Quantifying free energy pathways of competitive DNA-ligand interactions

DNA intercalators are considered for potential DNA targeted therapeutics. The kinetics of simple DNA intercalators are well-modeled and characterized in single molecules studies. However, DNA-ligand that have multi-binding modes including intercalation are often studied in equilibrium approach, which does not address the governing free energy pathways. It is crucial to map the transition pathways, in particular, for competitive DNA-ligand interactions that alter DNA extension, which provides insights for rational drug design. While intercalation elongates DNA, other binding modes can be distinguished by distinct rates of increase or decrease of DNA extension. For practical experimental conditions, a frame work is proposed here to analyze the kinetics of DNA-ligand multi-binding modes probed by time-dependent and force-dependent DNA extension. This enables characterizing and quantifying the free energy pathways in order to identify and further optimize the molecular binding mechanism.