyldimethylsilyl protection in RNA-synthesis: triethylamine trihydrofluoride (TEA, 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF). Nucleic Acids Res. 22, 2430–2431.
Song, Q. and Jones, R. A. (1999) Use of silyl ethers as fluoride scavengers in RNA synthesis. Tetrahedron Lett. 40, 4653–4654.
Vinayak, R., Andrus, A., and Hampel, A. (1995) Rapid desilylation of oligoribonucleotides at elevated temperatures: cleavage activity in ribozymesubstrate assays. Biomed. Pept. Proteins Nucleic Acids 1, 227–230.
Wincott, F., DiRenzo, A., Shaffer, C., et al. (1995) Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 23, 2677–2684.
Sproat, B., Colonna, F., Mullah, B., et al. (1995) An efficient method for the isolation and purification of oligoribonucleotides. Nucleosides Nucleotides 14, 255–273.
Pieles, U., Zürcher, W., Schär, M., and Moser, H. E. (1993) Matrix-assisted laser desorption ionization time-of flight mass spectrometry: a powerful tool for the mass and sequence analysis of natural and modified oligonucleotides. Nucleic Acids Res. 21, 3191–3196.
Sproat, B. S. and Gait, M. J. (1984) Solid-phase synthesis of oligodeoxyribonucleotides by the phosphotriester method. In Oligonucleotide Synthesis: A Practical Approach (Gait, M. J., ed.), IRL Press, Oxford, UK, p. 92.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Sproat, B.S. (2005). RNA Synthesis Using 2′-O-(Tert-Butyldimethylsilyl) Protection. In: Herdewijn, P. (eds) Oligonucleotide Synthesis. Methods in Molecular Biology, vol 288. Humana Press. https://doi.org/10.1385/1-59259-823-4:017
Download citation
DOI: https://doi.org/10.1385/1-59259-823-4:017
Publisher Name: Humana Press
Print ISBN: 978-1-58829-233-9
Online ISBN: 978-1-59259-823-6
eBook Packages: Springer Protocols