An Unnatural Amino Acid-based Chromatin Isolation Method (UChIMe) for the Study of Cancer-associated Protein-DNA Interactions

项目总结/摘要 蛋白质-DNA相互作用在所有已知生物的许多基本生物过程中起着关键作用。许多 人类疾病如癌症是由蛋白质-DNA相互作用的失调引起的。长期 其目标是精确绘制单细胞中全基因组和疾病相关蛋白质-DNA相互作用。 染色质免疫沉淀（ChIP）结合高通量测序（ChIP-Seq）已被广泛应用于 用于绘制蛋白质占据的DNA位点。与大多数免疫沉淀实验一样， 抗体的亲和力是ChIP分析中最关键的因素。据估计，只有不到20%的测试 抗体适用于ChIP分析。更重要的是，将随机性最小化仍然具有挑战性。 来自抗体非特异性结合的背景噪声。一种新的全基因组DNA解析方法 迫切需要具有改进的准确性和灵敏度的蛋白质结合位点。因此，这一目标 项目是开发一种新的基于非天然氨基酸（unAA）的染色质分离方法（UChIMe）， 以更高的准确性和灵敏度分析全基因组蛋白质-DNA相互作用。更具体地说，两个 目标将实现：1）开发UChIMe以探测Pol II结合位点，2）分析 使用UChIMe-seq的MEIS（髓样异位病毒整合位点）蛋白。含有炔的unAA 官能团将位点特异性地掺入活细胞中的靶蛋白中。这种非蛋白原性的 炔基将作为化学把手与含叠氮化物的生物素分子反应。目标 因此，蛋白质将用生物素标记，并准备好使用链霉亲和素树脂分离。相比 抗体依赖性ChIP方法，UChIMe将不需要使用抗体，导致大幅度减少 实验成本和时间。UChIMe还将使unAA能够在目标的任何位点并入 蛋白质，因此将比表位标记CHIP-seq方法更灵活。而且 unAA引发的化学反应的精密度将最大限度地减少由非特异性结合引起的背景， 该方法将利用抗体的高亲和力提高检测限， 生物素-链霉亲和素相互作用，其比表位与其抗原决定簇之间的相互作用大103-106倍。 特异性抗体总的来说，UChIMe将能够分析较小的样品（细胞数量）， 准确性和灵敏度，因此将扩大可以研究的样品类型。成功 该方法的发展将广泛应用于研究全基因组DNA结合位点， 靶蛋白。该项目符合内布拉斯加州综合生物分子通信中心的主题， 通过开发新的生物分子策略， 转录因子的DNA结合位点的全基因组作图。这一多学科项目将有助于 中心的协作环境和核心服务并为其提供便利。

PROJECT SUMMARY/ABSTRACT Protein-DNA interactions play key roles in many essential biological processes of all known organisms. Many human diseases, such as cancers, result from the misregulation of protein-DNA interactions. The long-term goal is to accurately map genome-wide and disease-associated protein-DNA interactions in single cells. Chromatin immunoprecipitation (ChIP) coupled to high-throughput sequencing (ChIP-Seq) has been widely used to map DNA sites of protein occupancy. Like most immunoprecipitation experiments, the specificity and affinity of antibodies is the most critical factor in ChIP analysis. It is estimated that fewer than 20% of tested antibodies are suitable for ChIP analyses. More importantly, it is still challenging to minimize random background noises from non-specific binding of antibodies. A novel method to elucidate genome-wide DNA binding sites of proteins with improved accuracy and sensitivity is critically needed. As such, the goal of this project is to develop a novel Unnatural Amino Acid (unAA)-based Chromatin Isolation Method (UChIMe) to analyze genome-wide protein-DNA interactions with improved accuracy and sensitivity. More specifically, two aims will be achieved: 1) develop UChIMe to probe Pol II binding sites and 2) analyze global binding sites of MEIS (myeloid ectopic viral integration site) proteins using UChIMe-seq. An unAA containing an alkyne functional group will be site-specifically incorporated into the target protein in live cells. This nonproteinogenic alkyne group will serve as a chemical handle to react with an azide-containing biotin molecule. The target protein will therefore be labeled with biotin and be ready for isolation using streptavidin resin. In comparison to the antibody-dependent ChIP method, UChIMe will not require the use of antibody, leading to major reductions in experimental cost and time. UChIMe will also enable the incorporation of unAA at any site of the target protein and will therefore be more flexible than the epitope tagging CHIP-seq method. Furthermore, the precision of an unAA-initiated chemical reaction will minimize background caused by non-specific binding of the antibody, and the proposed method will improve the detection limit by taking advantage of the high-affinity biotin-streptavidin interaction, which is 103-106 times greater than interactions between epitopes and their specific antibodies. Overall, UChIMe will enable the analysis of smaller samples (cell numbers) with improved accuracy and sensitivity and therefore will broaden the types of samples that can be studied. Successful development of the method will have broad applications to the studies of genome-wide DNA binding sites of target proteins. This project fits the Nebraska Center for Integrated Biomolecular Communication’s thematic focus on unraveling signal pathway shifts in complex diseases by developing novel biomolecular strategies for genome-wide mapping of DNA binding sites of transcription factors. This multidisciplinary project will contribute to and be facilitated by the Center’s collaborative environment and core services.