Abstract
In this chapter, methods for designing metal ion sensors using fluorophore- and quencher-labeled DNAzymes are discussed. In contrast to the classical molecular beacon method based on binding, the methods described here utilize catalytic cleavage to release the fluorophore for detection and quantification, making it possible to take advantage of catalytic turnovers for signal amplification. Unlike classical molecular beacons that detect only nucleic acids, catalytic molecular beacons can be applied to different DNAzymes to detect a broad range of analytes. The methods described are based on the finding that almost all known trans-cleaving DNAzymes share a similar structure comprised of a catalytic DNAzyme core flanked by two substrate recognition arms. Using a typical DNAzyme called the “8-17” DNAzyme as an example, the design of highly sensitive and selective Pb2+ sensors is described in detail. The initial design employs a single fluorophore-quencher pair in close proximity, with the fluorophore on the 5′-end of the substrate and the quencher on the 3′-end of the enzyme. Although this sensor is highly sensitive and selective at 4°C, high background fluorescence is observed at higher temperatures. Therefore a new design with an additional quencher attached to the 3′-end of the substrate is employed to suppress background fluorescence. The dual quencher method allows the sensor to perform at ambient temperatures with a high signal-to-noise ratio.
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References
Breaker, R. R. and Joyce, G. F. (1994) A DNA enzyme that cleaves RNA. Chem. Biol. 1, 223–229.
Breaker, R. R. (1997) DNA enzymes. Nat. Biotechnol. 15, 427–431.
Sen, D. and Geyer, C. R. (1998) DNA enzymes. Curr. Opin. Chem. Biol. 2, 680–687.
Lu, Y. (2002) New transition metal-dependent DNAzymes as efficient endonucleases and as selective metal biosensors. Chem. Eur. J. 8, 4588–4596.
Li, J., Zheng, W., Kwon, A. H., and Lu, Y. (2000) In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme. Nucleic Acids Res. 28, 481–488.
Brown, A. K., Li, J., Pavot, C. M. B., and Lu, Y. (2003) A lead-dependent DNAzyme with a two-step mechanism. Biochemistry 42, 7152–7161.
Santoro, S. W., Joyce, G. F., Sakthivel, K., Gramatikova, S. and Barbas, C. F., III. (2000) RNA cleavage by a DNA enzyme with extended chemical functionality. J. Am. Chem. Soc. 122, 2433–2439.
Cuenoud, B. and Szostak, J. W. (1995) A DNA metalloenzyme with DNA ligase activity Nature 375, 611–614.
Mei, S. H. J., Liu, Z., Brennan, J. D., and Li, Y. (2003) An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling. J. Am. Chem. Soc. 125, 412–420.
Bruesehoff P. J., Li J., Augustine A. J., and Lu Y. (2002) Improving metal ion specificity during in vitro selection of catalytic DNA. Comb. Chem. High T. Scr. 5, 327–335.
Wang, Y. and Silverman, S. K. (2003) Deoxyribozymes that synthesize branched and lariat RNA. J. Am. Chem. Soc. 125, 6880–6881.
Li, J. and Lu, Y. (2000) A highly sensitive and selective catalytic DNA biosensor for lead ions. J. Am. Chem. Soc. 122, 10,466–10,467.
Liu, J. and Lu, Y. (2003) Improving fluorescent DNAzyme biosensors by combining inter-and intramolecular quenchers. Anal. Chem. 75, 6666–6672.
Lu, Y., Liu, J., Li, J., Bruesehoff, P. J., Pavot, C. M. B., and Brown, A. K. (2003) New highly sensitive and selective catalytic DNA biosensors for metal ions. Biosens. Bioelectron. 18, 529–540.
Tyagi, S. and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308.
Santoro, S. W. and Joyce, G. F. (1997) A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 94, 4262–4266.
Faulhammer, D. and Famulok, M. (1996) The Ca2+ ion as a cofactor for a novel RNA-cleaving deoxyribozyme. Angew. Chem. Int. Ed. Engl. 35, 2837–2841.
Jenne, A., Gmelin, W., Raffler, N., and Famulook, M. (1999) Real-time characterization of ribozymes by fluorescence resonance energy transfer (FRET). Angew. Chem. Int. Ed. 38, 1300–1303.
Nowakowski, J., Shim, P. J., Prasad, G. S., Stout, C. D., and Joyce, G. F. (1999) Crystal structure of an 82-nucleotide RNA-DNA complex formed by the 10-23 DNA enzyme. Nat. Struct. Biol. 6, 151–156.
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Liu, J., Lu, Y. (2006). Fluorescent DNAzyme Biosensors for Metal Ions Based on Catalytic Molecular Beacons. In: Didenko, V.V. (eds) Fluorescent Energy Transfer Nucleic Acid Probes. Methods in Molecular Biology™, vol 335. Humana Press. https://doi.org/10.1385/1-59745-069-3:275
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DOI: https://doi.org/10.1385/1-59745-069-3:275
Publisher Name: Humana Press
Print ISBN: 978-1-58829-380-0
Online ISBN: 978-1-59745-069-0
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