Fluorescence assay of polyamide-DNA interactions
Fluorescence assay of polyamide-DNA interactions
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DOI：
10.1016/j.ab.2012.01.017
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发表时间：
2012-04-01
影响因子：
2.9
通讯作者：
He, Gaofei
中科院分区：
文献类型：
--
作者：
Dupureur, Cynthia M.;Bashkin, James K.;He, Gaofei
聚酰胺（PA）是结合小沟并能够序列选择性识别的DNA的偏端霉素型配体。这种能力为它们作为治疗剂的开发提供了可行的途径。本文介绍了一种简便的PA-DNA结合荧光分析法。将PA滴定到发夹DNA的样品中，该发夹DNA的特征在于塔姆拉染料连接到PA结合位点附近的内部dU。在6种PA的研究中，PA结合导致荧光强度的稳定可再现降低，可用于生成结合等温线。该测定法同样适用于短（6-至8-环）和长（14-环）PA，并且使用简单的单色器或过滤器配置容易获得约1 nM至至少140 nM的K-d值。竞争试验提供了一种评估可能的染料干扰的方法，其可以忽略不计。该测定也可用于确定PA消光系数和测量结合动力学，因此，它是一种可访问的和通用的工具，用于研究PA特性和PA-DNA相互作用。(C)2012 Elsevier Inc. All rights reserved.
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.