Non-canonical DNA structures, such as the G-quadruplex (G4) and i-motif (iM), are formed at guanine- and cytosine-rich sequences, respectively, in living cells and involved in regulating various biological processes during the cell cycle. Therefore, the formation and resolution of these non-canonical structures must be dynamically regulated by physiological conditions or factors that can bind G4 and iM structures. Although many G4 binding proteins responsible for tuning the G4 structure have been discovered, the structural regulation of iM by iM-binding proteins remains enigmatic. In this study, we developed a protein-labeling DNA probe bearing an alkyne moiety through a reactive linker, for proximity-labeling of nucleic acid-binding proteins, and searched for new iM-binding proteins. Alkyne-modified proteins in the nuclear extract of HeLa cells were labeled with biotin via a click reaction and then captured with streptavidin-coated magnetic beads. This fingerprint-targeting enrichment, followed by proteome analyses, identified new candidate proteins that potentially bind to the iM structure, in addition to the reported iM-binding proteins. Among the newly identified candidates, we characterized a nucleolar protein, nucleolin, that binds to the iM structure and relaxes it, while nucleolin stabilizes the G4 structure.

中文翻译：

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i 基序结合蛋白的分析揭示了核仁素在高阶 DNA 结构调节中的功能作用。


非经典 DNA 结构，如 G 四链体 （G4） 和 i 基序 （iM），在活细胞中分别以富含鸟嘌呤和胞嘧啶的序列形成，并参与调节细胞周期中的各种生物过程。因此，这些非经典结构的形成和分辨率必须由可以结合 G4 和 iM 结构的生理条件或因素动态调节。尽管已经发现了许多负责调节 G4 结构的 G4 结合蛋白，但 iM 结合蛋白对 iM 的结构调节仍然是个谜。在这项研究中，我们开发了一种通过反应性接头携带炔烃部分的蛋白质标记 DNA 探针，用于核酸结合蛋白的邻近标记，并寻找新的 iM 结合蛋白。通过点击反应用生物素标记 HeLa 细胞核提取物中的炔烃修饰蛋白，然后用链霉亲和素包被的磁珠捕获。除了报道的 iM 结合蛋白外，这种指纹靶向富集，然后进行蛋白质组分析，确定了可能与 iM 结构结合的新候选蛋白。在新确定的候选者中，我们表征了一种核仁蛋白 nucleolin，它与 iM 结构结合并使其松弛，而核仁素稳定 G4 结构。