CEITEC - Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic.
Sci Rep. 2016 Dec 23;6:39653. doi: 10.1038/srep39653.
Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions.
许多基本的生物过程都依赖于蛋白质和核酸之间复杂的相互作用网络，对这些相互作用进行定量描述对于理解控制 DNA 复制、转录或翻译的细胞机制非常重要。在这里，我们提出了一种快速定量评估蛋白质/核酸（NA）相互作用的通用方法。该方法基于蛋白质诱导的荧光增强（PIFE）现象，即蛋白质结合增加 Cy3 样染料的荧光。PIFE 主要用于单分子研究以检测蛋白质与 DNA 或 RNA 的结合。在这里，我们通过使用微孔板荧光读取器（mwPIFE）将 PIFE 应用于稳态定量测定蛋白质/NA 相互作用。我们展示了 mwPIFE 在检查 BamHI 限制酶和 Ku 和 XPF/ERCC1 等 DNA 修复复合物等各种方面的蛋白质/DNA 相互作用的一般适用性。这些包括确定序列和结构结合特异性、离解常数、检测弱相互作用以及蛋白质沿 DNA 易位的能力。mwPIFE 是一种简单且高通量的方法，不需要蛋白质标记，可应用于涉及蛋白质/NA 相互作用的广泛应用。
