Super-Resolution Detection of DNA Nanostructures Using a Nanopore.

High-resolution analysis of biomolecules has brought unprecedented insights into fundamental biological processes and dramatically advanced biosensing. Notwithstanding the ongoing resolution revolution in electron microscopy and optical imaging, only a few methods are presently available for high-resolution analysis of unlabeled single molecules in their native states. Here, label-free electrical sensing of structured single molecules with a spatial resolution down to single-digit nanometers is demonstrated. Using a narrow solid-state nanopore, the passage of a series of nanostructures attached to a freely translocating DNA molecule is detected, resolving individual nanostructures placed as close as 6 nm apart and with a surface-to-surface gap distance of only 2 nm. Such super-resolution ability is attributed to the nanostructure-induced enhancement of the electric field at the tip of the nanopore. This work demonstrates a general approach to improving the resolution of single-molecule nanopore sensing and presents a critical advance towards label-free, high-resolution DNA sequence mapping, and digital information storage independent of molecular motors.

Multi-resolution simulation of DNA transport through large synthetic nanostructures.

Modeling and simulation has become an invaluable partner in development of nanopore sensing systems. The key advantage of the nanopore sensing method - the ability to rapidly detect individual biomolecules as a transient reduction of the ionic current flowing through the nanopore - is also its key deficiency, as the current signal itself rarely provides direct information about the chemical structure of the biomolecule. Complementing experimental calibration of the nanopore sensor readout, coarse-grained and all-atom molecular dynamics simulations have been used extensively to characterize the nanopore translocation process and to connect the microscopic events taking place inside the nanopore to the experimentally measured ionic current blockades. Traditional coarse-grained simulations, however, lack the precision needed to predict ionic current blockades with atomic resolution whereas traditional all-atom simulations are limited by the length and time scales amenable to the method. Here, we describe a multi-resolution framework for modeling electric field-driven passage of DNA molecules and nanostructures through to-scale models of synthetic nanopore systems. We illustrate the method by simulating translocation of double-stranded DNA through a solid-state nanopore and a micron-scale slit, capture and translocation of single-stranded DNA in a double nanopore system, and modeling ionic current readout from a DNA origami nanostructure passage through a nanocapillary. We expect our multi-resolution simulation framework to aid development of the nanopore field by providing accurate, to-scale modeling capability to research laboratories that do not have access to leadership supercomputer facilities.

Translocation of DNA through Ultrathin Nanoslits.

2D nanoslit devices, where two crystals with atomically flat surfaces are separated by only a few nanometers, have attracted considerable attention because their tunable control over the confinement allows for the discovery of unusual transport behavior of gas, water, and ions. Here, the passage of double-stranded DNA molecules is studied through nanoslits fabricated from exfoliated 2D materials, such as graphene or hexagonal boron nitride, and the DNA polymer behavior is examined in this tight confinement. Two types of events are observed in the ionic current: long current blockades that signal DNA translocation and short spikes where DNA enters the slits but withdraws. DNA translocation events exhibit three distinct phases in their current-blockade traces-loading, translation, and exit. Coarse-grained molecular dynamics simulation allows the different polymer configurations of these phases to be identified. DNA molecules, including folds and knots in their polymer structure, are observed to slide through the slits with near-uniform velocity without noticeable frictional interactions of DNA with the confining graphene surfaces. It is anticipated that this new class of 2D-nanoslit devices will provide unique ways to study polymer physics and enable lab-on-a-chip biotechnology.

High-Fidelity Capture, Threading, and Infinite-Depth Sequencing of Single DNA Molecules with a Double-Nanopore System.

Nanopore sequencing of nucleic acids has an illustrious history of innovations that eventually made commercial nanopore sequencing possible. Nevertheless, the present nanopore sequencing technology leaves much room for improvement, especially with respect to accuracy of raw reads and detection of nucleotide modifications. Double-nanopore sequencing-an approach where a DNA molecule is pulled back and forth by a tug-of-war of two nanopores-could potentially improve single-molecule read accuracy and modification detection by offering multiple reads of the same DNA fragment. One principle difficulty in realizing such a technology is threading single-stranded DNA through both nanopores. Here, we describe and demonstrate through simulations a nanofluidic system for loading and threading DNA strands through a double-nanopore setup with nearly 100% fidelity. The high-efficiency loading is realized by using hourglass-shaped side channels that not only deliver the molecules to the nanopore but also retain molecules that missed the nanopore at the first passage to attempt the nanopore capture again. The second nanopore capture is facilitated by an orthogonal microfluidic flow that unravels the molecule captured by the first nanopore and delivers it to the capture volume of the second nanopore. We demonstrate the potential utility of our double-nanopore system for DNA sequencing by simulating repeat back-and-forth motion-flossing-of a DNA strand through the double-nanopore system. We show that repeat exposure of the same DNA fragments to the nanopore sensing volume considerably increases accuracy of the nucleotide sequence determination and that correlated displacement of ssDNA through the two nanopores may facilitate recognition of homopolymer fragments.