A competition assay for DNA binding using the fluorescent probe ANS.
Fluorescence spectroscopy is a technique frequently employed to study protein-nucleic acid interactions. Often, the intrinsic fluorescence emission spectrum of tryptophan residues in a nucleic-acid-binding protein is strongly perturbed upon interaction with a target DNA or RNA. These spectral changes can then be exploited in order to construct binding isotherms and the extract equilibrium association constant together with the stoichiometry of an interaction. However, when a protein contains many tryptophan residues that are not located in the proximity of the nucleic-acid-binding site, changes in the fluorescence emission spectrum may not be apparent or the magnitude too small to be useful. Here, we make use of an extrinsic fluorescence probe, the environmentally sensitive fluorophore 1-anilinonaphthalene-8-sulphonic acid (1,8-ANS). Displacement by DNA of 1,8-ANS molecules from the nucleic-acid-binding site of the Type I modification methylase EcoR124I results in red shifting and an intensity decrease of the 1,8-ANS fluorescence emission spectrum. These spectral changes have been used to investigate the interaction of EcoR124I with DNA target recognition sequences.
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, St Michael's Building, University of Portsmouth, Portsmouth PO1 2DT, UK.
Ian A
IA
Taylor
G Geoff
GG
Kneale
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D011485:Protein Binding; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013696:Temperature; D017186:Titrimetry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D012997:Solvents; C027132:1-anilino-8-naphthalenesulfonate; D004247:DNA; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific