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

## TL;DR

The paper introduces G4-Flame, a new fluorescent biosensor for visualizing G4-DNA in living cells, revealing its role in the cell cycle and cancer.

## Contribution

G4-Flame is a novel genetically encoded biosensor enabling real-time, high-resolution visualization of G4-DNA dynamics in living systems.

## Key findings

- Nuclear G4-DNA levels peak during the S phase of the cell cycle.
- Mitochondrial G4-DNA suppresses the expression of mitochondrial-encoded genes.
- Cancer patients have significantly higher serum G4-DNA levels than healthy controls.

## Abstract

G-quadruplex DNA (G4-DNA), a noncanonical tetrahelical nucleic acid structure stabilized by stacked G-quartets via Hoogsteen hydrogen bonding, plays critical roles in genomic regulation and disease pathogenesis. Current methodologies for detecting these structures face limitations in specificity, spatiotemporal resolution, and live-cell applicability. To address these challenges, we engineered G4-Flame, a genetically encoded fluorescent biosensor utilizing circularly permuted fluorescent protein technology. By strategically positioning a G4-specific binding domain proximal to the fluorophore of circularly permuted YFP (cpYFP), G4-Flame achieves real-time, high-resolution visualization of G4-DNA dynamics in living systems, with specificity across diverse G4 conformations. Experimental validation revealed distinct spatiotemporal patterns of G4-DNA during the cell cycle: nuclear G4-DNA levels peaked during the S phase, while mitochondrial G4-DNA was found to suppress the expression of mitochondrial-encoded genes. Clinically, serum analysis revealed significantly elevated G4-DNA levels in cancer patients compared to healthy controls. This work establishes G4-Flame as a transformative tool for investigating G4-DNA spatiotemporal regulation and advances its potential as a biomarker for early cancer detection, bridging fundamental research with clinical translation.

Graphical Abstract