Abstract
A novel method for DNA enzymatic cleavage assays using molecular beacons (MBs) as the substrate for nuclease is described. An MB is a hairpin-shaped DNA probe that is labeled with a fluorescent dye at one end and a quencher at the other end. The loop sequence of the MB can be used as the substrate for single-stranded specific nucleases, whereas the stem of the MB can be designed as the substrate for restriction enzymes. The enzymatic cleavage breaks the MB into fragments and leads to the distance separation of the quencher and the fluorophore, resulting in an increase in the fluorescent signal. Up to an 80-fold signal-to-noise ratio was observed when these probes were cleaved by nucleases. Taking advantage of the MB’s detection-without-separation property, this method allows for the real-time detection of DNA cleavage, which is useful for the characterization of DNA nuclease activity as well as the study of steady-state cleavage reaction kinetics. With its simplicity, convenience, high sensitivity, and excellent reproducibility, this method has the potential to be used in the study of both natural and artificial nucleic acid-cleaving enzymes.
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
Sambrook, J., Fritsch, E. F., and Manistis, T. (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Mclaughlin, L. W., Benseler, F., Graeser, E., Piel, N., and Scholtissek, S. (1987) Effects of functional-group changes in the ecori recognition site on the cleavage reaction catalyzed by the endonuclease. Biochemistry 26, 7238–7245.
Alves, J., Ruter, T., Geiger, R., Fliess, A., Maass, G., and Pingoud, A. (1989) Changing the hydrogen-bonding potential in the DNA-binding site of ecori by site-directed mutagenesis drastically reduces the enzymatic-activity, not, however, the preference of this restriction endonuclease for cleavage within the site-GATTC-. Biochemistry 28, 2678–2684.
Waters, T. R. and Connolly, B. A. (1992) Continuous spectrophotometric assay for restriction endonucleases using synthetic oligodeoxynucleotides and based on the hyperchromic effect. Anal. Biochem. 204, 204–209.
Lee, S. P. and Han, M. K. (1997) Fluorescence assays for DNA cleavage. Methods Enzymol. 278, 343–363.
Ghosh, S. S., Eis, P. S., Blumeyer, K., Fearon, K., and Millar, D. P. (1994) Real-Time Kinetics of Restriction-Endonuclease Cleavage Monitored by Fluorescence Resonance Energy-Transfer. Nucleic Acids Res. 22, 3155–3159.
Lee, S. P., Censullo, M. L., Kim, H. G., Knutson, J. R., and Han, M. K. (1995) Characterization of endonucleolytic activity of HIV-1 integrase using a fluorogenic substrate. Anal. Biochem. 227, 295–301.
Lee, S. P., Porter, D., Chirikjian, J. G., Knutson, J. R., and Han, M. K. (1994) Fluorometric assay for DNA cleavage reactions characterized with bamhi restriction-endonuclease. Anal. Biochem. 220, 377–383.
Li, J. J., Geyer, R., and Tan, W. (2000) Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA. Nucleic Acids Res. 28, 52.
Tyagi, S. and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308.
Bonnet, G., Krichevsky, O., and Libchaber, A. (1998) Kinetics of conformational fluctuations in DNA hairpin-loops. Proc. Natl. Acad. Sci. USA 95, 8602–8606.
Sokol, D. L., Zhang, X. L., Lu, P. Z., and Gewitz, A. M. (1998) Real time detection of DNA RNA hybridization in living cells. Proc. Natl. Acad. Sci. USA 95, 11,538–11,543.
Liu, X. J. and Tan, W. H. (1999) A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons. Anal. Chem. 71, 5054–5059.
Matsuo, T. (1998) In situ visualization of messenger RNA for basic fibroblast growth factor in living cells. Biochim. Biophys. Acta 1379, 178–184.
Rodriguez, L. C., Jen, K. Y., Gifford, L. K., Zhang, X. L., Lu, P. Z., and Gewirtz, A. M. (1999) Probing mRNA structure with molecular beacons (MB): a novel strategy for optimizing design of "e;antisense (AS)"e; nucleic acid molecules. Blood 94, 178A.
Tsuji, A., Koshimoto, H., Sato, Y., et al. (2000) Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope. Biophys. J. 78, 3260–3274.
Perlette, J. and Tan, W. H. (2001) Real-time monitoring of intracellular mRNA hybridization inside single living cells. Anal. Chem. 73, 5544–5550.
Tyagi, S., Bratu, D. P., and Kramer, F. R. (1998) Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16, 49–53.
Wiegand, R. C., Godson, G. N., and Radding, C. M. (1975) Specificity of the S1 nuclease from Aspergillus oryzae. J. Biol. Chem. 250, 8848–8855.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Yang, C.J., Li, J.J., Tan, W. (2006). Using Molecular Beacons for Sensitive Fluorescence Assays of the Enzymatic Cleavage of Nucleic Acids. In: Didenko, V.V. (eds) Fluorescent Energy Transfer Nucleic Acid Probes. Methods in Molecular Biology™, vol 335. Humana Press. https://doi.org/10.1385/1-59745-069-3:71
Download citation
DOI: https://doi.org/10.1385/1-59745-069-3:71
Publisher Name: Humana Press
Print ISBN: 978-1-58829-380-0
Online ISBN: 978-1-59745-069-0
eBook Packages: Springer Protocols