Development and application of G4-Flame as a visual biosensor for G4-DNA

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• G-quadruplex DNA (G4-DNA) is a crucial noncanonical DNA structure involved in genomic regulation and disease.
• Existing G4-DNA detection methods lack specificity, spatiotemporal resolution, and live-cell compatibility.

Purpose of the Study:

• To develop a novel biosensor for real-time, high-resolution visualization of G4-DNA dynamics in living systems.
• To investigate the spatiotemporal distribution and function of G4-DNA during the cell cycle and in disease states.

Main Methods:

• Engineered G4-Flame, a genetically encoded fluorescent biosensor using circularly permuted YFP (cpYFP) and a G4-specific binding domain.
• Utilized G4-Flame to visualize G4-DNA dynamics in living cells and analyzed serum samples.

Main Results:

• G4-Flame demonstrated real-time, high-resolution visualization of G4-DNA with specificity across conformations.
• Observed distinct nuclear and mitochondrial G4-DNA patterns during the cell cycle, with nuclear G4-DNA peaking in S phase.
• Mitochondrial G4-DNA was found to suppress mitochondrial-encoded gene expression.
• Significantly elevated serum G4-DNA levels were detected in cancer patients compared to healthy controls.

Conclusions:

• G4-Flame is a transformative tool for studying G4-DNA spatiotemporal regulation in live systems.
• G4-DNA holds potential as a biomarker for early cancer detection, bridging basic research and clinical applications.