[Paper Review] Controlling DNA Tug-of-War in a Dual Nanopore Device

Xu Liu, Yuning Zhang+3 more|arXiv (Cornell University)|Nov 27, 2018

Nanopore and Nanochannel Transport Studies4 references4 citations

TL;DR

This paper presents a dual nanopore device that uses active, opposing voltage control to create a 'tug-of-war' state, slowing DNA translocation by up to two orders of magnitude while suppressing folds. By balancing electrophoretic forces, the method enables prolonged, diffusively controlled sliding of DNA between pores, increasing translocation time and enabling high-fidelity, sequential sensing of labeled features on a single molecule.

ABSTRACT

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.

Motivation & Objective

• To address the critical challenge of rapid DNA translocation in solid-state nanopores, which limits sensing resolution and signal-to-noise ratio.
• To develop a method for active, real-time control of DNA translocation speed and conformation using two independently controlled nanopores.
• To suppress DNA folding during translocation by applying opposing forces that tauten the molecule between pores.
• To enable high-fidelity, sequential detection of physical labels (e.g., streptavidin) on a single DNA molecule by synchronously monitoring ionic current in both pores.
• To demonstrate that translocation slow-down can be quantitatively modeled using 1D first-passage theory for sub-diffusive processes.

Proposed method

• Two nanopores are fabricated on a single insulating membrane using wafer-scale processes, enabling independent voltage control and ionic current sensing.
• A Field-Programmable Gate Array (FPGA)-based control logic detects initial capture in one pore and triggers reversal of voltage in the second pore to initiate a tug-of-war.
• The opposing voltages create a net force near zero, causing DNA to slide diffusively between pores, with translocation speed controlled by voltage tuning.
• Co-capture efficiency is 76.8% for molecules initially detected by one pore, with the tug-of-war state sustained by balancing electrophoretic forces.
• Ionic current signals from both pores are used to detect sub-events, such as mono-streptavidin tags, by identifying current blockades below mean open-pore current minus 5σ.
• Translocation dynamics are modeled using 1D first-passage theory for sub-diffusive motion, explaining the observed resonance-like peak in tug-of-war lifetime.

Experimental results

Research questions

• RQ1 Can active control via opposing voltages in a dual nanopore system significantly slow DNA translocation while suppressing folding?
• RQ2 What voltage tuning results in the longest-lived tug-of-war state, and is this state consistent with diffusive sliding?
• RQ3 Can the ionic current from both pores be used to synchronously detect sequence-specific labels on a single DNA molecule?
• RQ4 How well does first-passage theory for sub-diffusive processes describe the observed translocation dynamics in the tug-of-war regime?
• RQ5 What is the efficiency of co-capture and sustained tug-of-war formation in a dual nanopore system?

Key findings

• The dual nanopore system achieves a maximum two-order-of-magnitude increase in average translocation time compared to single-pore translocation.
• Co-capture and sustained tug-of-war state formation occurs with 76.8% efficiency for molecules initially captured by one pore.
• The peak lifetime of the tug-of-war state occurs when opposing forces are balanced, indicating a regime dominated solely by diffusive sliding.
• Translocation slow-down is quantitatively described by 1D first-passage theory for sub-diffusive processes, validating the theoretical model.
• The ionic current from both pores enables sequential detection of physical labels, such as mono-streptavidin, with blockades identified as signals below mean current minus 5σ.
• The method enables conformational control by preventing DNA folding and ensures a linear, extended state during translocation for improved sensing.