Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry Chinese Academy of Sciences, 100190 Beijing, PR China; University of Chinese Academy of Sciences, Beijing, 100049, PR China.
Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry Chinese Academy of Sciences, 100190 Beijing, PR China.
Biochim Biophys Acta Gen Subj. 2018 May;1862(5):1101-1106. doi: 10.1016/j.bbagen.2018.01.022. Epub 2018 Feb 2.
Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking.
Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM).
Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal.
The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator.
The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells.
由于 G-四链体与生物功能有关的重要作用，直接检测人类细胞中的 G-四链体已成为一个重要问题。尽管已经开发了几种用于检测细胞质或整个细胞中 G-四链体的探针，但仍缺乏用于监测核仁 G-四链体的探针。
通过使用圆二色性（CD）光谱法确证核仁 G-四链体结构的形成。通过荧光光谱法和聚丙烯酰胺凝胶电泳（PAGE）分别测量噻唑橙（ThT）在溶液和凝胶系统中与各种 DNA/RNA 基序结合的亲和力和选择性。通过使用共焦激光扫描显微镜（CLSM）直接捕获活细胞中的 G-四链体成像。
体外证明了 rDNA 和 rRNA G-四链体结构的形成。发现 ThT 对这些 G-四链体结构在溶液或凝胶系统中比对其他核酸基序具有更高的亲和力和选择性。使用 ThT 作为荧光探针可以可视化活细胞中的核仁 G-四链体。活细胞中的 G-四链体配体处理导致 ThT 信号急剧下降。
使用 ThT 作为指示剂直接可视化活细胞核仁中的 G-四链体结构的自然存在。
该研究为 rRNA G-四链体结构的形成提供了实质性证据，并为直接可视化活细胞中的核仁 G-四链体提供了一种有效探针。
