Label Type Influence on DNA Translocation Velocity in Solid-State Nanopores.

Abstract

Solid-state nanopores enable single-molecule detection of long double-stranded nucleic acids and can resolve the position of site-specific molecular labels attached to a DNA carrier. These labels are employed in many applications, such as molecular barcoding, protein mapping, and structure-based DNA data storage. Often, it is implicitly assumed that these labels do not significantly perturb molecular transport through the pore. However, the magnitude of such perturbations and their potential impact on positional readout remain largely unquantified. Here, we systematically quantify how dense molecular labeling affects the translocation time of DNA carriers in solid-state nanopores using glass nanopipettes with diameters of 8-12 nm, exceeding the physical size of the labels. We employ multiple 7.2 kbp DNA carriers, each bearing up to 60 labels of a given type, including DNA nanostructures, monovalent streptavidin, and 20 kDa polyethylene glycol (PEG). Despite carrier-level differences in mass of up to 83% and charge of up to 23%, all labels produce only modest changes in global translocation time, remaining within ±15%, which is below intrameasurement variability (∼20%). This corresponds to a total velocity change of <0.25% per label. Analysis of the timing of label-associated spikes reveals that the velocity profile throughout the translocation is preserved across label types. It also indicates that 40-70% of the observed global translocation-time shift occurs while labeled regions pass through the pore, despite these regions comprising only 20% of the carrier. However, because global translocation times change only weakly and relative label positions remain largely unaffected, molecular barcoding and protein-positioning assays can generally be performed without label-specific velocity corrections under the conditions studied.