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

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.