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Molecular beacons for detecting DNA binding proteins

Nature Biotechnology volume 20, pages 171176 (2002) | Download Citation

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Abstract

We report here a simple, rapid, homogeneous fluorescence assay, the molecular beacon assay, for the detection and quantification of sequence-specific DNA-binding proteins. The central feature of the assay is the protein-dependent association of two DNA fragments each containing about half of a DNA sequence defining a protein-binding site. Protein-dependent association of DNA fragments can be detected by any proximity-based spectroscopic signal, such as fluorescence resonance energy transfer (FRET) between fluorochromes introduced into these DNA molecules. The assay is fully homogeneous and requires no manipulations aside from mixing of the sample and the test solution. It offers flexibility with respect to the mode of signal detection and the fluorescence probe, and is compatible with multicolor simultaneous detection of several proteins. The assay can be used in research and medical diagnosis and for high-throughput screening of drugs targeted to DNA-binding proteins.

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References

  1. 1.

    Genes VII (Oxford University Press, New York; 2000).

  2. 2.

    & p53—puzzle and paradigm. Genes Dev. 10, 1054–1072 (1996).

  3. 3.

    Transcription therapy for cancer. Oncogene 20, 3116–3127 (2001).

  4. 4.

    & Equilibria and kinetics of lac repressor–operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505–6525 (1981).

  5. 5.

    & DNase footprinting: a simple method for the detection of protein–DNA binding specificity. Nucleic Acid Res. 5, 3157–3170 (1978).

  6. 6.

    & Fluorescence approaches to study of protein–nucleic acid complexation. Methods Enzymol. 278, 390–416 (1997).

  7. 7.

    Principles of fluorescence spectroscopy (Kluwer Academic/Plenum Press, New York; 1999).

  8. 8.

    Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999).

  9. 9.

    & Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).

  10. 10.

    & Transcription activation by catabolite activator protein (CAP). J. Mol. Biol. 293, 199–213 (1999).

  11. 11.

    , & Consensus DNA site for the Escherichia coli catabolite activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site. Nucleic Acids Res. 17, 10295–10305 (1989).

  12. 12.

    & Direct recognition of the trp operator by the trp holorepressor—a review. Gene 150, 1–8 (1994).

  13. 13.

    & Lactose repressor protein: functional properties and structure. Progress Nucleic Acid Res. Mol. Biol. 58,127–164 (1998).

  14. 14.

    , & Thermodynamics of ligand binding to trp repressor. Biochemistry 32, 7302–7309 (1993).

  15. 15.

    & Equilibria and kinetics of lac repressor–operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505–6525 (1981).

  16. 16.

    & Protein–DNA binding correlates with structural thermostability for the full-length human p53 protein. Biochemistry 40, 3847–3858 (2001).

  17. 17.

    , , & p53CP is p51/p63, the third member of the p53 gene family: partial purification and characterization. Carcinogenesis 22, 295–300 (2001).

  18. 18.

    , , & Anti-cooperative biphasic equilibrium binding of transcription factor upstream stimulatory factor to its cognate DNA monitored by protein fluorescence changes. J. Biol. Chem. 270, 19325–19329 (1995).

  19. 19.

    , , , & Affinity and specificity of trp repressor–DNA interactions studied with fluorescent oligonucleotides. J. Mol. Biol. 273, 572–585 (1997).

  20. 20.

    , & Kinetic mechanism of DNA binding and DNA-induced dimerization of the Escherichia coli Rep helicase. Biochemistry 35, 2268–2282 (1996).

  21. 21.

    , & Binding of XPA and RPA to damaged DNA investigated by fluorescence anisotropy. Biochemistry 40, 2901–2910 (2001).

  22. 22.

    et al. Interaction between the Escherichia coli regulatory protein TyrR and DNA: a fluorescence footprinting study. Biochemistry 34, 15802–15812 (1995).

  23. 23.

    , & Simultaneous binding and bending of promoter DNA by the TATA binding protein: real time kinetic measurements. Biochemistry 35, 7459–7465 (1996).

  24. 24.

    , , , & Fluorescence study of the multiple binding equilibria of the galactose repressor. Biochemistry 37, 41–50 (1998).

  25. 25.

    , , , & Equilibrium binding of single-stranded DNA with herpes simplex virus type I–coded single-stranded DNA-binding protein, ICP8. J. Biol. Chem. 275, 10864–10869 (2000).

  26. 26.

    , & DNA tightens the dimeric DNA-binding domain of human papillomavirus E2 protein without changes in volume. Proc. Natl. Acad. Sci. USA 97, 14289–14294 (2000).

  27. 27.

    et al. Equilibrium binding of estrogen receptor with DNA using fluorescence anisotropy. J. Biol. Chem. 272, 30405–30411 (1997).

  28. 28.

    & Application of fluorescence energy transfer and polarization to monitor E. coli cAMP receptor protein and lac promoter interaction. Proc. Natl. Acad. Sci. USA 87, 1744–1748 (1990).

  29. 29.

    Fluorescence energy transfer as a spectroscopic ruler. Ann. Rev. Biochem. 47, 819–846 (1978).

  30. 30.

    , & Luminescence energy transfer. J. Am. Chem. Soc. 116, 6029–6030 (1994).

  31. 31.

    , & Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16, 49–53 (1998).

  32. 32.

    , , & Characterization of fluorescence quenching in bifluorophoric protease. Biophys. Chem. 67, 167–176 (1997).

  33. 33.

    & A homogeneous immunoassay for cyclic nucleotides based on chemiluminescence energy transfer. Biochem. J. 216, 185–194 (1983).

  34. 34.

    , & A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. USA 96, 151–156 (1999).

  35. 35.

    & Scintillation proximity assay (SPA)—a new method of immunoassay. Direct and inhibition mode detection with human albumin and rabbit antihuman albumin. Mol. Immunol. 16, 265–267 (1979).

  36. 36.

    Simultaneous binding of two DNA duplexes to the NtrC–enhancer complex studied by two-color fluorescence cross-correlation spectroscopy. Biochemistry 39, 2131–2139 (2000).

  37. 37.

    & The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions. Nat. Biotechnol. 16, 633–638 (1998).

  38. 38.

    , , & Quantitation of RNA polymerase II and its transcription factors in HeLa cell: little soluble holoenzyme but significant amounts of polymerase attached to the nuclear substructure. Mol. Cell. Biol. 19, 5383–5392 (1999).

  39. 39.

    , , & Characterization of mediator complexes from HeLa cell nuclear extract. Mol. Cell. Biol. 21, 4604–4613 (2001).

  40. 40.

    , , & Non-specific DNA binding of genome regulating proteins as a biological control mechanism: 1. The lac operon: equilibrium aspects. Proc. Natl. Acad. Sci. USA 71, 4808–4812 (1974).

  41. 41.

    , & Intracellular Trp repressor levels in E. coli. J. Bacteriol. 167, 272–278 (1986).

  42. 42.

    & Thiol reactive luminescence europium chelates. Luminescence probes for resonance energy transfer distance measurements in biomolecules. Anal. Biochem. 248, 216–227 (1997).

  43. 43.

    & E. coli cAMP receptor protein: evidence for three conformational states with different promoter binding affinities. Biochemistry 28, 6914–6924 (1989).

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Acknowledgements

This work was partially supported by a NIH grant GM 50514. We thank M. Brenowitz for the gift of purified lacR protein and K.S. Matthews and N.M. Nichols for the gift of purified p53 protein.

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  1. Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.

    • Tomasz Heyduk
    •  & Ewa Heyduk

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Correspondence to Tomasz Heyduk.

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DOI

https://doi.org/10.1038/nbt0202-171

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