Abstract
The sequence specificity of the “10–23” RNA-cleaving DNA enzyme can be utilized to discriminate between subtle differences in nucleic acid sequence. We examined this potential by comparing the cleavage activity of DNAzymes that target sequences derived from a relatively conserved segment of the L1 gene from different human papillomavirus (HPV) genotypes. DNAzyme activity was found to be highly sensitive to mismatches between its binding domain and substrate sequences containing polymorphisms. Type-specific DNAzyme-cleavable substrates can also be generated by genomic PCR using a chimeric primer containing three bases of RNA. The RNA component enables each amplicon to be cleavable in the presence of its matching DNAzyme. In this format, the specificity of DNAzyme cleavage is defined by Watson-Crick interactions between one substrate-binding domain (arm I) and the polymorphic sequence that is amplified during polymerase chain reaction (PCR). DNAzyme-mediated cleavage of amplicons generated by this method was used to examine the HPV status of genomic DNA derived from Caski cells, which are known to be positive for HPV16. This method is applicable to many types of nucleic acid sequence variation, including single-nucleotide polymorphisms (SNPs).
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
Santoro, S. W. and Joyce, G. F. (1997) A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 94, 4262–4266.
Santoro, S. W. and Joyce, G. F. (1998) Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry 37, 13,330–13,342.
Wu, Y., Yu, L., McMahon, R., Rossi, J. J., Forman, S. J., and Snyder, D. S. (1999) Inhibition of bcr-abl oncogene expression by novel deoxyribozymes (DNAzymes). Hum. Gene Ther. 10, 2847–2857.
Santiago, F. S., Kavurma, M. M., Lowe, H. C., Chesterman, C. N., Baker, A., Atkins, D. G., and Khachigian, L. M. (1999) New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury. Nat Med. 5, 1264–1269.
Cairns, M. J., Saravolac, E. G., and Sun, L. Q. (2002) Catalytic DNA: a novel tool for gene suppression. Curr. Drug Targets 3, 269–279.
Sun, L. Q, Cairns, M. J., Saravolac, E. G., Baker, A., and Gerlach, W. L. (2000) Catalytic nucleic acids: from lab to applications. Pharmacol Rev. 52, 325–347.
Cairns, M. J., Hopkins, T. M., Witherington, C., Wang, L., and Sun, L. Q. (1999) Target site selection for an RNA-cleaving catalytic DNA. Nature Biotechnol. 17, 480–486.
Sioud, M. and Leirdal, M. (2000) Design of nuclease resistant protein kinase calpha DNA enzymes with potential therapeutic application. J. Mol. Biol. 296, 937–947.
Todd, A. V., Fuery, C. J., Impey, H. L., Applegate, T. L., and Haughton, M. A. (2000) DzyNA-PCR: use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format. Clin Chem. 46, 625–630.
Cairns, M. J., Hopkins, T. M., Whitherington, C., and Sun, L. Q. (2000) The influence of arm length asymmetry and base substitution on the activity of the “10–23” DNA enzyme. Antisense Nucleic Acid Drug. Dev. 10, 323–332.
Cairns, M. J., King, A., and Sun, L. Q. (2000) Nucleic acid mutation analysis using catalytic DNA. Nucleic Acids Res. 28, e9.
Sugimoto, N., Nakano, S., Katoh, M., Matsumura, A., Nakamuta, H., Ohmichi, T., et al. (1995) Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34, 11,211–11,216.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Cairns, M.J., Sun, LQ. (2004). Nucleic Acid Sequence Analysis Using DNAzymes. In: Sioud, M. (eds) Ribozymes and siRNA Protocols. Methods in Molecular Biology™, vol 252. Humana Press. https://doi.org/10.1385/1-59259-746-7:291
Download citation
DOI: https://doi.org/10.1385/1-59259-746-7:291
Publisher Name: Humana Press
Print ISBN: 978-1-58829-226-1
Online ISBN: 978-1-59259-746-8
eBook Packages: Springer Protocols