Not a lot is known about G-quadruplexes – four-stranded DNA complexes. However, scientists have now developed a new technique to detect how G-quadruplexes act in living cells.
Guanine-rich sequences of DNA can fold into tetra-stranded helical assemblies. These are known as G-quadruplexes (G4s). These structures have been implicated in a number of essential biological processes such as telomere maintenance, transcription, translation and replication. Although their exact roles are still under significant scrutiny, it is commonly accepted that the formation of G4s can lead to DNA damage. For example, G4s appear in cancer cells at much higher rates than healthy cells, and have recently been characterised in breast cancer heterogeneity. As a result, there has been significant interest in developing tools to detect and visualise G4 DNA structures in cells.
Visualising dynamics live
In a study, published in Nature Communications, researchers used a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) to identify G4s within nuclei of live and fixed cells. The team measured the brightness of the fluorescence (which depends on the concentration of DNA molecules) and also tracked how long this fluorescence was emitted. This enabled the team to see how different molecules interacted with the G4s in living cells. For example, when a molecule bound to the DNA structure, it would displace the DAOTA-M2 molecule. This would cause the light to go out faster.
From this, the team identified two helicases – FancJ and RTEL1 – that were able to unwind the G4 complexes and jumpstart the process of their degradation. Specifically, they found that reduction of FancJ and RTEL1 expression in mammalian cells increased the DAOTA-M2 lifetime. This implies that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.
This FLIM-based cellular assay can be used to study the interaction of non-fluorescent small molecules with G4s. Most importantly, this assay can be applied to a wide range of drug candidates, enabling scientists to design drugs that bind to DNA.
Ramon Vilar, Professor of Medicinal Inorganic Chemistry at Imperial College London, stated:
“Many researchers have been interested in the potential of G-quadruplex-binding molecules as potential drugs for diseases such as cancers.
Our method will help to progress our understanding of these potential new drugs.”
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