Abstract
G-quadruplex (G4) structures have recently received increasing attention as a potential target for cancer research. We used time-gated fluorescence lifetime imaging microscopy (FLIM) with a G4 fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), to measure the number of o-BMVC foci, which may represent G4 foci, in cells as a common signature to distinguish cancer cells from normal cells. Here, the decrease in the number of o-BMVC foci in the pretreatment of cancer cells with TMPyP4, BRACO-19 and BMVC4 suggested that they directly bind to G4s in cells. In contrast, the increase in the number of o-BMVC foci in the pretreatment of cells with PDS and Hoechst 33258 (H33258) suggested that they do not inhabit the binding site of o-BMVC to G4s in cells. After the H33258 was removed, the gradual decrease of H33258-induced G4 foci may be due to DNA repair. The purpose of this work is to introduce o-BMVC foci as an indicator not only to verify the direct binding of potential G4 ligands to G4 structures but also to examine the possible effect of some DNA binding ligands on DNA integrity by monitoring the number of G4 foci in cells.
Highlights
IntroductionA high-throughput sequencing-based method has detected more than 700,000 G4-induced polymerase-stalling sites in the human genome [3]
G-quadruplexes (G4s) are four-stranded DNA structures that are formed by the stacking ofG-quartets with Hoogsteen hydrogen bonding of four guanines under physiological conditions [1,2].A high-throughput sequencing-based method has detected more than 700,000 G4-induced polymerase-stalling sites in the human genome [3]
More G4 foci in the pyridostatin (PDS) pretreated human U2OS cancer cells were detected using the BG4 antibody. This is because PDS can induce DNA damage at sites enriched in G4 motifs [8]
Summary
A high-throughput sequencing-based method has detected more than 700,000 G4-induced polymerase-stalling sites in the human genome [3]. One major concern is the chromatin integrity [4,5]. It is believed that there is very little opening for G4 formation in closed chromatin [6]. Biffi et al [7] provided convincing evidence to illustrate the existence of G4s in cells by immunofluorescence microscopy using a G4-specific antibody BG4. More G4 foci in the pyridostatin (PDS) pretreated human U2OS cancer cells were detected using the BG4 antibody. This is because PDS can induce DNA damage at sites enriched in G4 motifs [8]
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