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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