Abstract
As a result of both the therapeutrc (xc1) and structural (2) interest in RNA, there has been an increased demand for larger amounts of oligoribonucleotides. Although great progress has been made in improving the methods for DNA synthesis, such that production of large amounts has become commonplace, similar quantities of RNA are still difficult to obtain.
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References
Cech, T. (1992) Ribozyme engineering. Curr. Opinion Struct. Biol. 2, 605–609.
Usman, N. and Cedergren, R. J. (1992) Exploiting the chemical synthesis of RNA. Trends Biochem. Sci. 17, 334–339.
Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, N. (1995) Synthesis, deprotectton, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 23, 2677–2684.
Usman, N., Ogilvie, K. K., Jiang, M.-Y., and Cedergren, R. J. (1987) Automated chemical synthesis of long oligoribonucleotides using 2′-O-silyl-ribonucleoside-3′-O-phosphoramidites on a controlled-pore glass 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.
Scaringe, S. A., Francklyn, C., and Usman, N. (1990) Chemical synthesis of biologically active oligoribonucleotides using β-cyanoethyl protected ribonucleoside phosphoramidites. Nucleic Acids Res. 18, 5433–5341.
Andrus, A., Beaucage, S., Ohms, J., and Wert, K. (1986) A serves of 5′-substituted tetrazoles for phosphoramidite activation during oligonucleotide synthesis. American Chemical Society Meeting, New York, April 1986, Organic Division, Abstract 333.
Sproat, B., Colonna, F., Mullah, B., Tsou, D., Andrus, A., Hampel, A., and Vinayak, R. (1995) An efficient method for the isolation and purification of oligoribonucleotides. Nucleosides & Nucleotides 14, 255–273.
Gasparutto, D., Livache, T., Bazin, H., Duplaa, A.-M., Guy, A., Khorlin, A., Molko, D., Roget, A., and Téoule, R. (1992) Chemical synthesis of a biologically active natural tRNA with its minor bases. Nucleic Acids Res. 20, 5159–5166.
Wu, T., Ogilvie, K. K., and Pon, R. T. (1988) N-Phenoxyacetylated guanosine and adenosine phosphoramidites in the solid phase syntheses of oligoribonucleotides: synthesis of a ribozyme sequence. Tetrahedron Lett. 34, 4249–4252.
Chaix, C., Duplaa, A. M., Molko, D., and Téoule, R. (1989) Solid phase synthesis of the 5′-half of the initiator t-RNA from B. subtilis. Nucleic Acids Res. 17, 7381–7393.
Sinha, N. D., Davis, P., Usman, N., Pérez, J., Hodge, R., Kremsky, J., and Casale, R. (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.
Reddy, M. P., Hanna, N. B., and Farooqui, F. (1994) Fast cleavage and deprotection of oligonucleotides. Tetrahedron Lett. 55, 4311–4314.
Westman, E. and Stromberg, 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.
Vinayak, R., Andrus, A., and Hampel, A. (1995) Rapid desilylation of oligoribo-nucleotides at elevated temperatures: cleavage activity in ribozyme-substrate assays. Biomedical Peptides, Proteins and Nucleic Acids 1, 227–230.
Odai, O., Hiroaki, H., Sakata, T., Tanaka, T., and Uesugi, S. (1990) The role of a conserved guanosine residue in the hammerhead-type RNA enzyme. FEBS Lett. 267, 150–152.
Koizumi, M. and Ohtsuka, E. (1991) Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding. Biochemistry 30, 5145–5150.
Usman, N., Egli, M., and Rich, A. (1992) Large scale chemical synthesis, purification and crystallization of RNA-DNA chimeras. Nucleic Acids Res. 20, 6695–6699.
Slim, G. and Gait, M. J. (1991) Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res. 19, 1183–1188.
Hall, K. B. and McLaughlin, L. W. (1992) Properties of pseudouridine N1 amino protons located in the major groove of an A-form RNA duplex. Nucleic Acids Res. 20, 1883–1889.
Fu, D.-J. and McLaughlin, L. W. (1992) Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding. Biochemistry 31, 10,941–10,949.
Pon, R. T., Usman, N., and Ogilvie, K. K. (1988) Derivitization of controlled pore glass beads for solid phase oligonucleotide synthesis. BioTechniques 6, 768–775.
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Wincott, F.E., Usman, N. (1997). A Practical Method for the Production of RNA and Ribozymes. In: Turner, P.C. (eds) Ribozyme Protocols. Methods in Molecular Biology™, vol 74. Humana Press. https://doi.org/10.1385/0-89603-389-9:59
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DOI: https://doi.org/10.1385/0-89603-389-9:59
Publisher Name: Humana Press
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