Probing the DNA-Binding Affinity and Specificity of Designed Zinc Finger Proteins
Probing the DNA-Binding Affinity and Specificity of Designed Zinc Finger Proteins
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DOI：
10.1016/j.bpj.2009.11.021
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发表时间：
2010-03-03
影响因子：
3.4
通讯作者：
Berg, Jeremy M.
中科院分区：
文献类型：
--
作者：
Jantz, Derek;Berg, Jeremy M.
基于设计的Cys（2）His（2）锌指结构域的工程化转录因子和核酸内切酶已被证明是用于基因定向调控和修饰的有效工具。将该技术引入研究和临床环境中需要开发快速准确的方法来评估设计的锌指结构域的结合亲和力和结合特异性。使用荧光各向异性为基础的DNA结合试验，我们研究了DNA结合特性的两个工程化的锌指蛋白，不同的一个氨基酸。我们证明了对特定DNA位点具有最高亲和力的蛋白质不一定是以最高特异性结合该位点的蛋白质。此外，通过比较这两种蛋白质在不同盐浓度下的结合特性，我们发现离子强度对亲和力和特异性都有显著和可变的贡献。这些结果对锌指设计具有重要意义，因为它们突出了在设计用于特定应用的DNA结合结构域时考虑亲和力、特异性和环境要求的重要性。
Engineered transcription factors and endonucleases based on designed Cys(2)His(2) zinc finger domains have proven to be effective tools for the directed regulation and modification of genes. The introduction of this technology into both research and clinical settings necessitates the development of rapid and accurate means of evaluating both the binding affinity and binding specificity of designed zinc finger domains. Using a fluorescence anisotropy-based DNA-binding assay, we examined the DNA-binding properties of two engineered zinc finger proteins that differ by a single amino acid. We demonstrate that the protein with the highest affinity for a particular DNA site need not be the protein that binds that site with the highest degree of specificity. Moreover, by comparing the binding characteristics of the two proteins at varying salt concentrations, we show that the ionic strength makes significant and variable contributions to both affinity and specificity. These results have significant implications for zinc finger design as they highlight the importance of considering affinity, specificity, and environmental requirements in designing a DNA-binding domain for a particular application.