Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
A Convenient Fluorescence Assay of Polyamide-DNA Interactions
Polyamides (PA) are distamycin-type ligands of DNA that bind the minor groove and are capable of sequence selective recognition (1). This capability provides a viable route to their development as therapeutics. PAs have limited intrinsic fluorescence behavior, making this signal poorly suited for binding assays, and the introduction of a dye to this structure requires additional synthetic steps and can change uptake for cell-based assays (2). The most commonly performed assays of DNA binding by PAs involve complex techniques like calorimetry (3), surface plasmon resonance (3), and footprinting (4). Presented here is a simple and convenient fluorescence assay for polyamide-DNA binding. PAs are titrated into a sample of a hairpin DNA featuring a TAMRA dye attached to an internal dU a few nt away from the PA binding site. In a study of 12 polyamides, PA binding leads to a steady, reproducible decrease in fluorescence intensity that can be used to generate binding isotherms. The assays works equally well with both short (6-8 ring) and long (14 ring) polyamides, and Kd values ranging from 0.5 to at least 300 nM (depending on PA composition and target sequence), were readily obtained using a simple monochrometer configuration. Stronger Kd values were confirmed with a PCR fragment and quantitative footprinting analysis. Competition assays indicate that the effects of the dye are typically negligible when the dye-labeled dU residue is outside the binding site. When the dye is placed within the predicted binding site, binding experiments show disturbances in the classical binding trend. This effect provides a means to confirm the binding site in DNAs with more than one potential binding site.
This research is supported by NIH R01 AI083803-02
Cynthia M. Dupureur
Department of Chemistry