The Translocation of Circular DNA with Variable Single/Double Strand Ratios through Solid-State Nanopores: Transition from Entropic Barriers to Crowding-Dominated Kinetics

Solid-state nanopores serve as powerful single-molecule analytical platforms for characterizing the physicochemical signatures of nucleic acids and proteins, yet prior work has focused almost exclusively on linear DNA, leaving circular DNA translocation poorly understood, particularly how alternating single-/double-stranded (ss/ds) segments modulate transport kinetics. Here, a series of circular DNAs with discrete ssDNA fractions (0%, 33%, 66%, and 100%) was constructed and systematically interrogated for their voltage-dependent translocation through solid-state nanopores. Under fixed bias, ionic current blockade increases linearly with ss content, whereas dwell time scales exponentially with ss proportion. Notably, two voltage-dependent regimes emerge across a critical ss fraction threshold: constructs with high ss content (66% and 100%) exhibit dwell time decaying exponentially with voltage (t ∼ e-v/v0), consistent with entropy-limited uncoiling governed by entropic barriers; conversely, low-ss constructs (0% and 33%) show nearly voltage-invariant translocation, dominated by barrier-free electrophoretic drift due to rigid ds backbones and minimal entanglement. Weibull analysis of event charge deficit distributions confirms this kinetic transition, and ECD values correlate exponentially with ss fraction at all tested voltages, an effect attributed to enhanced excluded-volume crowding at the nanopore orifice by flexible ss segments, switching the driving force from entropic restriction to macromolecular crowding. Collectively, these results elucidate the interplay of hybrid ss/ds architecture and applied field on circular DNA transport, establishing a quantitative physical framework for semiflexible biopolymer transport under nanoconfinement and informing the design of nanopore sensors targeting topologically complex nucleic acids.