Imaging G-Quadruplex Nucleic Acids in Live Cells Using Thioflavin T and Fluorescence Lifetime Imaging Microscopy

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• Guanine-rich oligonucleotides form G-quadruplex (G4) structures in vivo.
• These noncanonical DNA structures are implicated in crucial biological processes.
• Direct visualization of G4s in live cells is essential for understanding their function.

Purpose of the Study:

• To explore the use of common fluorescent dyes for G4 visualization.
• To adapt fluorescence lifetime imaging microscopy (FLIM) for G4 detection using accessible reagents.
• To establish a quantitative method for imaging G4s in live cells.

Main Methods:

• Utilized fluorescence lifetime imaging microscopy (FLIM).
• Investigated the fluorescence lifetime of Thioflavin T (ThT) and Thiazole Orange (TO) bound to different DNA topologies.
• Applied FLIM with ThT to visualize G4s in live U2OS cells under varying conditions.

Main Results:

• The fluorescence lifetime of ThT and TO is sensitive to DNA topology, with G4s exhibiting distinct, long decay times.
• Successfully visualized G4 structures in live U2OS cells using FLIM with ThT.
• Demonstrated G4 detection upon manipulation of G4 levels via competitive binding or nuclease treatment.

Conclusions:

• Commercially available dyes ThT and TO can serve as effective probes for G4 structures based on their fluorescence lifetime.
• FLIM using ThT provides a robust and accessible method for quantitative G4 imaging in live cells.
• This approach simplifies G4 visualization, facilitating further research into their biological roles.