Abstract
Ribonucleases H are complex enzymes whose functions are not clearly understood, further compounded by the fact that multiple forms of the enzyme are present in various organisms. They are known to recognize and degrade the ribonucleic acid (RNA) strand of numerous deoxyribonucleic acid (DNA)-RNA duplex substrates, and so may provide a unique mode of therapeutic intervention at the genetic level of virtually any disease. We have therefore set out detailed procedures for conducting routine assays with almost any one of this family of enzymes by a straightforward assay aimed at identifying novel enzyme-activating antisense oligonucleotides (AONs). The procedures described herein should enable easy identification of potent AON molecules, provided that the RNA is appropriately labeled for subsequent visualization following the guidelines set forth in this protocol.
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
Walder, R. Y. and Walder, J. A. (1978) Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 75, 5011–5015.
Turchi, J. J., Huang, L., Murante, R. S., Kim, Y., and Bambara, R. A. (1994) Enzymatic completion of mammalian lagging-strand DNA replication. Proc. Natl. Acad. Sci. USA 91, 9803–9807.
Arudchandran, A., Cerritelli, S. M., Narimatsu, S. K., et al. (2000) The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair. Genes Cells 5, 789–802.
Rydberg, B. and Game, J. (2002) Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts. Proc. Natl. Acad. Sci. USA 99, 16,654–16,659.
Moelling, K., Bolognesi, D. P., Bauer, H., Büsen, W., Plassmann, H. W., and Hausen, P. (1971) Association of viral reverse transcriptase with an enzyme degrading the RNA moiety of RNA-DNA hybrids. Nat. New Biol. 234, 240–243.
Stein, H. and Hausen, P. (1969) Enzyme from calf thymus degrading the RNA moiety of DNA-RNA hybrids: effect on DNA-dependent RNA polymerase. Science 166, 393–395.
Eder, P. S. and Walder, J. A. (1991) Ribonuclease H from K562 human erythroleukemia cells: purification, characterization and substrate specificity. J. Biol. Chem. 266, 6472–6479.
Frank, P., Albert, S., Cazenave, C., and Toulmé, J.-J. (1994) Purification and characterization of human ribonuclease HII. Nucleic Acids Res. 22, 5247–5254.
Frank, P., Braunshofer-Reiter, C., Wintersberger, U., Grimm, R., and Büsen, W. (1998) Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII. Proc. Natl. Acad. Sci. USA 95, 12,872–12,877.
Frank, P. Braunshofer-Reiter, C., Pöltl, A., and Holzmann, K. (1998) Cloning, subcellular localization and functional expression of human RNase HII. Biol. Chem. 379, 1404–1412.
Johnson, M. S., McClure, M. A., Feng, D. F., Gray, J., and Doolittle, R. F. (1986) Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc. Natl. Acad. Sci. USA 83, 7648–7652.
Frank, P., Braunshofer-Reiter, C., and Wintersberger, U. (1998) Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII. FEBS Lett. 421, 23–26.
Berkower, L., Leis, J., and Hurwitz, J. (1973) Isolation and characterization of an endonuclease from Escherichia coli specific for ribonucleic acid in ribonucleic acid-deoxyribonucleic acid hybrid structures. J. Biol. Chem. 248, 5914–5921.
Crouch, R. J. and Dirksen, M. L. (1982) Ribonuclease H. In Linn, S. M. and Roberts, R. J. (Eds.), Nucleases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 211–241.
Minshull, J. and Hunt, T. (1986) The use of single-stranded DNA and RNase H to promote quantitative “hybrid arrest of translation“ of mRNA-DNA hybrids in reticulocyte lysate cell-free translations. Nucleic Acids Res. 14, 6433–6451.
Crooke, S. T. (1999) Molecular mechanism of action of antisense drugs. Biochim. Biophys. Acta 1489, 31–44.
Damha, M. J., Wilds, C. J., Noronha, A., et al. (1998) Hybrids of RNA and arabinonucleic acids (ANA and 2′F-ANA) are substrates of ribonuclease H. J. Am. Chem. Soc. 120, 12,976–12,977.
Noronha, A. M., Wilds, C. J., Lok, C.-N., Viazovkina, K., Arion, D., Parniak, M. A., and Damha, M. J. (2000) Synthesis and biophysical properties of arabinonucleic acids (ANA): circular dichroic spectra, melting temperatures and ribonuclease H susceptibility of ANA:RNA hybrid duplexes. Biochemistry 39, 7050–7062.
Wilds, C. J. and Damha M. J. (2000) 2′-deoxy-2′-fluoro-β-D-arabinonucleosides and oligonucleotides (2′F-ANA): synthesis and physicochemical studies. Nucleic Acids Res. 28, 3625–3635.
Wang, J., Verbeure, B., Luyten, I., et al. (2000) Cyclohexene nucleic acids (CeNA): serum stable oligonucleotides that activate RNase H and increase duplex stability with complementary RNA. J. Am. Chem. Soc. 122, 8595–8602.
Trempe, J. F., Wilds, C. J., Denisov, A. Y., Pon, R. T., Damha, M. J., and Gehring, K. (2001) NMR solution structure of an oligonucleotide hairpin with a 2′F-ANA-RNA stem: implications for RNase H specificity toward DNA-RNA hybrid duplexes. J. Am. Chem. Soc. 123, 4896–4903.
Damha, M. J., Noronha, A. M., Wilds, C. J., Trempe, J.-F., Denisov, A., and Gehring, K. (2001) Properties of arabinonucleic acids (ANA & 2′F-ANA): implications for the design of antisense therapeutics that invoke RNase H cleavage of RNA. Nucleosides Nucleotides Nucleic Acids 20, 429–440.
Minasov, G., Teplova, M., Nielsen, P., Wengel, J., and Egli, M. (2000) Structural basis of cleavage by RNase H of hybrids of arabinonucleic acids and RNA. Biochemistry 39, 3525–3532.
Mangos, M. M. and Damha, M. J. (2002) Flexible and frozen sugar-modified nucleic acids—modulation of biological activity through furanose ring dynamics in the antisense strand. Curr. Top. Med. Chem. 2, 1147–1171.
Mangos, M. M., Min, K.-L., Viazovkina, E., et al. (2003) Efficient Rnase H-directed cleavage of RNA promoted by antisense DNA or 2′F-ANA constructs containing acyclic nucleotide inserts. J. Am. Chem. Soc. 125, 651–659.
Roberts, G. C., Dennis, E. A., Meadows, D. H., Cohen, J. S., and Jardetzky, O. (1969) The mechanism of action of ribonuclease. Proc. Natl. Acad. Sci. USA 62, 1151–1153.
Yazbeck, D. R., Min, K.-L., and Damha, M. J. (2002) Molecular requirements for degradation of a modified sense RNA strand by Escherichia coli ribonuclease H. Nucleic Acids Res. 30, 3015–3025.
Blackburn, P. and Moore, S. (1982) The Enzymes. Academic Press, New York.
Blumberg, D. D. (1987) Creating a ribonuclease-free environment. Methods Enzymol. 152, 20–24.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY.
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
Galarneau, A., Min, KL., Mangos, M.M., Damha, M.J. (2005). Assay for Evaluating Ribonuclease H-Mediated Degradation of RNA-Antisense Oligonucleotide Duplexes. In: Herdewijn, P. (eds) Oligonucleotide Synthesis. Methods in Molecular Biology, vol 288. Humana Press. https://doi.org/10.1385/1-59259-823-4:065
Download citation
DOI: https://doi.org/10.1385/1-59259-823-4:065
Publisher Name: Humana Press
Print ISBN: 978-1-58829-233-9
Online ISBN: 978-1-59259-823-6
eBook Packages: Springer Protocols