Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• Guanine-rich DNA sequences form G-quadruplexes (G4s), crucial structures in cellular processes.
• Directly observing G4s in live cells is challenging, limiting understanding of their in vivo functions.

Purpose of the Study:

• To develop and validate a method for visualizing and studying G-quadruplexes in live cells.
• To assess the interaction of small molecules with G4s and their stability in a cellular context.

Main Methods:

• Utilized a novel fluorescent probe, DAOTA-M2, combined with fluorescence lifetime imaging microscopy (FLIM).
• Developed a FLIM-based cellular assay for studying G4s and their interactions with drug candidates.
• Investigated the role of FancJ and RTEL1 by observing changes in G4s upon expression reduction.

Main Results:

• DAOTA-M2 and FLIM successfully identified G-quadruplexes within the nuclei of live and fixed cells.
• The FLIM assay enabled the study of small molecule interactions with G4s and G4 stability.
• Reduced expression of FancJ and RTEL1 led to increased DAOTA-M2 lifetime, indicating more G4s and suggesting their role in G4 resolution.

Conclusions:

• DAOTA-M2 in conjunction with FLIM provides a powerful tool for visualizing and analyzing G-quadruplexes in live cells.
• This method facilitates the study of G4-interacting drugs and G4 dynamics.
• FancJ and RTEL1 are implicated in the resolution of G-quadruplex structures in mammalian cells.