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Nanopore-Based, Real-Time Single-Molecule Probing of i-Motif Structural Dynamics and Targeted PNA Disruption.

Adina Cimpanu1, Jonggwan Park2, Loredana Mereuta1

  • 1Department of Physics, Alexandru I. Cuza University, 700506 Iasi, Romania.

Nano Letters
|February 2, 2026
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Summary

We developed a nanopore method to study DNA i-motifs, revealing distinct folding behaviors. This technique allows monitoring i-motif dynamics and interactions, paving the way for new therapeutic strategies targeting gene regulation.

Keywords:
Nanopore SensingPeptide Nucleic Acid (PNA)i-MotifpH-responsive Nanostructures

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Area of Science:

  • Biophysics
  • Nanotechnology
  • Genomics

Background:

  • The i-motif is a noncanonical DNA structure critical for gene regulation.
  • Probing i-motif dynamics and interactions remains a significant challenge in molecular biology.

Purpose of the Study:

  • To develop a single-molecule method for sensing human telomeric i-motif folding and dynamics.
  • To investigate the interaction of peptide nucleic acids (PNAs) with i-motifs.

Main Methods:

  • Utilized a wild-type α-hemolysin (α-HL) nanopore to detect i-motif folding events.
  • Characterized two distinct sensing paradigms: transient current signatures from collisions and quasi-permanent blockades from vestibule entrapment.
  • Employed a 6-mer peptide nucleic acid (PNA) as an antisense switch to destabilize the i-motif.

Main Results:

  • Demonstrated two distinct nanopore sensing modes for i-motif dynamics: collision and vestibule-first entry.
  • Achieved high-resolution (nm³ scale) monitoring of pH-dependent volumetric changes within the nanopore.
  • Showcased PNA's ability to controllably invade and destabilize i-motifs, particularly when binding precedes folding.

Conclusions:

  • Established a powerful single-molecule tool for investigating i-motif structures and their interactions.
  • Highlighted PNA design principles for targeting i-motif-mediated gene regulatory elements.
  • Opened new avenues for developing therapeutic strategies based on i-motif modulation.