Fluorescence assay of polyamide-DNA interactions
- 作者：Cynthia M. Dupureur ; cdup@umsl.edu ; James K. Bashkin ; Karl Aston ; Kevin J. Koeller ; Kimberly R. Gaston ; Gaofei He
- 关键词：Polyamide ; DNA ; Fluorescence ; Binding
- 刊名：Analytical Biochemistry
- 出版年：2012
- 期刊代码：93_00032697
- 类别：imm
- 出版时间：1 April, 2012
- 卷：423
- 期：1
- 页码：178-183
- 文件大小：409 K
摘要
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 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.