Engineered Ratiometric Near‐Infrared Probes Enable Dual‐Organelle Visualization of G‐Quadruplex in Living Cells

ABSTRACT G‐quadruplexes (G4s) participate in nuclear genome regulation and mitochondrial metabolism, but tools for monitoring both compartments in the same living cell remain limited. Here, we report PEG‐INR‐Me, a ratiometric near‐infrared (NIR) probe designed for simultaneous visualization of nuclear and mitochondrial G4‐associated signals. G4 binding enhances the long‐wavelength emission, whereas the short‐wavelength channel serves as an operational normalization channel under matched acquisition conditions. Accordingly, cellular Channel 640 /Channel 560 values are interpreted as relative readouts within a defined compartment and experiment, rather than as absolute comparisons of G4 abundance between organelles. PEG‐INR‐Me revealed parallel cell‐cycle‐associated changes in nuclear and mitochondrial signals, higher signals in cancer cells than in noncancerous cells, and concurrent decreases during cisplatin treatment followed by partial recovery after caspase inhibition. These observations establish temporal concordance between mitochondria and nucleus. Following direct local administration, the probe also distinguished 4T1 tumors from a contralateral subcutaneous control site. PEG‐INR‐Me therefore provides a dual‐compartment imaging platform for investigating nuclear and mitochondrial G4‐associated dynamics, and their mechanistic relationship deserves to be further investigated.