Reversible DNA translocation as a molecular caliper to probe the nanoscale asymmetry of glass nanopores

Studying DNA conformation is crucial for understanding gene regulation, chromatin structure, and genomic stability. Nanopores have proven to be excellent label-free, high-throughput tools for studying conformational changes in various biomolecular structures. Conical glass nanopores are widely used in solid-state nanopore studies due to their simple and cost-effective fabrication. However, their inherent geometric asymmetry introduces distinct characteristics in the translocation dynamics of DNA. Here, we demonstrate bi-directional translocation of multiple DNA lengths through a conical nanopore to understand the role of pore asymmetry. We show a quantitative comparison of various parameters, such as the conductance drop (Δ<i>G</i>), translocation time (Δ<i>t</i>), event charge deficit (ECD) and percentage of DNA folding in both directions. A natural output of our ECD-based analysis is the estimation of the effective sensing length of our conical pore geometry. In the nanoscale regime, sensing length controls the spatial resolution of the nanopore detector and is challenging to measure. Our study reveals significant experimental insights into the dependence of DNA length, translocation directionality, and applied voltage on the translocation mechanism, contributing to the broader utilization of glass nanopores in sensing technologies.