William B. Dunbar | Searcharxiv

arXiv2022

Discriminating protein tags on dsDNA constructs using a dual Nanopore device

We report a novel simulation strategy that enables us to identify key parameters controlling the experimentally measurable characteristics of structural protein tags on dsDNA construct translocating through a double nanopore setup. First, we validate the scheme in silico by reproducing and explaining the physical origin of the experimental dwell time distributions of the Streptavidin markers on a 48 kbp long dsDNA. These studies reveal the important differences in the characteristics of the protein tags compared to the dynamics of dsDNA segments, immediately providing clues on how to improve the measurement protocols to decipher the unknown genomic lengths accurately. Of particular importance is the in silico studies on the effect of electric field inside and beyond the pores which we find is critical to discriminate protein tags based on their effective charges and masses revealed through a generic power-law dependence of the average dwell time at each pore. The simulation protocols enable to monitor piecewise dynamics of the individual monomers at a sub-nanometer length scale and provide an explanation of the disparate velocity variation from one tag to the other using the nonequilibrium tension propagation theory, - a key element to decipher genomic lengths accurately. We further justify the model and the chosen simulation parameters by calculating the Peclet number which is in close agreement with the experiment. Analysis of our simulation results from the CG model has the capability to refine the accuracy of the experimentally obtained genomic lengths and carefully chosen simulation strategies can serve as a powerful tool to discriminate different types of neutral and charged tags of different origins on a dsDNA construct in terms of their physical characteristics and can provide insights to increase both the efficiency and accuracy of an experimental dual-nanopore setup.

cond-mat.soft↗

arXiv2018

Controlling DNA Tug-of-War in a Dual Nanopore Device

Methods for reducing and directly controlling the speed of DNA through a nanopore are needed to enhance sensing performance for direct strand sequencing and detection/mapping of sequence-specific features. We have created a method for reducing and controlling the speed of DNA that uses two independently controllable nanopores operated with an active control logic. The pores are positioned sufficiently close to permit co-capture of a single DNA by both pores. Once co-capture occurs, control logic turns on constant competing voltages at the pores leading to a `tug-of-war' whereby the molecule is pulled from both ends by opposing forces. These forces exert both conformational and speed control over the co-captured molecule, removing folds and reducing the translocation rate. When the voltages are tuned so that the electrophoretic force applied to both ends of the molecule comes into balance, the life-time of the tug-of-war state is limited purely by diffusive sliding of the DNA between the pores. We are able to produce a tug-of-war state on 76.8% of molecules that are captured with a maximum two-order of magnitude increase in average pore translocation time relative to the average time for single-pore translocation. Moreover, we quantify the translocation slow-down as a function of voltage tuning and show that the slow-down is well described by a first passage analysis for a one-dimensional sub-diffusive process. The ionic current of each nanopore provides an independent sensor that synchronously measures a different region of the same molecule, enabling sequential detection of physical labels, such as mono-streptavidin tags. With advances in devices and control logic, future dual-pore applications include genome mapping and enzyme-free sequencing.

physics.bio-ph↗