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

Abstract

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 (tau 1/2 <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.

Rapid and Highly Efficient Separation of i-Motif DNA Species by CE-UV and Multivariate Curve Resolution

“…15 Also, labeling the i-motif forming sequences with appropriate fluorescent and quencher moieties may be used as pH-sensors near the biological pH in cell and in vivo media. 16,17 The study of the conformational equilibria involving i-motif structures is usually carried out by means of spectroscopic and separation techniques. Circular dichroism (CD) and ultraviolet (UV) absorption spectroscopies are used to monitor unfolding induced by temperature changes.…”

“…Hence, as an example, the folding/unfolding equilibria of i-motif species has been applied to create nanopores, the size of which is depending on pH . Also, labeling the i-motif forming sequences with appropriate fluorescent and quencher moieties may be used as pH-sensors near the biological pH in cell and in vivo media. , …”

“… 15 Also, labeling the i-motif forming sequences with appropriate fluorescent and quencher moieties may be used as pH-sensors near the biological pH in cell and in vivo media. 16 , 17 …”