Development and Application of G4-Flame as a Visual Biosensor for G4-DNA

G4-DNA, a non-canonical 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 (CPFP) 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. Overall design: The experimental design of this study systematically investigates genome-wide G4-DNA recognition and its cell cycle dynamics by combining genomic mapping and cellular imaging approaches. The core of the methodology employs CUT&Tag to perform a genome-wide consistency analysis, comparing the occupancy profiles obtained using the engineered probe G4-Flame (detected via an anti-HA antibody) with those from the endogenous G4-specific BG4 antibody. To study cell cycle-dependent changes, HEK293T cells expressing an NLS-tagged G4-Flame probe were synchronized at the G1/S boundary using a thymidine double-block protocol. The dynamics of nuclear G4-DNA were then directly visualized and compared between G1 and S phases through fluorescence microscopy imaging, thus integrating genome-wide comparative data with single-cell observational analysis.