Ribozymes pp 297-316

Part of the Methods in Molecular Biology book series (MIMB, volume 848) | Cite as

In Vitro Selection of Metal Ion-Selective DNAzymes



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.

Key words

DNAzyme In vitro selection Functional DNA Deoxyribozyme Catalytic DNA Metal ions Bioinorganic chemistry 


  1. 1.
    Breaker R, Joyce G (1994) A DNA Enzyme that Cleaves RNA. Chem Biol 1:223-229.PubMedCrossRefGoogle Scholar
  2. 2.
    Li Y, Sen D (1996) A Catalytic DNA for Porphyrin Metallation. Nat Struct Biol 3:743–747.PubMedCrossRefGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    Li Y, Liu Y, Breaker R (2000) Capping DNA with DNA. Biochem 39:3106–3114.CrossRefGoogle Scholar
  5. 5.
    Schlosser K, Li Y (2009) Biologically Inspired Synthetic Enzymes Made from DNA. Chem Biol 16:311–322.PubMedCrossRefGoogle Scholar
  6. 6.
    Silverman S (2008) Catalytic DNA (Deoxyribozymes) for Synthetic Applications—Current Abilities and Future Prospects. Chem Commun 3467–3485.Google Scholar
  7. 7.
    Lu Y, Liu J (2006) Functional DNA Nanotechnology: Emerging Applications of DNAzymes and Aptamers. Curr Opion Biotech 17:580–588.PubMedCrossRefGoogle Scholar
  8. 8.
    Franzen S (2010) Expanding the Catalytic Repertoire of Ribozymes and Deoxyribozymes Beyond RNA Substrates. Curr Opin Mol Ther 12:223–232.PubMedGoogle Scholar
  9. 9.
    McManus S, Li Y (2010) The Structural Diversity of Deoxyribozymes. Molecules 15:6269–6284.PubMedCrossRefGoogle Scholar
  10. 10.
    Li J, Lu Y (2000) A Highly Sensitive and Selective Catalytic DNA Biosensor for Lead Ions. J Am Chem Soc 122:10466–10467.CrossRefGoogle Scholar
  11. 11.
    Liu J, Lu Y (2003) A Colorimetric Lead Biosensor Using DNAzyme-Directed Assembly of Gold Nanoparticles. J Am Chem Soc 125:6642–6643.CrossRefGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    Lan T, Furuya K, Lu Y (2010) A Highly Selective Lead Sensor Based on a Classic Lead DNAzyme. Chem Commun 46:3896–3898.CrossRefGoogle Scholar
  14. 14.
    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.CrossRefGoogle Scholar
  15. 15.
    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.PubMedCrossRefGoogle Scholar
  16. 16.
    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.CrossRefGoogle Scholar
  17. 17.
    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.Google Scholar
  18. 18.
    Schlosser K, Li Y (2009) DNAzyme-mediated Catalysis with Only Guanosine and Cytidine Nucleotides. Nucleic Acids Res 37:413–420.PubMedCrossRefGoogle Scholar
  19. 19.
    Lam J, Li Y (2010) Influence of Cleavage Site on Global Folding of an RNA-Cleaving DNAzyme. Chem Bio Chem 11: 1710–1719.PubMedGoogle Scholar
  20. 20.
    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.PubMedGoogle Scholar
  21. 21.
    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.PubMedCrossRefGoogle Scholar
  22. 22.
    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.PubMedCrossRefGoogle Scholar
  23. 23.
    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.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

Personalised recommendations