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Chemical profiling of DNA G-quadruplex-interacting proteins in live cells
Nature Chemistry ( IF 21.8 ) Pub Date : 2021-06-28 , DOI: 10.1038/s41557-021-00736-9
Xiaoyun Zhang 1 , Jochen Spiegel 2 , Sergio Martínez Cuesta 1, 2, 3 , Santosh Adhikari 1 , Shankar Balasubramanian 1, 2, 4
Affiliation  

DNA–protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.



中文翻译:

活细胞中 DNA G-四链体相互作用蛋白的化学分析

DNA-蛋白质相互作用调节关键的生物过程。识别与特定功能基因组位点结合的蛋白质对于了解分子水平的潜在调控机制至关重要。在这里,我们描述了一种共同结合介导的蛋白质分析 (CMPP) 策略来研究天然染色质中 DNA G-四链体 (G4s) 的相互作用组。CMPP 涉及可渗透细胞的功能化 G4 配体探针,这些探针结合内源性 G4 并随后原位交联到共结合 G4 相互作用蛋白。我们首先通过在体外对 G4 结合蛋白进行邻近标记来展示 CMPP 的稳健性。在活细胞中采用这种方法,我们随后从各种功能类别中鉴定出数百种推定的 G4 相互作用蛋白。下一个,我们在体外证实了几种新发现的 G4 相互作用物的高 G4 结合亲和力和选择性,并且我们使用独立方法验证了细胞染色质中功能重要的候选物的直接 G4 相互作用。我们的研究提供了一种化学策略来绘制活细胞中特定核酸特征的蛋白质相互作用。

更新日期:2021-06-28
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