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

## TL;DR

This paper introduces a nanopore-based method to study the dynamic structure of i-motifs and how PNA can disrupt them, offering new insights for therapeutic design.

## Contribution

A novel single-molecule approach using α-HL nanopores to monitor i-motif dynamics and PNA disruption in real time.

## Key findings

- Two distinct nanopore sensing modes reveal i-motif structural dynamics and pH-dependent volumetric changes.
- A 6-mer PNA acts as a reversible antisense switch to destabilize i-motifs, especially when binding precedes folding.
- The method provides insights into i-motif interactions and potential therapeutic PNA design principles.

## Abstract

The i-motif, a crucial noncanonical DNA structure, is
prevalent
in gene regulatory regions, yet its dynamics is challenging to probe.
Here, we employ a wild-type α-hemolysin nanopore (α-HL)
to sense the folding of a human telomeric i-motif. We demonstrate
two distinct sensing paradigms: reversible i-motif collisions at the
nanopore’s β-barrel, producing transient current signatures,
versus vestibule-first entry, yielding quasi-permanent blockades.
The collision mode enables continuous i-motif dynamics monitoring,
while vestibule entrapment provides ground for resolving pH-dependent
volumetric changes in nanoconfinement with ∼nm3 resolution.
We show that a short 6-mer peptide nucleic acid (PNA) complementary
to the C-rich strand acts as a reversible antisense switch, capable
of controllably invading and destabilizing the i-motifan effect
that is particularly pronounced when PNA binding precedes pH-induced
folding. This work establishes a powerful single-molecule tool for
investigating i-motif interactions and highlights new design principles
for therapeutic PNAs by targeting i-motif-mediated regulatory structures.