Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, 5290002, Israel.
Blavatnik School of Computer Science, Tel-Aviv University, Tel-Aviv, 69978, Israel.
Sci Rep. 2016 Sep 15;6:33351. doi: 10.1038/srep33351.
Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.
转录因子 (TFs) 通过与 DNA 的序列特异性相互作用，响应环境变化来改变基因表达。这些相互作用最好被描绘为 TF 结合亲和力的景观。目前研究序列特异性结合偏好的方法存在动态范围有限、序列偏好、缺乏特异性和通量有限等问题。我们开发了一种基于微流控的 SELEX 亲和力图谱 (SELMAP) 方法，用于 TF 结合，该方法允许同时平行测量 16 种蛋白质。我们使用它来测量 Pho4、AtERF2 和 Btd 全长蛋白对数百万种不同 DNA 结合位点的相对亲和力，并在平衡条件下检测到高亲和和低亲和相互作用，生成了所有可能的 DNA 6- mers 的相对 TF 亲和力的综合图谱，甚至在增加测序深度的情况下，还可以生成 DNA10-mers 的图谱。TF 和 DNA 寡聚物的少量用量足以获得高质量的结果，显著降低了实验成本。SELMAP 允许对数百种 TF 进行深入筛选，并为更好地理解控制基因表达的调控过程提供了一种手段。
