Fluorescence assay of polyamide–DNA interactions

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 tit...

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Bibliographic Details
Published in:Analytical biochemistry 2012-04, Vol.423 (1), p.178-183
Main Authors: Dupureur, Cynthia M., Bashkin, James K., Aston, Karl, Koeller, Kevin J., Gaston, Kimberly R., He, Gaofei
Format: Article
Language:English
Subjects:
absorption
Base Sequence
Binding
binding sites
Biological Assay - methods
DNA
DNA - chemistry
DNA - metabolism
Fluorescence
Kinetics
ligands
Nylons - chemical synthesis
Nylons - chemistry
Nylons - metabolism
Polyamide
polyamides
Rhodamines - chemistry
Spectrometry, Fluorescence
therapeutics
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Summary: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 Kd values ranging from approximately 1nM to at least 140nM 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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2012.01.017