Diversifying i-motif-based pH sensors: Labeling patterns tune the intracellular localization

Intercalated DNA motifs (iMs) provide a convenient scaffold for the design of biocompatible pH sensors. Among unimolecular iM-based sensors, only those labeled with conventional fluorophores or fluorophore/quencher pairs at the 3′ and 5′ termini for FRET/quenching upon pH-dependent iM folding have been tested in cells and tended to accumulate in the nuclei. Here, we used cytosine mimics as internal iM labels and synthesized a new phenoxazine-based non-fluorescent nucleoside analog, tC°Azo, which quenches the fluorescence of a known cytosine mimic, tC°. Incorporation of the tC°/tC°Azo pair into a genomic iM-forming sequence C5T resulted in a high-contrast pH sensor with an increased pH transition point and a working range compatible with physiological conditions. Importantly, unlike the known nuclei-specific C5T-based sensors with conventional labels that provide a fluorescent signal in the green/red channels, the new sensor localized mainly in the cytoplasm and allowed pH monitoring based on the tC° signal in the blue channel. As the labeling scheme was the only unique feature of the new sensor, it must account for the unique distribution pattern, i.e., the accumulation of the sensor in the cytoplasm. These findings highlight the importance of the labeling scheme of unimolecular iM-based pH sensors and open the way for multiplexed pH monitoring in different cellular compartments.