Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP)

Area of Science:

• Biophysics
• Molecular Biology
• Cell Biology

Background:

• Fluorescence imaging is crucial for understanding DNA and RNA quadruplexes in cellular and tissue contexts.
• The photophysical properties of fluorescent probes limit imaging modalities and equipment choices.
• Developing versatile probes is essential for advancing quadruplex-based diagnostics and therapies.

Purpose of the Study:

• To characterize a new generation of quadruplex dye, G4-REP (quadruplex-specific red-edge probe).
• To demonstrate the dye's versatility and applicability across different imaging techniques and cell types.
• To promote a novel approach for designing efficient and convenient quadruplex-reporting dyes.

Main Methods:

• Characterization of the photophysical properties of the G4-REP prototype.
• Cell imaging using confocal and multiphoton microscopy.
• Real-time live-cell imaging over extended periods in human cell lines (non-cancerous and cancerous).

Main Results:

• G4-REP exhibits fluorescence responses independent of excitation wavelength due to the red-edge effect.
• The dye is compatible with diverse imaging modalities and facilities.
• Successful imaging and quantification of quadruplexes in both non-cancerous and cancerous human cell lines were achieved.

Conclusions:

• G4-REP represents a versatile, efficient, and convenient dye for tracking higher-order nucleic acid structures in living cells.
• The red-edge effect offers a new strategy for designing adaptable fluorescent probes.
• This advancement facilitates mechanistic insights for quadruplex-directed molecular diagnostics and therapies.