Journal
ANALYTICAL BIOCHEMISTRY
Volume 423, Issue 1, Pages 178-183Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.01.017
Keywords
Polyamide; DNA; Fluorescence; Binding
Funding
- National Institutes of Health (NIH) [R01 AI083803-02]
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Polyamides (PAs) are distamycin-type ligands of DNA that bind the minor groove and are capable of sequence selective recognition. This capability provides a viable route to their development as therapeutics. Presented here is a simple and convenient fluorescence assay for PA-DNA binding. PAs are titrated into a sample of a hairpin DNA featuring a TAMRA dye attached to an internal dU near the PA binding site. In a study of 6 PAs, PA binding leads to a steady reproducible decrease in fluorescence intensity that can be used to generate binding isotherms. The assay works equally well with both short (6- to 8-ring) and long (14-ring) PAs, and K-d values ranging from approximately 1 nM to at least 140 nM were readily obtained using a simple monochromator or filter configuration. Competition assays provide a means to assessing possible dye interference, which can be negligible. The assay can also be used to determine PA extinction coefficients and to measure binding kinetics; thus, it is an accessible and versatile tool for the study of PA properties and PA-DNA interactions. (C) 2012 Elsevier Inc. All rights reserved.
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