Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
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

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Cynthia M. Dupureur
James K. Bashkin
Karl Aston
Kevin J. Koeller
Kimberly R. Gaston
and Gaofei He

Department of Chemistry
Center for Nanoscience
University of Missouri St. Louis
St. Louis, MO 63121 USA

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