title: Fluorescence assay of polyamide-DNA interactions.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry and Center for Nanoscience, University of Missouri-St. Louis, St. Louis, MO 63121, USA. cdup@umsl.edu
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Dupureur
authors.forename: James K
authors.initials: JK
authors.lastname: Bashkin
authors.forename: Karl
authors.initials: K
authors.lastname: Aston
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Koeller
authors.forename: Kimberly R
authors.initials: KR
authors.lastname: Gaston
authors.forename: Gaofei
authors.initials: G
authors.lastname: He
mesh_terms: D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009757:Nylons / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D009757:Nylons; D012235:Rhodamines; D004247:DNA