Department of Biochemistry and Physiology, School of Pharmacy and Food sciences, and IN2UB, University of Barcelona, 08028 Barcelona, Spain.
Instituto de Química y Física 'Rocasolano', CSIC, Serrano 119, 28006 Madrid, Spain.
Int J Mol Sci. 2020 Jul 16;21(14):5028. doi: 10.3390/ijms21145028.
Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5'UTR of the gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS.
胸苷酸合成酶（TYMS）酶是一种抗癌靶点，因为它在 DNA 生物合成中发挥作用。TYMS 抑制剂（例如 5-氟尿嘧啶）可能会通过 TYMS 的自动调节机制导致其过度表达而产生耐药性。由于 G-四链体（G4）可以调节基因表达，我们在 基因中搜索可能被多聚嘌呤反向 Hoogsteen 发夹（PPRH）靶向的假定 G4 形成序列（G4FS）。使用四链体形成 G-丰富序列映射器检测 基因中的 G4 结构，并通过使用合成寡核苷酸的圆二色性和 NMR 等光谱方法进行确认。通过 EMSA 和硫代黄素 T 染色研究 G4FS 与 TYMS 蛋白或 G4FS 与靶向该序列的 PPRH（HpTYMS-G4-T）之间的相互作用。我们在 DNA 和 RNA 中都鉴定出 基因 5'UTR 中的一个 G4FS，它能够与 TYMS 蛋白相互作用。PPRH 结合其对应的靶 dsDNA，促进 G4 形成。在癌细胞中，HpTYMG-G4-T 降低了 TYMS mRNA 和蛋白水平，导致细胞死亡，并与 5-氟尿嘧啶联合使用时表现出协同作用。这些结果表明 基因中存在 G4 基序，可能参与 TYMS 表达的自动调节，以及针对 G4FS 的 PPRH 的治疗潜力。
