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

用于研究癌症相关蛋白-DNA 相互作用的基于非天然氨基酸的染色质分离方法 (UChIMe)

• 批准号: 10271843
• 负责人: Wei Niu
• 金额: $ 11.88万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10271843
• 关键词: Address; Affinity; Alkynes; Amber; Antibodies; Antibody Affinity; Azides; Basic Science; Binding; Binding Sites; Bioinformatics; Biological Process; Biological Sciences; Biomedical Research; Biotin; Cell Count; Cells; ChIP-seq; Chemicals; Chromatin; Codon Nucleotides; Communication; Complex; Coupled; DNA; DNA Binding; DNA Polymerase II; DNA mapping; DNA-Binding Proteins; DNA-Directed RNA Polymerase; DNA-Protein Interaction; Data Science; Data Set; Detection; Development; Disease; Engineering; Epitopes; Event; Gene Expression Regulation; Genetic Fingerprintings; Genome; Genomic DNA; Goals; High-Throughput Nucleotide Sequencing; Immunoprecipitation; Individual; Investigation; Label; Libraries; MCF7 cell; Malignant Neoplasms; Maps; Methodology; Methods; Myelogenous; Nature; Nebraska; Noise; Organism; Outcome; Performance; Plant Resins; Play; Positioning Attribute; Production; Protein Engineering; Proteins; Protocols documentation; Quadruplet Multiple Birth; Reagent; Research; Role; Sample Size; Sampling; Secure; Services; Signal Pathway; Signal Transduction; Site; Source; Specificity; Streptavidin; Terminator Codon; Time; Virus Integration; antibody test; base; chemical reaction; chromatin immunoprecipitation; collaborative environment; cost; crosslink; detection limit; experimental study; flexibility; functional group; genetic regulatory protein; genome-wide; genome-wide analysis; human disease; improved; integration site; interest; metabolic engineering; method development; microbial; multidisciplinary; novel; protein complex; success; synthetic biology; transcription factor; tumor progression; unnatural amino acids

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.

项目摘要/摘要 蛋白质-DNA相互作用在所有已知生物体的许多基本生物学过程中发挥着关键作用。许多 人类的疾病，如癌症，是由蛋白质-DNA相互作用的错误调节造成的。长期的 目标是准确地绘制单细胞中全基因组和与疾病相关的蛋白质-DNA相互作用图。 染色质免疫沉淀(ChIP)结合高通量测序技术(ChIP-Seq)已得到广泛应用 用于绘制蛋白质占位的DNA位置图。像大多数免疫沉淀实验一样，特异性和 抗体的亲和力是芯片分析中最关键的因素。据估计，只有不到20%的受试者 抗体适用于芯片分析。更重要的是，将随机性降至最低仍具有挑战性 背景噪音来自抗体的非特异性结合。一种阐明全基因组DNA的新方法 迫切需要提高精确度和灵敏度的蛋白质结合部位。因此，这一目标是 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)来 分析全基因组的蛋白质-DNA相互作用，提高了准确性和灵敏度。更具体地说，两个 目的：1)开发UChIMe来探测POL II结合位点；2)分析Pol II的全球结合位点 利用UChIMe-seq.一种含有炔烃的UNAA 功能基团将被特异性地整合到活细胞中的靶蛋白中。这种不产生蛋白质的物质 炔基将作为化学手柄与含叠氮的生物素分子反应。目标是 因此，蛋白质将被生物素标记，并准备好使用链霉亲和素树脂进行分离。与之相比 抗体依赖的芯片方法，UChIMe将不需要使用抗体，导致大幅减少 在实验成本和时间上。UChIMe还将使UNAA能够在目标的任何地点合并 因此将比表位标记芯片-SEQ方法更灵活。此外， UNAA发起的化学反应的精确度将最大限度地减少由非特异性结合引起的背景 该方法充分利用了抗体的高亲和力，提高了检测限。 生物素-链霉亲和素相互作用，比表位和它们的表位之间的相互作用大103-106倍 特定的抗体。总体而言，UChIMe将支持对较小样本(细胞数量)的分析，并改进了 精确度和灵敏度，因此将扩大可研究的样本类型。成功 该方法的发展将在全基因组DNA结合位点的研究中有广泛的应用 靶蛋白。该项目适合内布拉斯加州集成生物分子通信中心的主题 通过开发新的生物分子策略来解开复杂疾病中的信号通路转移 转录因子DNA结合位点的全基因组图谱。这一多学科项目将有助于 为中心的协作环境和核心服务提供便利。