Label-free monitoring of DNA-ligand interactions.
We report on the label- and isotope-free monitoring of DNA interactions with low-molecular-weight ligands. An optical technique based on interference at thin layers was used to monitor in real time binding of ligands at DNA which was immobilized by Coulomb interactions at a positively charged surface. Approximately 2 ng DNA/m2 was irreversibly bound to the surface, which remained stable over several days. This result 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, interactions were monitored by reflectometric interference spectroscopy. Binding effects between 10 and 400 pg/ mm2 were detected unambiguously. Nonspecific binding effects were excluded by using a negatively charged reference surface. Variation of intercalator concentration allowed the characterization of interaction with respect to kinetics and thermodynamics by the evaluation of binding rate and equilibrium coverage. The affinity constants were determined in the range between 10(5) and 10(6) M-1, in good agreement to those obtained by homogeneous phase assays. 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 determined by evaluation of the binding curves. Both the fast and simple test format and a universal applicability make the new technique described attractive for detecting and characterizing interaction of low-molecular-weight molecules with DNA.
Piehler
J
J
Institut für Physikalische Chemie, Tübingen, Germany. jacob.piehler@uni-tuebingen.de
Brecht
A
A
Gauglitz
G
G
Zerlin
M
M
Maul
C
C
Thiericke
R
R
Grabley
S
S
D004247:DNA / Q000737:chemistry*; D003609:Dactinomycin / Q000737:chemistry; D007364:Intercalating Agents / Q000737:chemistry; D007554:Isotopes; D008024:Ligands; D015394:Molecular Structure; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D007364:Intercalating Agents; D007554:Isotopes; D008024:Ligands; D003609:Dactinomycin; D004247:DNA