First generation of fluorescent labeled DNA bis-intercalators

Understanding biochemical functions at the most basic level promises greater understanding and control over cellular processes. Synthetic molecules that specifically bind double helical DNA have been pursued as a means of controlling gene expression as well as creating novel therapeutic agents. The Iverson group has developed a unique mode of DNA recognition that is referred to as threading polyintercalators. These threading polyintercalators are composed of amino acids and the electron deficient 1,4,5,8-naphthalenetetracarboxylic diimide linked in a head-to-tail fashion and they are constructed using Fmoc-based solid phase peptide synthesis. Dimer, trimer, tetramer, and octamer polyintercalators have been synthesized and studied. NMR structural analysis has been used to study the mode of DNA binding by a tetrameric threading polyintercalating molecule and its linkers were found to reside in alternating grooves in the order of minor-major-minor groove. The threading polyintercalation mode of DNA binding poses a number of interesting questions regarding the kinetics of association and dissociation. The attachment of carboxyfluorescein to a previously synthesized bis-intercalator will be a valuable tool with which to measure association and disassociation rates in the context of DNA binding. The interaction of the fluorophore with DNA can be monitored by UV-Vis and especially single molecule fluorescence. The idea will be to uncover the mechanism by which threading polyintercalators associates and dissociates from their preferred binding sites. A number of important questions come to mind including the way in which threading polyintercalator molecules might find their preferred binding sites as well as the order of events in polyintercalative binding. Mass spectrometry combined with high-pressure liquid chromatography verified isolation of the desired bis-intercalator conjugates. Preliminary studies have confirmed DNA binding to homopolymer DNA sequences, setting the stage for detailed kinetic studies