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.

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对于处于某种状态的给定细胞，哪些蛋白质与基因组 DNA 结合以及它们在基因组中的位置结合序列是理解细胞功能和疾病机制的基本问题。到为了解决这些问题，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。然而，大多数当前技术依赖于蛋白质的捕获通过甲醛交联形成 DNA 复合物。越来越多的证据表明甲醛固定可能是破坏当前方法有效性的一个主要问题。这很大程度上是由于甲醛对蛋白质造成高反应性和非特异性损伤，且无法交联 DNA 蛋白质。甲醛捕获的 DNA 不是与蛋白质共价连接，而是被固定在蛋白质中复合物，这可能导致捕获大量非特异性 DNA 片段，从而掩盖真实信号 ()。最近报道了蛋白质与 DNA 和 RNA 的直接紫外交联，但这些方法受到低交联效率和短波长 UVC（~250 nm）使用的限制破坏蛋白质和核酸。对分子工具和技术的研究需求亟待满足可以高效、选择性地捕获细胞中蛋白质-DNA 复合物的技术（即，仅靶向 DNA 结合蛋白）和稳定性（以实现蛋白质-DNA 复合物的稳健分离）后续分析）。拟议的研究旨在开发一类双功能光交联探针（BFPX）是细胞和核可渗透的，在没有紫外线的情况下对细胞分子呈惰性，结合并富集 DNA 或 RNA，并在长波长 UVA（~360nm）照射下被激活与 DNA 或 RNA 形成共价加合物，同时交联附近与 DNA 或 RNA 结合的蛋白质 RNA通过高效的光化学反应。这些分子探针将作为强大的工具在广泛的蛋白质原位研究中稳健捕获蛋白质-DNA 和蛋白质-RNA 复合物核酸相互作用。