Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe

Publication date: Available online 2 February 2018
Source:Biochimica et Biophysica Acta (BBA) - General Subjects
Author(s): Suge Zhang, Hongxia Sun, Hongbo Chen, Qian Li, Aijiao Guan, Lixia Wang, Yunhua Shi, Shujuan Xu, Meirong Liu, Yalin Tang
BackgroundDirect 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.MethodsFormation 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).ResultsFormation 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.ConclusionsThe natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator.General significanceThe 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.



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