Bi-functional photo-crosslinking (BFPX) for genome-wide study of protein-nucleic acid interactions

双功能光交联 (BFPX) 用于蛋白质-核酸相互作用的全基因组研究

基本信息

批准号：
10593666
负责人：
LIN CHEN
金额：
$ 24.75万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-16 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10593666
关键词：
Address Amines Antibodies Belief Binding Binding Proteins Binding Sites Biological Process Cell Count Cell Line Cell Nucleus Cell physiology Cells ChIP-seq Chemistry Complex Complex Analysis Crosslinker DNA DNA Binding DNA Binding Domain DNA-Binding Proteins DNA-Protein Interaction DNA-protein crosslink Data Data Set Diazomethane Disease Effectiveness Engineering Epitopes Formaldehyde GATA1 gene Gene Expression Genetic Transcription Genome Genomic DNA Genomics Hela Cells In Situ In Vitro K-562 Lighting Link Maps Masks Methods Modification Molecular Molecular Biology Molecular Probes Monitor Morphologic artifacts Names Noise Nuclear Nucleic Acids Permeability Photoaffinity Labels Post-Translational Protein Processing Proteins Protocols documentation RNA Reaction Reporting Repression Reproducibility Research Signal Transduction Site Source Specificity Surface Techniques Technology Testing Time Work adduct carbene cell fixation chromatin immunoprecipitation crosslink data quality design disuccinimidyl glutarate dosage established cell line functional group genetic regulatory protein genome-wide genome-wide analysis improved in vivo intercalation muscle enhancer factor-2A nitrene novel protein complex protein crosslink recruit sample fixation tool transcription factor

项目摘要

Bi-functional photo-crosslinking (BFPX) for genome-wide study of protein-nucleic acid interactions For a given cell at a certain state, what proteins bind to the genomic DNA and where they bind in the genomic sequence are fundamental questions to understanding cellular functions and disease mechanisms. To address these questions, a variety of techniques have been developed to capture protein-DNA complexes for analyses and identifications. However, most of the current technologies depend on the capture of protein- DNA complexes by formaldehyde crosslinking. Increasing evidence suggests that formaldehyde fixation could be a major problem undermining the effectiveness of the current approaches. This is largely due to the highly reactive and non-specific damages to proteins by formaldehyde and its inability to crosslink DNA to proteins. DNA captured by formaldehyde is not covalently linked to protein but trapped in fixed protein complexes, which can lead to the capture of a large amount of non-specific DNA fragments which will mask the real signals (). Direct UV crosslinking of protein to DNA and RNA have recently been reported, but these approaches are limited by the low crosslinking efficiency and the use of short wavelength UVC (~250 nm) that damages proteins and nucleic acids. There is a critical unmet research need for molecular tools and technologies that can capture protein-DNA complexes in cells with high efficiency, selectivity (i.e., only targeting DNA-bound proteins) and stability (to enable robust isolation of protein-DNA complexes for subsequent analyses). The proposed research seek to develop a class of bi-functional photo-crosslinking probes (BFPX) that are cell and nuclear permeable, inert to cellular molecules in the absence of UV, bind and enrich on DNA or RNA, and under illumination with long wavelength UVA (~360nm), become activated to form covalent adduct to DNA or RNA and at the same time to crosslink nearby proteins bound to DNA or RNA through highly efficient photochemical reactions. These molecular probes will serve as powerful tools for robust capture of protein-DNA and protein-RNA complexes in a wide range of in situ studies of protein- nucleic acid interactions.

用于全基因组核酸相互作用基因组研究的双功能光链接（BFPX）对于特定状态下的给定细胞，什么蛋白与基因组DNA结合及其在基因组中结合的位置序列是理解细胞功能和疾病机制的基本问题。到解决这些问题，已经开发了多种技术来捕获蛋白质-DNA复合物用于分析和识别。但是，当前大多数技术取决于蛋白质的捕获 DNA复合物通过甲醛交联。越来越多的证据表明甲醛固定可能是破坏当前方法的有效性的主要问题。这主要是由于甲醛对蛋白质的高反应性和非特异性损害及其无法交联的DNA到蛋白质。甲醛捕获的DNA与蛋白质无关，而是被困在固定蛋白中复合物，可以导致捕获大量的非特异性DNA片段，这些片段将掩盖真实信号（）。最近已经报道了蛋白质与DNA和RNA的直接紫外线交联，但这些方法受到低交联效率和短波长UVC（〜250 nm）的限制这会损害蛋白质和核酸。对分子工具和可以以高效率，选择性捕获细胞中蛋白质-DNA复合物的技术（即靶向DNA结合蛋白）和稳定性（以稳健地分离蛋白DNA复合物的蛋白质-DNA复合物随后的分析）。拟议的研究旨在开发一类双功能的照片连接在没有紫外线，结合和在DNA或RNA上富集，并用长波长UVA（〜360nm）照明，被激活为形成与DNA或RNA的共价加合物，同时与与DNA或通过高效的光化学反应RNA。这些分子探针将作为强大的工具在广泛的原位研究中，蛋白质-DNA和蛋白-RNA复合物的稳健捕获 - 核酸相互作用。