Dongchul Suh

Criteria for the Mode of Binding of DNA Binding Agents

A complete characterization of DNA binding agents requires that their mode of binding to DNA be established. In the absence of high resolution structural data, the mode of binding is, of necessity, usually inferred indirectly from various solution studies. The purpose of this study is to show that only certain methods can be used reliably to infer the DNA binding mode. Comparative fluorescence and hydrodynamic studies using the proven intercalator ethidium and the groove binder Hoechst 33258 are described. The results of our studies show that while fluorescence intensity, polarization, and quenching measurements can detect a binding interaction of the ligand with DNA, none are sensitive indicators of the binding mode. Fluorescence contact energy transfer studies can reliably indicate intercalation, as can viscosity measurements. Our results illustrate reliable criteria that may be used to distinguish intercalation from groove binding in the absence of high resolution structural data.

Determining the binding mode of DNA sequence specific compounds

Abstract The binding mode of DNA sequence specific compounds can be established by characterizing the binding agents completely. A variety of solution studies may indirectly be used for determining the binding mode. The sequence specificity of certain DNA binding drugs has been found to be related to the specific mode of binding. In this study, only certain methods are found to be reliable to infer the binding mode of DNA binding drugs. Comparative fluorescence and hydrodynamic studies have been done. The results in this study show that most fluorescence measurements are not sensitive indicators of the mode of binding. The fluorescence intensity, fluorescence polarization, and quenching measurements can detect the binding interaction of ligands with DNA. Fluorescence contact energy transfer studies can reliably indicate the different mode of binding. These results illustrate reliable criteria that may be used to distinguish the intercalation from the groove binding. They also infer the mode of binding in the absence of high-resolution structural data.

DAUNOMYCIN BINDING TO DEOXYPOLYNUCLEOTIDES WITH ALTERNATING SEQUENCES: COMPLETE THERMODYNAMIC PROFILES OF HETEROGENEOUS BINDING SITES

ABSTRACT Complete thermodynamic binding profiles for the interaction of the anticancer drug, daunomycin with natural DNA and synthetic deoxypolynucleotides were described. Fluorescence titration method was used to estimate the equilibrium binding constants. Binding isotherms were found to be surprisingly complex in some cases, presumably because there were heterogeneous sites even in simple deoxypolynucleotides of repeating sequence. Some polynucleotides consisting of alternating sequence contain at least two different binding sites for daunomycin. The binding affinity of the primary binding sites of alternating and non-alternating sequences was found to differ by two orders of magnitude. An isothermal microtitration calorimeter was used to directly measure the binding enthalpy at 25°C with a high sensitivity. The binding enthalpy of poly[d(A-T)] was found to be −5.5 Kcal/mol, which was much lower than any other polynucleotides, while the binding constant of the high affinity sites, was similar. In this report, the complete thermodynamic profiles of daunomycin binding to deoxypolynucleotides were reliably shown for the first time.