Cellular Detection of G‐Quadruplexes by Optical Imaging Methods

Abstract

G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex-selective fluorophores, the twice-as-smart quadruplex ligands named TASQ (for template-assembled synthetic G-quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real-time fluorescent image collection. © 2017 by John Wiley & Sons, Inc.

Selectivity of Terpyridine Platinum Anticancer Drugs for G-quadruplex DNA

“…A large amount of data provides evidence that such structures could form in cells and may play important roles in biology, such as genomic stability, replication, transcription, translation, and telomere maintenance [5,6]. Bioinformatics [7,8], cellular imaging [9,10,11,12,13], as well as high throughput sequencings of genomic DNA or RNA [14,15,16] have contributed to identifying G4 prevalence at specific key genomic sequences such as telomere and promoter regions of oncogenes. So far, they have been identified as potential drug targets, especially in cancer [17,18,19].…”

Structural insights and shedding light on preferential interactions of dietary flavonoids with G-quadruplex DNA structures: A new horizon

“…Several ligands with potential biophysical properties have been investigated in cells using chromosomal imaging [ 10 , 37 ]. While preferential staining in the nucleoli/nucleus has been found in a few experiments, the findings may not be taken as the presence of G-quadruplex-DNA complexes in vivo because of non-specific adherence to other proteins [ 40 , 41 ]. Furthermore, the cellular localisation of several ligands varies based upon when the cells were alive or fixed, as well as the form of fixation or crosslinking utilized.…”

Template-Assembled Synthetic G-Quartets (TASQs): multiTASQing Molecular Tools for Investigating DNA and RNA G-Quadruplex Biology

“…On this basis, we devised an experimental setup to use N-TASQ to label either RNA G4s (live-cell N-TASQ incubation before fixation and imaging) or DNA G4s (cell fixation before N-TASQ incubation and imaging) (Figure 5G). 54 We also developed a quantitative use of N-TASQ fluorescence to assess the modulation of the G4 landscape in cancer cells (e.g., HeLa) upon incubation with G4-interacting compounds; for instance, the G4-stabilizer BRACO-19 was found to increase it 55 or the G4-destabilizer PhpC 4 to decrease it (unpublished). N-TASQ was also found to be compatible with live-cell imaging systems, which enables the characterization in real time of the pace at which it enters cells (ca.…”

“…We used these properties to show that high-quality images could be collected using a classical confocal microscope, with lasers adjusted at 408, 488, and 555 nm (Figure F). On this basis, we devised an experimental setup to use N-TASQ to label either RNA G4s (live-cell N-TASQ incubation before fixation and imaging) or DNA G4s (cell fixation before N-TASQ incubation and imaging) (Figure G) . We also developed a quantitative use of N-TASQ fluorescence to assess the modulation of the G4 landscape in cancer cells ( e.g.…”