National R&D Center for Se-rich Agricultural Products Processing, Hubei Engineering Research Center for Deep Processing of Green Se-rich Agricultural Products, School of Modern Industry for Selenium Science and Engineering, Wuhan Polytechnic University, Wuhan 430023, China.
Department of Zoology, University of Narowal, Narowal, Punjab 51600, Pakistan.
Int J Biol Macromol. 2023 Jun 15;240:124442. doi: 10.1016/j.ijbiomac.2023.124442. Epub 2023 Apr 14.
G-quadruplexes are widely distributed in cells and are usually essential in mediating biological processes. The intracellular environment is often in a state of molecular crowding, and the current research considerably focuses on the effect of molecular crowding on the conformation of telomeric G-quadruplexes. However, G-quadruplex-forming oligonucleotides are primarily located in the promoter region of the proto-oncogene and on mRNA inside the cell and are reported to fold into parallel structures. Thus, studying the interaction mechanism between ligands and parallel structured G-quadruplexes under crowding conditions is crucial for the design of drugs targeting G-quadruplexes. In our study, molecular crowding was simulated through polyethylene glycol with an average molecular weight of 200 (PEG200) to investigate the parallel structure of the canonical G-quadruplexes c-KIT1, c-MYC, and 32KRAS and their interactions with ligands. Circular dichroism (CD) spectral scanning, fluorescence resonance energy transfer (FRET), and native polyacrylamide gel electrophoresis (PAGE) analysis revealed that molecular crowding failed to induce oligonucleotides to form parallel G-quadruplex structures in the explored model sequences while induced telomeric G-rich sequences to form antiparallel G-quadruplexes in solution without K. Molecular crowding did not induce changes in their parallel structures but promoted the formation of G-quadruplex aggregates. Moreover, to some extent, molecular crowding also induced a looser structure of the monomer G-quadruplexes. Further studies showed that molecular crowding did not alter the binding stoichiometry of the ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate (RHPS4) to c-KIT1, while it inhibited its interaction with parallel structured G-quadruplexes. This work provides new insights into developing anticancer drugs targeting parallel structured G-quadruplexes.
四链体广泛分布于细胞中，通常在介导生物过程中发挥重要作用。细胞内环境通常处于分子拥挤状态，目前的研究主要集中在分子拥挤对端粒 G-四链体构象的影响上。然而，形成 G-四链体的寡核苷酸主要位于原癌基因的启动子区域和细胞内的 mRNA 中，据报道它们折叠成平行结构。因此，研究拥挤条件下配体与平行结构 G-四链体的相互作用机制对于设计靶向 G-四链体的药物至关重要。在我们的研究中，通过平均分子量为 200 的聚乙二醇（PEG200）模拟分子拥挤，以研究典型 G-四链体 c-KIT1、c-MYC 和 32KRAS 的平行结构及其与配体的相互作用。圆二色性（CD）光谱扫描、荧光共振能量转移（FRET）和天然聚丙烯酰胺凝胶电泳（PAGE）分析表明，分子拥挤未能诱导寡核苷酸在研究的模型序列中形成平行 G-四链体结构，而在没有 K 的情况下，诱导富含端粒的 G-序列在溶液中形成反平行 G-四链体。分子拥挤不会改变其平行结构，但会促进 G-四链体聚集体的形成。此外，在某种程度上，分子拥挤也诱导单体 G-四链体结构变得更加松散。进一步的研究表明，分子拥挤不会改变配体 3,11-二氟-6,8,13-三甲基-8H-喹啉[4,3,2-kl]吖啶鎓甲磺酸盐（RHPS4）与 c-KIT1 的结合化学计量，但抑制其与平行结构 G-四链体的相互作用。这项工作为开发针对平行结构 G-四链体的抗癌药物提供了新的见解。
