LABEL-FREE MONITORING OF DNA-LIGAND INTERACTIONS

We report on the label- and isotope-free monitoring of DNA interaction s with low-molecular-weight ligands. An optical technique based on int erference at thin layers was used to monitor in real time binding of l igands at DNA which was immobilized by Coulomb interactions at a posit ively charged surface. Approximately 2 ng DNA/mm(2) was irreversibly b ound to the surface, which remained stable over several days. This res ult was confirmed by characterization of the layer using spectroscopic ellipsometry. During incubation of immobilized DNA with a variety of intercalators and other DNA-binding compounds in a flow system, intera ctions were monitored by reflectometric interference spectroscopy. Bin ding effects between 10 and 400 pg/mm(2) were detected unambiguously. Nonspecific binding effects were excluded by using a negatively charge d reference surface. Variation of intercalator concentration allowed t he characterization of interaction with respect to kinetics and thermo dynamics by the evaluation of binding rate and equilibrium coverage. T he affinity constants were determined in the range between 10(5) and 1 0(6) M-1, in good agreement to those obtained by homogeneous phase ass ays. Association rate constants between 10(3) and 10(5) M(-1)s(-1) and dissociation rate constants between 10(-1) and 10(-2) s(-1) were dete rmined by evaluation of the binding curves. Both the fast and simple t est format and a universal applicability make the new technique descri bed attractive for detecting and characterizing interaction of low-mol ecular-weight molecules with DNA. (C) 1997 Academic Press.