CytK is a newly characterized biological nanopore that establishes stable open-pore states with low baseline drift and long recording lifetimes, enabling reproducible single-molecule measurements. Most applications have focused on peptide and protein analytes, while DNA-level sensing remains underexplored. Here, we developed a framework to quantify the inner sensing profile of wild-type and engineered CytK mutants using an immobilization assay in which single-stranded DNA (ssDNA) was held at the pore entrance via a biotin-streptavidin tether. As benchmarks, poly-A/C/T homopolymers produced distinct residual current levels, confirming base-specific readout under immobilized conditions, differing from patterns seen in α-hemolysin and MspA. To map sensing zones, we constructed a positional-scanning library T n GGG T 22-n (0 ≤ n ≤ 22) that shifts a GGG motif stepwise along the strand. In wild-type CytK, the resulting profile showed a pronounced minimum near n ≈ 14, identifying the effective sensing zone. Furthermore, a secondary position led to an overall bimodal current distribution, suggesting two distinct luminal constrictions that supports previously reported work. We are extending this mapping approach across a panel of CytK mutants to determine how pore modifications influence the depth, position, and shape of the sensing response. This systematic analysis will provide a quantitative basis for engineering and improving CytK as a blocker-type nanopore for position-resolved DNA interrogation under varying orientations, voltages, and buffer conditions.
Wang et al. (Sun,) studied this question.
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