Probing the pH- and Voltage-Driven Intramolecular Folding Dynamics of the c -MycC52 Promoter i -Motif at the Single-Molecule Level

Abstract The human c-Myc proto-oncogene is a transcription factor that regulates protein expression and is controlled by GC-rich regulatory sequences capable of adopting non-canonical DNA secondary structures. Here, we use the α-hemolysin (α-HL) protein nanopore to probe pH- and voltage-dependent effects on the stability of the i-motif formed by the 52-nucleotide cytosine-rich c-Myc promoter (c-MycC52). Under acidic conditions, electrophoretically driven c-MycC52 i-motifs are transiently trapped within the α-HL vestibule, generating pH-dependent electrical current signatures that are volumetrically and kinetically distinct and resolvable at the single-molecule level. The larger molecular volume measured in the acidic pH range, compared with that of linearized DNA at neutral pH, is consistent with the formation of an i-motif. In contrast, the applied transmembrane voltage does not induce detectable changes in the topology of the captured i-motif fragments. Kinetic modeling of current fluctuations within a continuous-time Markov chain framework reveals a subtle coupling between the acidic pH-dependent compaction topology of the captured i-motif and its local interactions with the α-HL vestibule interior and constriction zone. Although molar KCl concentrations (1 and 3 M) yield c-MycC52 i-motif structures of comparably similar volumes at low pH, reduced charge screening at lower ionic strength enhances electrostatic repulsion along the negatively charged DNA backbone, rendering the vestibule-captured folded structure kinetically more stable and more effective at occluding the α-HL constriction. These findings establish α-HL electrophysiology as a sensitive platform for distinguishing subtle differences in i-motif conformational stability, with potential applications in DNA secondary structure dynamics and disease-targeted diagnostic strategies.