Department of Biological Engineering, Indian Institute of Technology, Gandhinagar 382355, India.
Department of Chemistry, Indian Institute of Technology, Gandhinagar, Gandhinagar 382355, India.
Molecules. 2021 Mar 5;26(5):1400. doi: 10.3390/molecules26051400.
G-quadruplexes (G4) are the most actively studied non-canonical secondary structures formed by contiguous repeats of guanines in DNA or RNA strands. Small molecule mediated targeting of G-quadruplexes has emerged as an attractive tool for visualization and stabilization of these structures inside the cell. Limited number of DNA and RNA G4-selective assays have been reported for primary ligand screening. A combination of fluorescence spectroscopy, AFM, CD, PAGE, and confocal microscopy have been used to assess a dimeric carbocyanine dye B6,5 for screening G4-binding ligands in vitro and in cellulo. The dye B6,5 interacts with physiologically relevant DNA and RNA G4 structures, resulting in fluorescence enhancement of the molecule as an in vitro readout for G4 selectivity. Interaction of the dye with G4 is accompanied by quadruplex stabilization that extends its use in primary screening of G4 specific ligands. The molecule is cell permeable and enables visualization of quadruplex dominated cellular regions of nucleoli using confocal microscopy. The dye is displaced by quarfloxin in live cells. The dye B6,5 shows remarkable duplex to quadruplex selectivity in vitro along with ligand-like stabilization of DNA G4 structures. Cell permeability and response to RNA G4 structures project the dye with interesting theranostic potential. Our results validate that B6,5 can serve the dual purpose of visualization of DNA and RNA G4 structures and screening of G4 specific ligands, and adds to the limited number of probes with such potential.
G-四链体（G4）是最活跃的研究非canonical 二级结构，由 DNA 或 RNA 链中连续重复的鸟嘌呤形成。小分子介导的 G-四链体靶向已成为在细胞内可视化和稳定这些结构的有吸引力的工具。已经报道了用于初级配体筛选的少数几种 DNA 和 RNA G4 选择性测定法。荧光光谱法、原子力显微镜、CD、PAGE 和共焦显微镜已被用于评估二聚体碳菁染料 B6,5 用于体外和细胞内筛选 G4 结合配体。染料 B6,5 与生理相关的 DNA 和 RNA G4 结构相互作用，导致分子荧光增强，作为 G4 选择性的体外读出。染料与 G4 的相互作用伴随着四链体的稳定化，从而扩展了其在 G4 特异性配体的初步筛选中的用途。该分子具有细胞通透性，并能够使用共焦显微镜可视化核仁中主导四链体的细胞区域。在活细胞中，染料被 quarfloxin 取代。该染料在体外具有显著的双链体到四链体选择性，以及对 DNA G4 结构的配体样稳定化。细胞通透性和对 RNA G4 结构的响应为该染料提供了有趣的治疗潜力。我们的结果验证了 B6,5 可以同时用于可视化 DNA 和 RNA G4 结构以及筛选 G4 特异性配体，这增加了具有这种潜力的探针的数量有限。
