DNA in the spotlight: visualization, quantification, and manipulation of DNA with small molecule intercalators

Fluorophores for staining nucleic acids have become an essential part of molecular and cell biology research. Recently, a wide range of DNA- and RNA-binding fluorophores has been developed for staining nucleic acids after in vitro separation in gels, in single molecule experiments, and in (living) cells for fluorescence imaging. For optimal staining, it is crucial to know the binding parameters. Here, I present three research projects investigating the molecular binding mechanisms between different dyes and DNA, how binding affects the photophysics and photochemistry of the dyes, and in turn the structural and mechanical properties of DNA. In the first part, I review the fluorescent dye SYBR Gold. I present its chemical structure and determine structural aspects of DNA binding. Using a combination of single molecule methods and bulk fluorescence measurements, I quantify the binding of SYBR Gold to DNA and show that SYBR Gold binds in an intercalative binding mode, which leads to a tremendous increase in fluorescence quantum efficiency. The second part focuses on the interactions between the ruthenium complex Ru(TAP)32+ [TAP = 1,3,5,8-tetraazaphenanthrene] and DNA. Using several single-molecule techniques, I show that Ru(TAP)32+ can bind to DNA with a strong binding mode via intercalation, and a weak binding mode that presumably occurs based on hydrogen bond formation. By combining both interactions, Ru(TAP)32+ can bind two DNA segments together in a non-covalent manner. Through bulk photophysical and photochemical experiments, I demonstrate how multivalent binding can accelerate the photochemistry of covalent adduct formation. In the third part of this thesis, I investigate DNA-ligand binding in a topologically closed system. The experimental investigation is based on the intercalators ethidium bromide, SYBR Gold, SYTOX Orange and trimethylpsoralene. In addition, I introduce a theoretical model that accurately describes the experimental data and allows the quantitative study of different DNA supercoiling densities both in vitro and in vivo