Ban Yuki, Ando Yuka, Terai Yuma, Matsumura Risa, Nakane Keita, Iwai Shigenori, Sato Shinichi, Yamamoto Junpei
Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, 6-3 Aramaki aza-Aoba, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
Nucleic Acids Res. 2024 Dec 11;52(22):13530-13543. doi: 10.1093/nar/gkae1001.
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.
非经典DNA结构，如G-四链体（G4）和i-基序（iM），分别在活细胞中富含鸟嘌呤和胞嘧啶的序列处形成，并参与细胞周期中各种生物过程的调控。因此，这些非经典结构的形成和解析必须受到生理条件或能够结合G4和iM结构的因子的动态调控。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的结构调控仍然是个谜。在本研究中，我们通过反应性连接子开发了一种带有炔基部分的蛋白质标记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。HeLa细胞核提取物中的炔基修饰蛋白通过点击反应与生物素标记，然后用链霉亲和素包被的磁珠捕获。这种指纹靶向富集，随后进行蛋白质组分析，除了已报道的iM结合蛋白外，还鉴定出了可能与iM结构结合的新候选蛋白。在新鉴定的候选蛋白中，我们鉴定了一种核仁蛋白——核仁素，它能与iM结构结合并使其松弛，而核仁素则能稳定G4结构。
