Abstract
A protocol for the straightforward preparation of small circular oligodeoxyribonucleotides (2–28 nt) is reported. The assembly of the oligonucleotide chain (standard phosphoramidite chemistry) and cyclization by the phosphotriester method take place on a tailor-made nucleotide-derivatized solid support. Although cyclization yields are moderate, the procedure exploits a synthesis design that allows selective cleavage of the circular oligonucleotide from the support, which facilitates isolation of the target molecule by simple filtration.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Kool, E. T. (1998) Recognition of DNA, RNA and proteins by circular oligonucleotides. Acc. Chem. Res. 31, 502–510.
Kool, E. T. (1996) Circular oligonucleotides: new concepts in oligonucleotide design. Annu. Rev. Biophys. Biomol. Struct. 25, 1–28.
Rumney, S., IV and Kool, E. T. (1992) DNA recognition by hybrid oligoetheroligodeoxynucleotide macrocycles. Angew. Chem. Int. Ed. Engl. 31, 1617–1619.
Prakash, G. and Kool. E. T. (1992) Structural effects in the recognition of DNA by circular oligonucleotides. J. Am. Chem. Soc. 114, 3523–3527.
Fire, A. and Xu, S. (1995) Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92, 4641–4645.
Liu, D., Daubendiek, S. L., Zillman, M. A., Ryan, K., and Kool, E. T. (1996) Rolling circle DNA synthesis: small circular oligonucleotides as efficient templates for DNA polymerases. J. Am. Chem. Soc. 118, 1587–1594.
Daubendiek, S. L. and Kool, E. T. (1997) Generation of catalytic RNAs by rolling transcription of synthetic DNA nanocircles. Nat. Biotechnol. 15, 273–277.
Frieden, M., Pedroso, E., and Kool, E. T. (1999) Tightening the belt on polymerases: evaluating the physical constraints on enzyme substrate size. Angew. Chem. Int. Ed. Engl. 38, 3654–3657.
Kuhn, H., Demidov, V. V., and Frank-Kamenetskii, M. D. (2002) Rolling-circle amplification under topological constraints. Nucleic Acids Res. 30, 574–580.
Nilsson, M., Krejci, K., Koch, J., Kwiatkowski, M., Gustavsson. P., and Landegren. U., (1997) Padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21. Nat. Genet. 16, 252–254.
Lizardi, P. M., Huang, X., Zhu, Z., Bray-Ward, P., Thomas, D. C., and Ward, D. C. (1998) Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225–232.
Clusel, C., Ugarte, E., Enjolras, N., Vasseur, M., and Blumenfeld, M. (1993) Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbbell oligonucleotides. Nucleic Acids Res. 21, 3405–3411.
Mazumder, A., Uchida, H., Neamati, N., et al. (1997) Probing interactions between viral DNA and human immunodeficiency virus type I integrase using dinucleotides. Mol. Pharmacol. 51, 567–575.
Abe, T., Takai, K., Nakada, S., Yokota, T., and Takaku, H. (1998) Specific inhibition of influenza virus RNA polymerase and nucleoprotein gene expression by circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides. FEBS Lett. 425, 91–96.
Rowley, P. T., Kosciolek, B. A., and Kool, E. T. (1999) Circular antisense oligonucleotides inhibit growth of chronic myeloid leukemia cells. Mol. Med. 5, 693–700.
Roulon, T., Hélène, C., and Escudé, C. (2001) A ligand-modulated padlock oligonucleotide for supercoiled plasmids. Angew. Chem. Int. Ed. Engl. 40, 1523–1526.
Ippel. J. H., Lanzotti, V., Galeone, A., et al. (1995) Slow conformational exchange in DNA minihairpin loops: a conformational study of the circular dumbbell d〈pCGCTTGCGTT〉. Biopolymers 36, 681–694.
Salisbury, S. A., Wilson, S. E., Powell, H. R., et al. (1997) The bi-loop, a new general four-stranded DNA motif. Proc. Natl. Acad. Sci. USA 94, 5515–5518.
Escaja, N., Pedroso, E., Rico, M., and González, C. (2000) Dimeric solution structure of two cyclic octamers: four-stranded DNA structures stabilized by A:T:A:T and G:C:G:C tetrads. J. Am. Chem. Soc. 122, 12,732–12,742.
Escaja, N., Gelpí, J. L., Orozco, M., Rico, M., Pedroso, E., and González, C. (2003) Four-stranded DNA structure stabilized by a novel G:C:A:T tetrad. J. Am. Chem. Soc. 125, 5654–5662.
Ashley, G. W. and Kushlan, D. M. (1991) Chemical synthesis of oligodeoxynucleotide dumbbells. Biochemistry 30, 2927–2933.
Dolinnaya, N. G., Blumenfeld, M., Merenkova, I. N., et al. (1993) Oligonucleotide circularization by template-directed chemical ligation. Nucleic Acids Res. 21, 5403–5407.
Maksimenko, A. V., Volkov, E. M., Bertrand, J.-R., et al. (2000) Targeting of single-stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides. Eur. J. Biochem. 267, 3592–3603.
Diegelman, A. M. and Kool, E. T. (2000) Chemical and enzymatic methods for preparing circular single-stranded DNAs. In Current Protocols in Nucleic Acid Chemistry, vol. 2 (Beaucage, S. et al., eds.). John Wiley, New York, Chapter 5.2.
Li, T., Liu, D., Chen, J., Lee, A. H. F., Qi, J., and Chan, A. S. C. (2001) Construction of circular oligodeoxyribonucleotides on the new structural basis of i-motif. J. Am. Chem. Soc. 123, 12,901–12,902.
Liu, D., Chen, J., Lee, A. H. F., Chow, L. M. C., Chan, A. S. C., and Li, T. (2003) Small circular oligodeoxynucleotides achieved from self-assembling entities. Angew. Chem. Int. Ed. Engl. 42, 797–799.
Capobianco, M., Carcuro, A., Tondelli, L., Garbesi, A., and Bonora, G. M. (1990) One pot solution synthesis of cyclic oligodeoxyribonucleotides. Nucleic Acids Res. 18, 2661–2669.
Vroom, E., Broxterman, H. J. G., Sliedregt, L. A. J. M., van der Marel, G. A., and van Boom, J. H. (1988) Synthesis of cyclic oligonucleotides by a modified phosphotriester approach. Nucleic Acids Res. 16, 4607–4620.
Reese, C. B. and Song, Q. (1999) The H-phosphonate approach to the solution phase synthesis of linear and cyclic oligoribonucleotides. Nucleic Acids Res. 27, 963–971.
De Napoli, L., Montesarchio, D., Piccialli, G., et al. (1991) Solid-phase synthesis of cyclic oligodeoxyribonucleotides: intermolecular versus intramolecular coupling. Gazz. Chim. Ital. 121, 505–508.
De Napoli, L., Galeone, A., Mayol, L., Messere, A., Montesarchio, D., and Picciali, G. (1995) Automated solid phase synthesis of cyclic oligonucleotides: a further improvement. Bioorg. Med. Chem. 3, 1325–1329.
Smietana, M. and Kool, E. T. (2002) Efficient and simple solid-phase synthesis of short cyclic oligodeoxynucleotides bearing a phosphorothioate linkage. Angew. Chem. Int. Ed. Engl. 41, 3704–3707.
Alazzouzi, E., Escaja, N., Grandas, A., and Pedroso, E. (1997) A straightforward solid-phase synthesis of cyclic oligonucleotides. Angew. Chem. Int. Ed. Engl. 36, 1506–1508.
Mazur, S. and Jayalekshmy, P. (1979) Chemistry of polymer-bound o-benzyne: frequency of encounter between substituents on cross-linked polystyrenes. J. Am. Chem. Soc. 101, 677–683.
Frieden, M., Grandas, A., and Pedroso, E. (1999) Making cyclic RNAs easily available. Chem. Commun. 1593, 1594.
Micura, R. (1999) Cyclic oligoribonucleotides (RNA) by solid-phase synthesis. Chem. Eur. J. 5, 2077–2082.
Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595–598.
Caruthers, M. H., Barone, A. D., Beaucage, S. L., et al. (1987) Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. Methods Enzymol. 154, 287–313.
Reese, C. B., Titmas, R. C., and Yau, L. (1978) Oximate ion promoted unblocking of oligonucleotide phosphotriester linkages Tetrahedron Lett. 2727–2730.
Brown T. and Brown D. J. S. (1990) Modern machine-aided methods of oligodeoxyribonucleotide synthesis. In Oligonucleotides and Analogues: A Practical Approach (Eckstein, F., ed.). IRL Press, Oxford, U.K., p. 20.
Adams, R. L. P., Knowler, J. T., and Leader, D. P. P. (1992) The Biochemistry of the Nucleic Acids, 11th ed. Chapman ⇐p; Hall, London, pp. 87–108.
Pieles, U., Zürcher, W., 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.
Zhu, Y. F., Chung, C. N., Taranenko, N. I., et al. (1996) The study of 2,3,4-trihydroxyacetophenone and 2,4,6-trihydroxyacetophenone as matrices for DNA detection in matrix-assisted laser desorption/ionization time-of-flight spectrometry. Rapid Commun. Mass Spectrom. 10, 383–388.
Tang, K., Fu, D., Köter, S., Cotter, R. J., Cantor, C. R., and Köster, H. (1995) Matrix-assisted laser desorption/ionisation mass spectrometry of immobilized duplex DNA probes. Nucleic Acids Res. 23, 3126–3131.
Patel, T. P., Chauncey, M. A., Millican, T. A., Bose, C. C., and Eaton, M. A. (1984) A rapid deprotection procedure for phosphotriester DNA synthesis. Nucleic Acids Res. 12, 6953–6859.
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
Pedroso, E., Escaja, N., Frieden, M., Grandas, A. (2005). Solid-Phase Synthesis of Circular Oligonucleotides. In: Herdewijn, P. (eds) Oligonucleotide Synthesis. Methods in Molecular Biology, vol 288. Humana Press. https://doi.org/10.1385/1-59259-823-4:101
Download citation
DOI: https://doi.org/10.1385/1-59259-823-4:101
Publisher Name: Humana Press
Print ISBN: 978-1-58829-233-9
Online ISBN: 978-1-59259-823-6
eBook Packages: Springer Protocols