Ratiometric i-Motif-Based Sensor for Precise Long-Term Monitoring of pH Micro Alterations in the Nucleoplasm and Interchromatin Granules.

DNA-intercalated motifs (iMs) are facile scaffolds for the design of various pH-responsive nanomachines, including biocompatible pH sensors. First, DNA pH sensors relied on complex intermolecular scaffolds. Here, we used a simple unimolecular dual-labeled iM scaffold and minimized it by replacing the redundant loop nucleosides with abasic or alkyl linkers. These modifications improved the thermal stability of the iM and increased the rates of its pH-induced conformational transitions. The best effects were obtained upon the replacement of all three native loops with short and flexible linkers, such as the propyl one. The resulting sensor showed a pH transition value equal to 6.9 ± 0.1 and responded rapidly to minor acidification (tau1/2 <1 s for 7.2 → 6.6 pH jump). We demonstrated the applicability of this sensor for pH measurements in the nuclei of human lung adenocarcinoma cells (pH = 7.4 ± 0.2) and immortalized embryonic kidney cells (pH = 7.0 ± 0.2). The sensor stained diffusely the nucleoplasm and piled up in interchromatin granules. These findings highlight the prospects of iMs in the studies of normal and pathological pH-dependent processes in the nucleus, including the formation of biomolecular condensates.

Phenoxazine pseudonucleotides in DNA i-motifs allow precise profiling of small molecule binders by fluorescence monitoring.

The lack of high throughput screening (HTS) techniques for small molecules that stabilize DNA iMs limits their development as perspective drug candidates. Here we showed that fluorescence monitoring for probing the effects of ligands on the iM stability using the FAM-BHQ1 pair provides incorrect results due to additional dye-related interactions. We developed an alternative system with fluorescent phenoxazine pseudonucleotides in loops that do not alter iM unfolding. At the same time, the fluorescence of phenoxazine residues is sensitive to iM unfolding that enables accurate evaluation of ligand-induced changes of iM stability. Our results provide the basis for new approaches for HTS of iM ligands.