Abstract
Exploring the properties of molecules that cleave DNA (i.e., enzymatic nucleases, chemical footprinting agents, and naturally occurring DNA cleaving antibiotics) has been an ongoing process with benefits extending toward both laboratory and clinical applications. Despite the progress that has been made toward understanding the mechanics of DNA cleavage, a simple and continuous assay for detecting DNA cleavage has been lacking. Herein, we describe the molecular break light assay, wherein a single oligonucleotide modified by a 5′-fluorophore-3′-quencher pair adopting a stem-loop structure with an appropriate DNA recognition site, provides for the rapid assaying of DNA cleavage with high sensitivity. Furthermore, the described methodology is highly convenient in that it is readily adaptable to common laboratory fluorometers and multi-well plate/ array systems, which may provide the basis for high-throughput screening of novel DNA cleaving agents. This assay may also be further extended to natural or “unnatural” transcription factor protection assay systems.
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References
Klostermeier, D. and Millar, D. P. (2002) Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids. Biopolymers 61, 159–179.
Brodie, S., Giron, J., and Latt, S. A. (1975) Estimation of accessibility of DNA in chromatin from fluorescence measurements of electronic excitation energy transfer. Nature 253, 470–471.
Yarbrough, L. R., Schlageck, J. G., and Baughman, M. (1979) Synthesis and properties of fluorescent nucleotide substrates for DNA-dependant RNA polymerases. J. Biol. Chem. 254, 12,069–12,073.
Wu, F. Y-H. and Tyagi, S. C. (1987) Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase. J. Biol. Chem. 262, 13,147–13,154.
Murchie, A. I. H., Clegg, R. M., von Kitzing, E., Duckett, D. R., Diekmann, S., and Lilley, D. M. J. (1989) Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules. Nature 341, 763–766.
Tyagi, S. and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308.
Biggins, J. B., Prudent, J. R., Marshall, D. J., Ruppen, M., and Thorson, J. S. (2000) A continuous assay for DNA cleavage: the application of &“break lights&” to enediynes, iron-dependent agents, and nucleases. Proc. Nat. Acad. Sci. USA 97, 13,537–13,542.
Myers, A. G., Cohen, S. B., and Kwon, B.M. (1994) A study of the reaction of calicheamicin γ1 with glutathione in the presence of double-stranded DNA. J. Am. Chem. Soc. 116, 1255–1271.
Burger, R. M., Horwitz, S. B., and Peisach, J. (1985) Stimulation of iron(II) bleomycin activity by phosphate-containing compounds. Biochemistry 24, 3623–3629.
Burger, R. M., Projan, S. J., Horwitz, S. B., and Peisach, J. (1985) The DNA cleavage mechanism of iron-bleomycin. Kinetic resolution of strand scission from base propenal release. J. Biol. Chem. 261, 15,955–15,959.
Roy, K. B., Vrushank, D., and Jayaram, B. (1994) Use of isotope-dilution phenomenon to advantage in the determination of kinetic constants KM and k cat for BamHI restriction endonuclease: an empirical and iterative approach. Anal. Biochem. 220, 160–164.
Hashimoto, S., Wang, B., and Hecht, S. M. (2001) Kinetics of DNA cleavage by Fe(II)-bleomycins. J. Am. Chem. Soc. 123, 7437–7438.
Jacobsen, E. N. and Finney, N. S. (1994) Synthetic and biological catalysts in chemical synthesis: how to assess practical utility. Chem. Biol. 1, 85–90.
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© 2006 Humana Press Inc., Totowa, NJ
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Biggins, J.B., Prudent, J.R., Marshall, D.J., Thorson, J.S. (2006). A Continuous Assay for DNA Cleavage Using Molecular Break Lights. In: Didenko, V.V. (eds) Fluorescent Energy Transfer Nucleic Acid Probes. Methods in Molecular Biology™, vol 335. Humana Press. https://doi.org/10.1385/1-59745-069-3:83
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DOI: https://doi.org/10.1385/1-59745-069-3:83
Publisher Name: Humana Press
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