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.

项目概要/摘要 蛋白质-DNA 相互作用在所有已知生物体的许多重要生物过程中发挥着关键作用。 人类疾病，例如癌症，是由蛋白质-DNA 相互作用的长期失调引起的。 目标是准确绘制单细胞中全基因组和疾病相关的蛋白质-DNA 相互作用图谱。 染色质免疫沉淀 (ChIP) 与高通量测序 (ChIP-Seq) 联用已得到广泛应用 与大多数免疫沉淀实验一样，用于绘制蛋白质占据的 DNA 位点。 抗体的亲和力是 ChIP 分析中最关键的因素，估计只有不到 20% 的测试对象。 抗体适合 ChIP 分析，更重要的是，最大限度地减少随机性仍然具有挑战性。 抗体非特异性结合的背景噪音。一种阐明全基因组 DNA 的新方法。 因此，迫切需要具有更高准确性和灵敏度的蛋白质结合位点。 该项目旨在开发一种新型的基于非天然氨基酸 (unAA) 的染色质分离方法 (UChIMe) 更具体地说，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。 将实现的目标：1) 开发 UChIMe 来探测 Pol II 结合位点，2) 分析 Pol II 结合位点的全局结合位点 使用 UChIMe-seq 的 MEIS（骨髓异位病毒整合位点）蛋白 这种非蛋白原性的功能基团将被位点特异性地整合到活细胞中的目标蛋白中。 炔基将作为与含叠氮化物的生物素分子反应的化学手柄。 因此，与相比，蛋白质将用生物素标记并准备好使用链霉亲和素树脂进行分离。 抗体依赖性 ChIP 方法，UChIMe 不需要使用抗体，从而大幅减少 UChIMe 还可以在目标的任何位点掺入 UNAA。 因此比表位标记 CHIP-seq 方法更灵活。 unAA 引发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 抗体的高亲和力，所提出的方法将提高检测限 生物素-链霉亲和素相互作用，比表位与其之间的相互作用大 103-106 倍 总体而言，UChIMe 将能够以改进的方式分析更小的样品（细胞数量）。 准确性和灵敏度，因此将扩大可成功研究的样品类型。 该方法的开发将在全基因组DNA结合位点的研究中具有广泛的应用 该项目符合内布拉斯加州综合生物分子通讯中心的主题。 通过开发新的生物分子策略，专注于揭示复杂疾病中的信号通路转变 这个多学科项目将有助于对转录因子的 DNA 结合位点进行全基因组图谱分析。 中心的协作环境和核心服务并受其促进。