A simple, high-resolution method for establishing DNA binding affinity and sequence selectivity
A simple, high-resolution method for establishing DNA binding affinity and sequence selectivity
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DOI：
10.1021/ja010041a
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发表时间：
2001-06-27
影响因子：
15
通讯作者：
Hedrick, MP
中科院分区：
文献类型：
--
作者：
Boger, DL;Fink, BE;Hedrick, MP
一个简单的，非破坏性的，高通量的方法建立DNA结合亲和力和序列选择性的发展的全部细节。该方法基于溴化乙锭或噻唑橙子从感兴趣的DNA中置换而导致的荧光损失，或者在选定的情况下，基于DNA结合剂本身内在荧光的变化，并且适用于评估相对或绝对DNA结合亲和力。收集包含以96孔格式展示的所有五个碱基对（512个发夹）或四个碱基对（136个发夹）序列的发夹脱氧寡核苷酸文库，产生化合物与所有可能序列结合的等级顺序，从而使用该荧光嵌入剂置换（FID）测定法对其序列选择性进行高分辨率定义。因此，该技术补充了足迹法或亲和切割法用于建立DNA结合选择性的使用，并以更高的分辨率提供信息。通过这种等级顺序结合生成的合并条形图提供了一种比较或描述DNA结合亲和力和选择性的定性方法。96孔格式测定（512个发夹）可以以最小的成本进行（目前约为100%）。100美元用于发夹脱氧寡核苷酸/用溴化乙锭或更少用噻唑橙子的测定），使用荧光板读数器快速读出（15分钟），并且适于自动化（Tecan Genesis Workstation 100机器人系统）。描述了其在产生包括偏端霉素A、netropsin、DAPI、Hoechst 33258和Berenil的几种药剂的DNA结合选择性的概况中的用途。建立从定量滴定结合常数的技术进行了比较，并建议使用Scatchard或曲线拟合分析的滴定结合曲线作为一种可靠的手段来定量的结合亲和力。
Full details of the development of a simple, nondestructive, and high-throughput method for establishing DNA binding affinity and sequence selectivity are described. The method is based on the loss of fluorescence derived from the displacement of ethidium bromide or thiazole orange from the DNA of interest or, in selected instances, the change in intrinsic fluorescence of a DNA binding agent itself and is applicable for assessing relative or absolute DNA binding affinities. Enlisting a library of hairpin deoxyoligonucleotides containing all five base pair (512 hairpins) or four base pair (136 hairpins) sequences displayed in a 96-well format, a compound's rank order binding to all possible sequences is generated, resulting in a high-resolution definition of its sequence selectivity using this fluorescent intercalator displacement (FID) assay. As such, the technique complements the use of footprinting or affinity cleavage for the establishment of DNA binding selectivity and provides the information at a higher resolution. The merged bar graphs generated by this rank order binding provide a qualitative way to compare, or profile, DNA binding affinity and selectivity. The 96-well format assay (512 hairpins) can be conducted at a minimal cost (presently ca. $100 for hairpin deoxyoligonucleotides/assay with ethiduim bromide or less with thiazole orange), with a rapid readout using a fluorescent plate reader (15 min), and is adaptable to automation (Tecan Genesis Workstation 100 robotic system). Its use in generating a profile of DNA binding selectivity for several agents including distamycin A, netropsin, DAPI, Hoechst 33258, and berenil is described. Techniques for establishing binding constants from quantitative titrations are compared, and recommendations are made for use of a Scatchard or curve fitting analysis of the titration binding curves as a reliable means to quantitate the binding affinity.