A Specific and Quantitative Fluorogenic Tag-Probe System for Live-Cell Imaging of R-loop Dynamics [CUT&Tag]

Their specific, dynamic, and quantitative visualization in living cells has remained a major methodological challenge. Here, we develop a bimodal fluorogenic sensor that integrates a high-fidelity targeting module, PYP3-tagged catalytically inactive RNase H1, with a conditionally activated synthetic probe, YOCNB.

The system enforces a two-step verification for signal generation: the probe must first bind the PYP3 tag localized at R-loop sites and then intercalate into the RNA-DNA hybrid, ensuring specificity and a near-zero background. This design decouples the signal from sensor concentration, enabling reliable quantification. The system's rigorous validation confirmed its accurate reporting of R-loop dynamics under both genetic perturbations and pharmacological treatments.