Fluorescence Assay Development for the Measurement of Binding of Weak DNA-Minor Groove Binders

Several compounds are being developed in our laboratory that can bind in the minor groove of DNA at specific sequences and alkylate the N-3 site on adenines. Alkylation of this site is known to cause cell death, and therefore such compounds can have potential applications in cancer chemotherapy. These compounds have been designed to be weak minor groove binders, and their binding affinity to targeted sequences is expected to be a critical determinant of the efficiency of alkylation. Our goal with this project is to develop an assay which will enable us to measure the binding constants of various compounds to the desired sequences on DNA. In order to achieve this goal, we have synthesized fluorescent compounds that can bind to the same sites on DNA as the other compounds being synthesized in our laboratory. It is expected that the appropriate fluorescent compound will exhibit different fluorescent properties when it is bound to the DNA as opposed to when it is free in solution. Therefore, the extent to which this fluorescent compound is displaced from the DNA by the other compounds being tested can be measured as a change in the observed fluorescence, and this would enable us to measure the binding constant of the various candidates being tested. The synthesis of the various candidate fluorescent compounds, and their evaluation for use in a binding assay are described here.