Molecular beacons for detecting DNA binding proteins.
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.
Heyduk
Tomasz
T
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, 1402 S. Grand Blvd., St. Louis, MO 63104, USA. heydukt@slu.edu
Heyduk
Ewa
E
D001483:Base Sequence; D001665:Binding Sites; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004345:Drug Industry / Q000191:economics / Q000379:methods; D019793:Fluorescein / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000191:economics / Q000379:methods; D013053:Spectrophotometry
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D019793:Fluorescein