Abstract
The discovery of DNAzymes that can catalyze a wide range of reactions in the presence of metal ions is important on both fundamental and practical levels; it advances our understanding of metal–nucleic acid interactions and allows for the design of highly sensitive and selective metal ion sensors. A crucial factor in this success is a technique known as in vitro selection, which can rapidly select metal-specific RNA-cleaving DNAzymes. In vitro selection is an iterative process where a DNA pool containing a random region is incubated with the target metal ion. Those DNA sequences that catalyze the preferred reaction (the “winners”) are amplified and carried on to the next step, where the selection is carried out under more stringent conditions. In this way, the selection pool becomes enriched with DNAzymes that exhibit desirable activity and selectivity. The method described can be applied to isolate DNAzymes selective to many different types of metal ions or different oxidation states of the same metal ion.
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
Breaker R, Joyce G (1994) A DNA Enzyme that Cleaves RNA. Chem Biol 1:223-229.
Li Y, Sen D (1996) A Catalytic DNA for Porphyrin Metallation. Nat Struct Biol 3:743–747.
Lu Y (2002) New Transition Metal Ion-Dependent Catalytic DNA and Their Applications as Efficient RNA Nucleases and as Sensitive Metal Ion Sensors. Chem Euro J 8:4588–4596.
Li Y, Liu Y, Breaker R (2000) Capping DNA with DNA. Biochem 39:3106–3114.
Schlosser K, Li Y (2009) Biologically Inspired Synthetic Enzymes Made from DNA. Chem Biol 16:311–322.
Silverman S (2008) Catalytic DNA (Deoxyribozymes) for Synthetic Applications—Current Abilities and Future Prospects. Chem Commun 3467–3485.
Lu Y, Liu J (2006) Functional DNA Nanotechnology: Emerging Applications of DNAzymes and Aptamers. Curr Opion Biotech 17:580–588.
Franzen S (2010) Expanding the Catalytic Repertoire of Ribozymes and Deoxyribozymes Beyond RNA Substrates. Curr Opin Mol Ther 12:223–232.
McManus S, Li Y (2010) The Structural Diversity of Deoxyribozymes. Molecules 15:6269–6284.
Li J, Lu Y (2000) A Highly Sensitive and Selective Catalytic DNA Biosensor for Lead Ions. J Am Chem Soc 122:10466–10467.
Liu J, Lu Y (2003) A Colorimetric Lead Biosensor Using DNAzyme-Directed Assembly of Gold Nanoparticles. J Am Chem Soc 125:6642–6643.
Xiao Y, Rowe A, Plaxco K (2007) Electrochemical Detection of Parts-per-billion Lead via an Electrode-Bound DNAzyme Assembly. J Am Chem Soc 129:262–263.
Lan T, Furuya K, Lu Y (2010) A Highly Selective Lead Sensor Based on a Classic Lead DNAzyme. Chem Commun 46:3896–3898.
Liu J, Brown A, Meng X, Cropek D, Istok J, Watson D, Lu Y (2007) A Catalytic Beacon Sensor for Uranium with Parts-per-trillion Sensitivity and Millionfold Selectivity. P Natl Acad Sci USA 104:2056–2061.
Li J, Zheng W, Kwon A, Lu Y (2000) In Vitro Selection and Characterization of a Highly Efficient Zn(II)-dependent RNA-cleaving Deoxyribozyme. Nucleic Acids Res. 28:481–488.
Li Y, Breaker R (1999) Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2′-Hydroxyl Group. J Am Chem Soc 121: 5364–5372.
Vant-Hull B, Gold L, Zichi D (2000) Theoretical Principles of In Vitro Selection using Combinatorial Nucleic Acid Libraries. In: Egli M, Herdewijn, P, Matusda, A, Sangyi Y (ed) Current Protocols in Nucleic Acid Chemistry. Wiley, New York.
Schlosser K, Li Y (2009) DNAzyme-mediated Catalysis with Only Guanosine and Cytidine Nucleotides. Nucleic Acids Res 37:413–420.
Lam J, Li Y (2010) Influence of Cleavage Site on Global Folding of an RNA-Cleaving DNAzyme. Chem Bio Chem 11: 1710–1719.
Schlosser K, Li Y (2010) A Versatile Endoribonuclease Mimic Made of DNA: Characteristics and Applications of the 8–17 RNA-Cleaving DNAzyme. Chem Bio Chem 11:866–879.
Bruesehoff PJ, Li J, Augustine III, AJ, Lu Y (2002) Improving Metal Ion Specificity During In Vitro Selection of Catalytic DNA. Combinator Chem High Throughput Screening 5:327–335.
Schlosser K, Lam J, Li Y (2009) A Genotype-to-Phenotype Map of In Vitro Selected RNA-cleaving DNAzymes: Implications for Accessing the Target. Nucleic Acids Res 37:3545–3557.
Lam J, Withers J, Li Y (2010) A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine–Pyrimidine Junction. J Mol Bio 400:689–701.
Acknowledgments
The authors would like to thank Andrea K. Brown, Debapriya Mazumdar, Nandini Nagraj, Tian Lan, and Seyed-Fakhreddin Torabi for developing and fine-tuning this method. This work was supported by the US National Institutes of Health (ES016865) and Department of Energy (DE-FG02-08ER64568).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ihms, H.E., Lu, Y. (2012). In Vitro Selection of Metal Ion-Selective DNAzymes. In: Hartig, J. (eds) Ribozymes. Methods in Molecular Biology, vol 848. Humana Press. https://doi.org/10.1007/978-1-61779-545-9_18
Download citation
DOI: https://doi.org/10.1007/978-1-61779-545-9_18
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-544-2
Online ISBN: 978-1-61779-545-9
eBook Packages: Springer Protocols