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

Area of Science:

• Biochemistry
• Nanotechnology
• Molecular Biology

Background:

• DNA-intercalated motifs (iMs) serve as adaptable platforms for creating pH-responsive nanomachines.
• Previous DNA pH sensors utilized complex intermolecular scaffolds, limiting their efficiency.
• Developing biocompatible and efficient pH sensors is crucial for cellular studies.

Purpose of the Study:

• To design and optimize a simple, unimolecular DNA pH sensor based on iMs.
• To enhance the thermal stability and speed of pH-induced conformational transitions in iMs.
• To demonstrate the sensor's utility for intracellular pH measurements in living cells.

Main Methods:

• Minimized a dual-labeled iM scaffold by replacing loop nucleosides with abasic or alkyl linkers.
• Optimized linker composition, particularly using propyl linkers, for improved performance.
• Tested the sensor's response to pH changes and its applicability in human lung and kidney cells.

Main Results:

• The modified iM scaffold exhibited enhanced thermal stability and faster pH-induced transitions.
• The optimized sensor demonstrated a pH transition value of 6.9 ± 0.1.
• The sensor responded rapidly to minor acidification (τ_1/2 <1 s for a 7.2 → 6.6 pH jump).
• Successful intracellular pH measurements were performed in human lung adenocarcinoma and kidney cells (pH 7.4 ± 0.2 and 7.0 ± 0.2, respectively).

Conclusions:

• Optimized DNA-intercalated motifs provide a robust and efficient platform for biocompatible pH sensing.
• The developed sensor accurately measures intracellular pH in living cells, localizing within the nucleoplasm and interchromatin granules.
• These findings underscore the potential of iMs for investigating nuclear pH-dependent biological processes.