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

用于全基因组蛋白质-核酸相互作用研究的双功能光交联剂 对于处于特定状态的特定细胞，哪些蛋白质与基因组dna结合，以及它们在基因组中的什么位置结合？ 序列是理解细胞功能和疾病机制的基本问题。至 为了解决这些问题，已经开发了各种技术来捕获蛋白质-DNA复合体 用于分析和鉴定。然而，目前的大多数技术都依赖于捕获蛋白质- 以甲醛为交联剂的DNA络合物。越来越多的证据表明，甲醛固定 这可能是破坏当前办法有效性的一个主要问题。这在很大程度上是由于 甲醛对蛋白质的高度反应性和非特异性损伤及其不能将DNA与 蛋白质。甲醛捕获的DNA不与蛋白质共价连接，而是固定在固定的蛋白质中 复合体，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的直接紫外光交联，但这些 由于交联率低和短波紫外线(~250 nm)的使用，这些方法受到限制。 破坏蛋白质和核酸的物质。对分子工具和研究的迫切需求尚未得到满足 可以高效、选择性地捕获细胞中的蛋白质-DNA复合体的技术(即仅 以DNA结合蛋白为靶点)和稳定性(以实现蛋白质-DNA复合体的可靠分离 随后的分析)。拟议中的研究试图开发一类双功能光交联剂 探针(BFPX)是细胞和细胞核的渗透性探针，在没有紫外线的情况下对细胞分子是惰性的，结合和 富含DNA或RNA，在长波紫外线(~360 nm)照射下，激活 与DNA或RNA形成共价加合物，同时使与DNA或RNA结合的邻近蛋白质发生交联 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，对蛋白质-DNA和蛋白质-RNA复合体的可靠捕获 核酸相互作用。

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.