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Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.STATEMENT OF SIGNFICANCENanopore sequencing of DNA is a fast and cheap technology that uniquely delivers multi-kilobase reads. It is currently used world-wide in many applications such as genome sequencing, epigenetics, and surveillance of viral and bacterial pathogens and has started to revolutionize human lives in medicine, agriculture and environmental studies. However, the high base-calling error rates prevent nanopore DNA sequencing from reaching its full potential. In this study, we converted octameric MspA into a single-chain pore enabling asymmetric mutations to fine-tune the pore geometry and chemistry and address the shortcomings of nanopores. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

RNA fibers are a class of biomaterials that can be assembled using HIV-like kissing loop interactions. Because of the programmability of molecular design and low immunorecognition, these structures present an interesting opportunity to solve problems in nanobiotechnology and synthetic biology. However, the experimental tools to fully characterize and discriminate among different fiber structures in solution are limited. Herein, we utilize solid-state nanopore experiments and Brownian dynamics simulations to characterize and distinguish several RNA fiber structures that differ in their degrees of branching. We found that, regardless of the electrolyte type and concentration, fiber structures that have more branches produce longer and deeper ionic current blockades in comparison to the unbranched fibers. Experiments carried out at temperatures ranging from 20–60 °C revealed almost identical distributions of current blockade amplitudes, suggesting that the kissing loop interactions in fibers are resistant to heating within this range.

In contrast to sequence-specific techniques such as polymerase chain reaction, DNA sequencing does not require prior knowledge of the sample for surveying DNA. However, current sequencing technologies demand high inputs for a suitable library preparation, which typically necessitates DNA amplification, even for single-molecule sequencing methods. Here, electro-optical zero-mode waveguides (eZMWs) are presented, which can load DNA into the confinement of zero-mode waveguides with high efficiency and negligible DNA fragment length bias. Using eZMWs, highly efficient voltage-induced loading of DNA fragments of various sizes from ultralow inputs (nanogram-to-picogram levels) is observed. Rapid DNA fragment identification is demonstrated by burst sequencing of short and long DNA molecules (260 and 20 000 bp) loaded from an equimolar picomolar-level concentration mixture in just a few minutes. The device allows further studies in which low-input DNA capture is essential, for example, in epigenetics, where native DNA is required for obtaining modified base information.

Compared with conventional methods, single-molecule real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-nanogram quantities is impractical owing to inefficient delivery of DNA molecules into the confines of zero-mode waveguides-zeptolitre optical cavities in which DNA sequencing proceeds. Here, we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000-base-pair-long DNA template within seconds from a sub-nanogram input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.

Biological nanopores have been used as powerful platforms for label-free detection and identification of a range of biomolecules for biosensing applications and single molecule biophysics studies. Nonetheless, high limit of detection (LOD) of analytes due to inefficient biomolecular capture into biological nanopores at low voltage poses practical limits on their biosensing efficacy. Several approaches have been proposed to improve the voltage stability of the membrane, including polymerization and hydrogel coating, however, these compromise the lipid fluidity. Here, we developed a chip-based platform that can be massively produced on a wafer scale that is capable of sustaining high voltages of 350 mV with comparable membrane areas to traditional systems. Using this platform, we demonstrate sensing of DNA hairpins in α-hemolysin nanopores at the nanomolar regime under high voltage. Further, we have developed a workflow for one-pot enzymatic release of DNA hairpins with different stem lengths from magnetic microbeads, followed by multiplexed nanopore-based quantification of the hairpins within minutes, paving the way for novel nanopore-based multiplexed biosensing applications.

Label-free nanopore technology for sequencing biopolymers such as DNA and RNA could potentially replace existing methods if improvements in cost, speed, and accuracy are achieved. Solid-state nanopores have been developed over the past two decades as physically and chemically versatile sensors that mimic biological channels, through which transport and sequencing of biomolecules have already been demonstrated. Of particular interest is the use of two-dimensional (2D) materials as nanopore substrates, since these can in theory provide the highest resolution readout (1 nm of a biopolymer segment) and opportunities for electronic multiplexed readout through their interesting electronic properties. In this work, we report on nanopores comprising atomically thin flakes of 2D transition metal carbides called MXenes. We demonstrate a high-yield (60%), contamination-free, and alignment-free transfer method that involves their self-assembly at a liquid-liquid interface to large-scale (mm-sized) films composed of sheets, followed by nanopore fabrication using focused electron beams. Our work demonstrates the feasibility of MXenes, a class of hydrophilic 2D materials with over 20 compositions known to date, as nanopore membranes for DNA translocation and single-molecule sensing applications.

We have recently shown that nanopore zero-mode waveguides are effective tools for capturing picogram levels of long DNA fragments for single-molecule DNA sequencing. Despite these key advantages, the manufacturing of large arrays is not practical due to the need for serial nanopore fabrication. To overcome this challenge, we have developed an approach for the wafer-scale fabrication of waveguide arrays on low-cost porous membranes, which are deposited using molecular-layer deposition. The membrane at each waveguide base contains a network of serpentine pores that allows for efficient electrophoretic DNA capture at picogram levels while eliminating the need for prohibitive serial pore milling. Here, we show that the loading efficiency of these porous waveguides is up to 2 orders of magnitude greater than their nanopore predecessors. This new device facilitates the scaling-up of the process, greatly reducing the cost and effort of manufacturing. Furthermore, the porous zero-mode waveguides can be used for applications that benefit from low-input single-molecule real-time sequencing.

We study the driven translocation of a semi-flexible polymer through a nanopore by means of a modified version of the iso-flux tension propagation theory, and extensive molecular dynamics (MD) simulations. We show that in contrast to fully flexible chains, for semi-flexible polymers with a finite persistence length $${\tilde{{\boldsymbol{\ell }}}}_{{\boldsymbol{p}}}$$ ℓ ˜ p the trans side friction must be explicitly taken into account to properly describe the translocation process. In addition, the scaling of the end-to-end distance RN as a function of the chain length N must be known. To this end, we first derive a semi-analytic scaling form for RN, which reproduces the limits of a rod, an ideal chain, and an excluded volume chain in the appropriate limits. We then quantitatively characterize the nature of the trans side friction based on MD simulations. Augmented with these two factors, the theory shows that there are three main regimes for the scaling of the average translocation time τ ∝ N α . In the rod $${\boldsymbol{N}}{\boldsymbol{/}}{\tilde{{\boldsymbol{\ell }}}}_{{\boldsymbol{p}}}{\boldsymbol{\ll }}1$$ N / ℓ ˜ p ≪ 1 , Gaussian $${\boldsymbol{N}}{\boldsymbol{/}}{\tilde{{\boldsymbol{\ell }}}}_{{\boldsymbol{p}}}\sim {\bf{1}}{{\bf{0}}}^{{\bf{2}}}$$ N / ℓ ˜ p ∼ 1 0 2 and excluded volume chain $${\boldsymbol{N}}{\boldsymbol{/}}{\tilde{{\boldsymbol{\kappa }}}}_{{\boldsymbol{p}}}$$ N / κ ˜ p ≫ 10 6 limits, α = 2, 3/2 and 1 + ν, respectively, where ν is the Flory exponent. Our results are in good agreement with available simulations and experimental data.

Nucleosomes are the fundamental repeating units of chromatin, and dynamic regulation of their positioning along DNA governs gene accessibility in eukaryotes. Although epigenetic factors have been shown to influence nucleosome structure and dynamics, the impact of DNA methylation on nucleosome packaging remains controversial. Further, all measurements to date have been carried out under zero-force conditions. In this paper, we present the first automated force measurements that probe the impact of CpG DNA methylation on nucleosome stability. In solid-state nanopore force spectroscopy, a nucleosomal DNA tail is captured into a pore and pulled on with a time-varying electrophoretic force until unraveling is detected. This is automatically repeated for hundreds of nucleosomes, yielding statistics of nucleosome lifetime vs electrophoretic force. The force geometry, which is similar to displacement forces exerted by DNA polymerases and helicases, reveals that nucleosome stability is sensitive to DNA sequence yet insensitive to CpG methylation. Our label-free method provides high-throughput data that favorably compares with other force spectroscopy experiments and is suitable for studying a variety of DNA–protein complexes.

Nucleic acids are central figures in many of life’s key molecular processes, e.g., enzymatic activity, epigenetics/gene regulation, viral replication, aging, cancer, and other diseases. Over the past two decades, nanopores have emerged as a new tool for studying the properties of nucleic acids at the single-molecule level. In this review, we summarize the use of nanopores as sensors of nucleic acid structure, particularly for studying chemically modified and damaged DNA, and for probing the interactions of small-molecule drugs with nucleic acid targets.

We present a strategy for designed self-assembly of peptides into two-dimensional monolayer crystals on the surface of graphene and graphite. As predicted by computation, designed peptides assemble on the surface of graphene to form very long, parallel, in-register β -sheets, which we call β -tapes. Peptides extend perpendicularly to the long axis of each β -tape, defining its width, with hydrogen bonds running along the axis. Tapes align on the surface to create highly regular microdomains containing 4-nm pitch striations. Moreover, in agreement with calculations, the atomic structure of the underlying graphene dictates the arrangement of the β -tapes, as they orient along one of six directions defined by graphene's sixfold symmetry. A cationic-assembled peptide surface is shown here to strongly adhere to DNA, preferentially orienting the double helix along β -tape axes. This orientational preference is well anticipated from calculations, given the underlying peptide layer structure. These studies illustrate how designed peptides can amplify the Angstrom-level atomic symmetry of a surface onto the micrometer scale, further imparting long-range directional order onto the next level of assembly. The remarkably stable nature of these assemblies under various environmental conditions suggests applications in enzymelike catalysis, biological interfaces for cellular recognition, and two-dimensional platforms for studying DNA-peptide interactions.

Modified DNA bases are widespread in biology. 5-Methylcytosine (mC) is a predominant epigenetic marker in higher eukaryotes involved in gene regulation, development, aging, cancer, and disease. Recently, 5-hydroxymethylcytosine (hmC) was identified in mammalian brain tissue and stem cells. However, most of the currently available assays cannot distinguish mC from hmC in DNA fragments. We investigate here the physical properties of DNA with modified cytosines, in efforts to develop a physical tool that distinguishes mC from hmC in DNA fragments. Molecular dynamics simulations reveal that polar cytosine modifications affect internal base pair dynamics, while experimental evidence suggest a correlation between the modified cytosine's polarity, DNA flexibility, and duplex stability. On the basis of these physical differences, solid-state nanopores can rapidly discriminate among DNA fragments with mC or hmC modification by sampling a few hundred molecules in the solution. Further, the relative proportion of hmC in the sample can be determined from the electronic signature of the intact DNA fragment.

Solid-state nanopores are sensors capable of analysing individual unlabelled DNA molecules in solution. Although the critical information obtained from nanopores (for example, DNA sequence) comes from the signal collected during DNA translocation, the throughput of the method is determined by the rate at which molecules arrive and thread into the pores. Here, we study the process of DNA capture into nanofabricated SiN pores of molecular dimensions. For fixed analyte concentrations we find an increase in capture rate as the DNA length increases from 800 to 8,000 base pairs, a length-independent capture rate for longer molecules, and increasing capture rates when ionic gradients are established across the pore. Furthermore, we show that application of a 20-fold salt gradient allows the detection of picomolar DNA concentrations at high throughput. The salt gradients enhance the electric field, focusing more molecules into the pore, thereby advancing the possibility of analysing unamplified DNA samples using nanopores.

We present a novel single-molecule method for rapidly evaluating small-molecule binding to individual DNA molecules using nanopores fabricated in ultrathin silicon membranes. A measurable shift in the residual ion current through a approximately 3.5 nm pore results from threading of a dye-intercalated DNA molecule, as compared to the typical residual current of native DNA. The average level of the residual current can be used to directly quantify the fraction of bound molecules to DNA, providing a new way to determine binding isotherms. Spatial sensitivity is also demonstrated by designing a two-segment DNA molecule that contains small-molecule binding sites in one of its two segments. Translocations of such molecules exhibit two current levels upon incubation with a DNA-binding dye, caused by selectively bound dye in one of the DNA segments. Our results, as shown here with four different dyes, coincide well with bulk fluorescence measurements performed under identical conditions. The nanopore approach for "reading-out" molecular binding along a DNA molecule, combined with the miniscule amounts of DNA required and the potential for scalability using nanopore arrays, provide a novel platform for future applications in analytical drug screening.

Synthetic nucleic acids offer rich potential to understand and engineer new cellular functions, yet an unresolved limitation in their production and usage is deleterious products, which restrict design complexity and add cost. Herein, we employ a solid-state nanopore to differentiate molecules of a gene synthesis reaction into categories of correct and incorrect assemblies. This new method offers a solution that provides information on gene synthesis reactions in near-real time with higher complexity and lower costs. This advance can permit insights into gene synthesis reactions such as kinetics monitoring, real-time tuning, and optimization of factors that drive reaction-to-reaction variations as well as open venues between nanopore-sensing, synthetic biology, and DNA nanotechnology.

Oxidation of a DNA thymine to 5-hydroxymethyluracil is one of several recently discovered epigenetic modifications. Here, we report the results of nanopore translocation experiments and molecular dynamics simulations that provide insight into the impact of this modification on the structure and dynamics of DNA. When transported through ultrathin solid-state nanopores, short DNA fragments containing thymine modifications were found to exhibit distinct, reproducible features in their transport characteristics that differentiate them from unmodified molecules. Molecular dynamics simulations suggest that 5-hydroxymethyluracil alters the flexibility and hydrophilicity of the DNA molecules, which may account for the differences observed in our nanopore translocation experiments. The altered physico-chemical properties of DNA produced by the thymine modifications may have implications for recognition and processing of such modifications by regulatory DNA-binding proteins.

We present here the synthesis and characterization of nanoscale materials with DNA binding properties. We have functionalized graphene quantum dots (GQDs) with graphite-binding peptides to obtain graphene/peptide conjugates. The conjugates form stable 2D crystalline beta-sheet domains on the graphene surface. Upon incubation with DNA, we observe that DNA binding to the domain is directional, and the DNA preferentially aligns to the peptide direction. Upon binding the peptide-functionalized GQDs to DNA we observe an architectural role of GQDs in global and local flexibility, looping, and compacting of DNA. We use worm like chain (WLC) model to determine the persistence length of DNA in the absence and in the presence of GQDs, confirming a decreased persistence length upon GQD binding. Further, we find that the presence of peptide augments the number of DNA binding sites, as compared with exposure of bare GQDs to DNA. Our results show that peptide-modified GQDs can be a potentially ideal nanomaterial mimic of DNA-binding proteins similar to nucleosomal structures and other DNA-metabolic enzymes.

Nanopores are a promising platform in next generation DNA sequencing. In this platform, an individual DNA strand is threaded into nanopore using an electric field, and enzyme-based ratcheting is used to move the strand through the detector. During this process the residual ion current through the pore is measured, which exhibits unique levels for different base combinations inside the pore. While this approach has shown great promise, accuracy is not optimal because the four bases are chemically comparable to one another, leading to small differences in current obstruction. Nucleobase-specific chemical tagging can be a viable approach to enhancing the contrast between different bases in the sequence. Herein we show that covalent modification of one or both of the pyrimidine bases by an osmium bipyridine complex leads to measureable differences in the blockade amplitudes of DNA molecules. We qualitatively determine the degree of osmylation of a DNA strand by passing it through a solid-state nanopore, and are thus able to gauge T and C base content. In addition, we show that osmium bipyridine reacts with dsDNA, leading to substantially different current blockade levels than exhibited for bare dsDNA. This work serves as a proof of principle for nanopore sequencing and mapping via base-specific DNA osmylation.

We have developed a hybrid nanopore/zero-mode waveguide device for single-molecule fluorescence and DNA sequencing applications. The device is a freestanding solid-state membrane with sub-5 nm nanopores that reversibly delivers individual biomolecules to the base of 70 nm diameter waveguides for interrogation. Rapid and reversible molecular loading is achieved by controlling the voltage across the device. Using this device we demonstrate protein and DNA loading with efficiency that is orders of magnitude higher than diffusion-based molecular loading.

Eukaryotic DNA is packaged into nucleosomes, each comprised of ~147 base pairs of DNA wrapped 1.7 turns around histone octamers.1 Nucleosome organization inherently limits the accessibility of regulatory proteins to genes, which serves as a sophisticated mechanism to control transcription, replication, and repair processes in a cell.2 While it is known that dynamic modulation of nucleosomal structures is achieved via epigenetic modifications of histone proteins and DNA3,4 and by ATP-dependent remodelers,5 the mechanisms by which these enzyme-assisted modifications affect intranucleosomal interactions remain elusive. Methods for fast, label-free measurements of DNA-histone interactions at the single nucleosome level can greatly accelerate our understanding of the factors that modulate nucleosome stability. While valuable insight has been obtained by Forster resonance energy transfer (FRET),6-8 atomic force microscopy,9 and optical tweezers,10-14 all of these methods require time-consuming sample labeling and/or surface immobilization. Recently, Soni and co-workers reported the use of nanopores for studying chromatin sub-structures such as histone monomers, tetramers, and octamers.15 While other DNA/protein systems have been studied using solid-state nanopores,16-18 in this paper we employ force spectroscopy for studying nucleosomal structures by choosing a pore size that applies an unraveling force to the wound DNA molecule. Figure 1a displays the schematic of our solid-state nanopore setup. A nanopore in an ultrathin silicon nitride membrane connects two chambers filled with an ionic buffer solution (0.265 M KCl, 0.0825 M NaCl, 1 mM EDTA, 10 mM tris buffered to pH 7.9) that is isotonic with the eukaryotic cell nucleus.19 Voltage applied across the membrane creates a steady-state trans-membrane ion current flux that sculpts a highly localized ( 300 mV. We hypothesize that these events correspond to nucleosome unraveling. To support this, we have analyzed >6,000 events for each voltage in the range 225-350 mV for the DNA/nucleosome sample. Surprisingly, during the course of our experiments our pores did not foul with nucleosomes, which may owe to the larger size of histones with respect to our 3 nm pore. Results for sample 2 are summarized in Figure 2, in which we plot two-dimensional color maps of normalized ΔI/Iopen and td for different applied voltages. Above each scatter plot we show log-normal dwell time histograms fit to multi-Gaussian distributions. Three distinct populations, labeled as Populations 1-3, are attributed to free DNA translocation, nucleosome collisions, and nucleosome unraveling events, respectively. A minor, fast population seen for V ≤ 225 mV (td ~5 μs, ΔI/Iopen ~ 0.6) is attributed to signal artifacts due to fast DNA translocation. Figure 2 Scatter plots of current blocked vs. dwell time in the voltage range 225-350 mV (n = 6400 for each voltage) collected from a single pore, and corresponding log(dwell time) histograms. Log-normal fits (red dashed lines) are shown and errors are indicated ... Our assignment of Population 1 as free DNA translocations is supported by the systematic decrease in dwell times with voltage, with log(td) regularly declining from 1.71 to 0.65 (corresponding to td 51.3 and 4.5 μs). Increased broadening of ΔI/I in the maps for V > 250mV is due to coincidence of our signal durations with the minimum time resolution (~5 μs). While in similar experiments with pure DNA only Population 1 was observed, Populations 2 and 3 only appeared for the nucleosome sample., Population 2 appears at V = 300 mV and gradually disappears at larger voltages. Two observations suggest that events in Population 2 represent nucleosome collisions with the pore: 1) Mean td values do not decrease with increasing force (78 μs at 300 mV vs. 85 μs at 325 mV), and 2) The rate of events in Population 2 decreases with voltage. Population 3, which becomes pronounced at voltages 325 mV and above, is attributed to nucleosome unraveling by the pore. This is supported by an increase in the frequency of events in Population 3 with voltage, and a decrease in mean event duration (1.29 ms for 325 mV, 1.07 ms for 340 mV, 0.96 ms for 350 mV, 0.78 ms for 360 mV, and 0.71 ms for 370 mV see SI). To provide further rationale for our hypothesis, we have estimated the force acting on the nucleosomal DNA in the nanopore, and compared it to the literature value of the total rupture force required for complete disruption of DNA from the histone octamers (see SI for a detailed description of our assumptions). To provide an estimate of force, we have compiled a set of reported DNA translocation times23-29 and related the observed velocities to directly measured forces.30,31 Our approach yields nucleosome rupturing forces of ~10 pN for V = 325 mV and ~18 pN for V = 350 mV, in good agreement with previous reports.9,11-13 In a study by Gemmen et al,12 DNA-histone assemblies were stretched using optical tweezers at a constant rate, in which rupture occurred with sub-10 ms kinetics, similar to our rupturing rates. Mean nucleosome rupture forces decreased from 31 pN to 24 pN as monovalent salt concentrations increased from 5 mM to 100 mM. Extrapolation to our monovalent salt regime of 350 mM yields a nucleosome rupture force of ~6 pN, consistent with our study. We note that in contrast to most AFM and optical tweezers measurements, the loading rate in a typical nanopore experiment is unknown and can exceed that of established single-molecule techniques because of μs-timescale loading of a protein-DNA assembly into a pore. Molecular dynamics simulations have recently provided an in-depth description of this process,32 suggesting that direct comparison of nanopore force measurements to other single-molecule studies can be difficult. Future use of active voltage control techniques can provide more precise control over loading rates. In summary, we have shown here the reproducible (see SI) capture and unraveling of individual nucleosomes using a 3 nm diameter pore. Three force regimes were observed, as shown in Figure 3: Below 300 mV ( 7 pN), nucleosomes are captured and unraveled by the pore. Nanopore-based measurements of histone-DNA interactions are label-free and convenient. Since samples contain a resolvable mixture of free DNA and nucleosomes, unraveling forces are easily calibrated based on free DNA velocities during the experiment. This self-calibration is important in solid-state nanopore experiments, where pore size variability can substantially affect forces within the pore. Future studies can focus on mechanisms that control transcription, replication, and repair processes in a cell through modulation of DNA-histone interactions, as well as in diagnosis of diseases with abnormal patterns of DNA and histone modifications. In addition, further work will be carried out to obtain a more accurate force determination, as well as an analytical model that accounts for DNA-pore interactions and hydrodynamics. Figure 3 Scheme outlining the force-dependent interactions of nucleosomes with a nanopore.

We present a study of double- and single-stranded DNA transport through nanopores fabricated in ultrathin (2–7 nm thick) free-standing hafnium oxide (HfO2) membranes. The high chemical stability of ultrathin HfO2 enables long-lived experiments with 50 000 DNA translocations with no detectable pore expansion. Mean DNA velocities are slower than velocities through comparable silicon nitride pores, providing evidence that HfO2 nanopores have favorable physicochemical interactions with nucleic acids that can be leveraged to slow down DNA in a nanopore.

Single-molecule techniques are becoming more practical and useful in academic research and commercial healthcare devices. A prime example of this is single-molecule DNA sequencing, which is now achievable using either nanopores or single-molecule real time (SMRT) methods. In these platforms, read lengths exceed those of bulk sequencing methods by orders of magnitude, and sensitivity to epigenetic modifications is allowed in cases where DNA amplification is not necessary. However, since sequencing and epigenetic analysis require native DNA fragments, a critical part of accessing this information is loading of the DNA into a readout device with high sensitivity. While typical DNA library input requirements are in the 100's of ng, ideally one would want to probe single cell amounts, or pg-level DNA. In this work, we have utilized micro and nano fabrication to construct zero-mode waveguides that contain nanopores in their base, and show that these devices can be used for extremely sensitive DNA capture and sequencing. The device, a nanopore-zero-mode waveguide (NZMW), allows sub nanogram levels of DNA to be efficiently drawn for analysis in seconds, and does not exhibit a bias towards low-molecular weight DNA as in diffusion-based methods. We will discuss the implications of such a device on DNA sequencing, RNA sequencing, as well as other single-molecule biophysical studies where only precious samples are available.

Nanopore technology employs a nanoscale hole in an insulating membrane to stochastically sense with high throughput individual biomolecules in solution. The generality of the nanopore detection principle and the ease of single-molecule detection suggest many potential applications of nanopores in biotechnology. Recent progress has been made with nanopore fabrication and sophistication, as well as with applications in DNA/protein mapping, biomolecular structure analysis, protein detection, and DNA sequencing. In addition, concepts for DNA sequencing devices have been suggested, and computational efforts have been made. The state of the nanopore field is maturing and given the right type of nanopore and operating conditions, nearly every application could revolutionize medicine in terms of speed, cost, and quality. In this review, we summarize progress in nanopores for biotechnological applications over the past 2–3 years.

Much more than ever, nucleic acids are recognized as key building blocks in many of life's processes, and the science of studying these molecular wonders at the single-molecule level is thriving. A new method of doing so has been introduced in the mid 1990's. This method is exceedingly simple: a nanoscale pore that spans across an impermeable thin membrane is placed between two chambers that contain an electrolyte, and voltage is applied across the membrane using two electrodes. These conditions lead to a steady stream of ion flow across the pore. Nucleic acid molecules in solution can be driven through the pore, and structural features of the biomolecules are observed as measurable changes in the trans-membrane ion current. In essence, a nanopore is a high-throughput ion microscope and a single-molecule force apparatus. Nanopores are taking center stage as a tool that promises to read a DNA sequence, and this promise has resulted in overwhelming academic, industrial, and national interest. Regardless of the fate of future nanopore applications, in the process of this 16-year-long exploration, many studies have validated the indispensability of nanopores in the toolkit of single-molecule biophysics. This review surveys past and current studies related to nucleic acid biophysics, and will hopefully provoke a discussion of immediate and future prospects for the field.

Nanopore sensors have attracted considerable interest for high-throughput sensing of individual nucleic acids and proteins without the need for chemical labels or complex optics. A prevailing problem in nanopore applications is that the transport kinetics of single biomolecules are often faster than the measurement time resolution. Methods to slow down biomolecular transport can be troublesome and are at odds with the natural goal of high-throughput sensing. Here we introduce a low-noise measurement platform that integrates a complementary metal-oxide semiconductor (CMOS) preamplifier with solid-state nanopores in thin silicon nitride membranes. With this platform we achieved a signal-to-noise ratio exceeding five at a bandwidth of 1 MHz, which to our knowledge is the highest bandwidth nanopore recording to date. We demonstrate transient signals as brief as 1 μs from short DNA molecules as well as current signatures during molecular passage events that shed light on submolecular DNA configurations in small nanopores.

Nanopores have emerged as single-molecule analytic tools for fundamental biophysical characterization of nucleic acids as well as for future genomic applications. The enormous interest in single-molecule analysis has spurred the development of many different approaches to nanopore fabrication. Of these, ultrathin solid-state membranes are the most promising substrates, combining exceptional robustness and control over pore size and shape with an inherently planar geometry that enables parallel detection with nanopore arrays. Moreover, nanopores with diameters in the range of 1–5 nm represent an important size regime for studying nucleic acids, as these pores can translocate long DNA and RNA molecules in a linear fashion, enabling readout of local nucleic acid structure with unparalleled read-length. In this review, we focus on two fundamental aspects of nucleic acid analysis using nanopores, namely the process of DNA capture and the subsequent translocation dynamics. We compile here a multi-parametric study of double-stranded DNA molecules of lengths ranging from 50 to 50,000 bp, and discuss the influence of DNA length, applied voltage, temperature, and salt buffer concentrations on the capture and translocation processes.

Small RNA molecules have an important role in gene regulation and RNA silencing therapy, but it is challenging to detect these molecules without the use of time-consuming radioactive labelling assays or error-prone amplification methods. Here, we present a platform for the rapid electronic detection of probe-hybridized microRNAs from cellular RNA. In this platform, a target microRNA is first hybridized to a probe. This probe:microRNA duplex is then enriched through binding to the viral protein p19. Finally, the abundance of the duplex is quantified using a nanopore. Reducing the thickness of the membrane containing the nanopore to 6 nm leads to increased signal amplitudes from biomolecules, and reducing the diameter of the nanopore to 3 nm allows the detection and discrimination of small nucleic acids based on differences in their physical dimensions. We demonstrate the potential of this approach by detecting picogram levels of a liver-specific miRNA from rat liver RNA.

We demonstrate a purely electrical method for the single-molecule detection of specific DNA sequences, achieved by hybridizing double-stranded DNA (dsDNA) with peptide nucleic acid (PNA) probes and electrophoretically threading the DNA through sub-5 nm silicon nitride pores. Bis-PNAs were used as the tagging probes in order to achieve high affinity and sequence specificity. Sequence detection is performed by reading the ion current traces of individual translocating DNA molecules, which display a characteristic secondary blockade level, absent in untagged molecules. The potential for barcoding DNA is demonstrated through nanopore analysis of once-tagged and twice-tagged DNA at different locations on the same genomic fragment. Our high-throughput, long-read length method can be used to identify key sequences embedded in individual DNA molecules, without the need for amplification or fluorescent/radio labeling. This opens up a wide range of possibilities in human genomics as well as in pathogen detection for fighting infectious diseases.

Manipulating the drag force acting on charged biopolymers, such as DNA, during their passage through solid-state nanopores is critical for various single-molecule applications for biotechnology (e.g., sequencing, genotyping, DNA/protein interactions). We have recently shown for solid-state nanopores in silicon nitride (SiN) that small variations in the nanopore diameter, temperature, and voltage, strongly affect the biopolymer's average sliding velocity, indicating that in this size regime DNA transport is governed by DNA/pore attraction[1]. However, the exact nature of these interactions has remained to date unknown. To elucidate the character of these nanopore/DNA interactions, we have performed two independent types of experiments: First, we investigated the capture rate of DNA as a function of temperature. Surprisingly, our temperature measurements reveal anti-Arrhenius behavior, suggesting a two-step capture process. Second, we mapped the spatial distribution of charged biopolymer in the vicinity of the SiN membrane, using a custom confocal microscope equipped with a nano-positioning stage. Our measurements reveal a salt-dependent enhancement of DNA concentration in the vicinity of the SiN. These combined results suggest that electrostatic DNA/pore and DNA/membrane interactions are present between the weakly positive SiN surface and the negatively-charged DNA. In this presentation, these results are discussed and a model that explains these and previously reported observations is presented.[1] Wanunu, M. Sutin, J. McNally, B. Chow, A. and A. Meller (2008) DNA Translocation Governed by Interactions with Solid State Nanopores, Biophys. J., 95 (11), in press.

Nanopores have recently emerged as high-throughput tools for probing and manipulating nucleic acid secondary structure at the single-molecule level. While most studies to date have utilized protein pores embedded in lipid bilayers, solid-state nanopores offer many practical advantages which greatly expand the range of applications in life sciences and biotechnology. Using sub-2 nm solid-state nanopores, we show for the first time that the unzipping kinetics of individual DNA duplexes can be probed by analyzing the dwell-time distributions. We performed high-bandwidth electrical measurements of DNA duplex unzipping as a function of their length, sequence, and temperature. We find that our longer duplexes (>10 bp) follow Arrhenius dependence on temperature, suggesting that unzipping can be approximated as a single-barrier crossing, but the unzipping kinetics of shorter duplexes do not involve a barrier, due to the strong biasing electrical force. Finally, we show that mismatches in the duplex affect unzipping times in a position-sensitive manner. Our results are a crucial step towards sequence variability detection and our single-molecule nanopore sequencing technology, which rely on parallel detection from nanopore arrays.

We present experimental and theoretical results on the voltage-dependent escape dynamics of DNA hairpins of different orientations threaded into an alpha -hemolysin channel. Using a coarse-grained formulation, we map the motion of the polymer in the pore to that of a biased single-particle random walk along the translocation coordinate. By fitting the escape probability distributions obtained from theory to experimental data, we extract the voltage-dependent diffusion constants and bias-induced velocities. Using our two-parameter theory, we obtain excellent agreement with experimentally measured escape time distributions. Further, we find that the ratio of mean escape times for hairpins of different orientations is strongly voltage dependent, with the ratio of 3' - to 5' -threaded DNA decreasing from approximately 1.7 to approximately 1 with increasing assisting voltages V(a) . We also find that our model describes 5' -threaded DNA escape extremely well, while providing inadequate fits for 3' escape. Finally, we find that the escape times for both orientations are equal for high assisting voltages, suggesting that the interactions of DNA with the alpha -hemolysin channel are both orientation and voltage dependent.

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for approximately $1,000 in approximately 24 h.

Nanopores are being hailed as a potential next-generation DNA sequencer that could provide cheap, high-throughput DNA analysis. In this review we present a detailed summary of the various sensing techniques being investigated for use in DNA sequencing and mapping applications. A crucial impasse to the success of nanopores as a reliable DNA analysis tool is the fast and stochastic nature of DNA translocation. We discuss the incorporation of biological motors to step DNA through a pore base-by-base, as well as the many experimental modifications attempted for the purpose of slowing and controlling DNA transport.

Voltage-driven transport of double-stranded DNA through nanoscale pores holds much potential for applications in quantitative molecular biology and biotechnology, yet the microscopic details of translocation have proven to be challenging to decipher. Earlier experiments showed strong dependence of transport kinetics on pore size: fast regular transport in large pores (> 5 nm diameter), and slower yet heterogeneous transport time distributions in sub-5 nm pores, which imply a large positional uncertainty of the DNA in the pore as a function of the translocation time. In this work, we show that this anomalous transport is a result of DNA self-interaction, a phenomenon that is strictly pore-diameter dependent. We identify a regime in which DNA transport is regular, producing narrow and well-behaved dwell-time distributions that fit a simple drift-diffusion theory. Furthermore, a systematic study of the dependence of dwell time on DNA length reveals a single power-law scaling of 1.37 in the range of 35–20,000 bp. We highlight the resolution of our nanopore device by discriminating via single pulses 100 and 500 bp fragments in a mixture with >98% accuracy. When coupled to an appropriate sequence labeling method, our observation of smooth DNA translocation can pave the way for high-resolution DNA mapping and sizing applications in genomics.

A zero-mode waveguide (ZMW) is a nanoscale optical waveguide driven at a frequency below its cut-off. In this mode, the electric field, instead of traveling down the axis of the conducting cavity, decays exponentially. By fabricating waveguides with sub-wavelength diameters and illuminating them with laser light, the electric field in the waveguide is confined enough to enable single-molecule optical detection at micromolar concentration [1]. Immobilizing single DNA polymerases in ZMWs and using special phosphate-fluorescently labeled dNTPs form the basis for single-molecule real-time DNA sequencing, one of the most promising next-generation sequencing platforms [2]. In this method, the polymerase replicates the sample DNA, and as it incorporates new bases into the product strand, the labeled dNTPs emit a burst of light before the phosphate is cleaved off. The sequence of colors corresponds to the DNA sequence (see Figure 1 below from Eid et al., 2009). Because the ZMW aperture’s diameter is sub-diffracti...

We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7–5nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150–3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.

We report on DNA translocations through nanopores created in graphene membranes. Devices consist of 1-5 nm thick graphene membranes with electron-beam sculpted nanopores from 5 to 10 nm in diameter. Due to the thin nature of the graphene membranes, we observe larger blocked currents than for traditional solid-state nanopores. However, ionic current noise levels are several orders of magnitude larger than those for silicon nitride nanopores. These fluctuations are reduced with the atomic-layer deposition of 5 nm of titanium dioxide over the device. Unlike traditional solid-state nanopore materials that are insulating, graphene is an excellent electrical conductor. Use of graphene as a membrane material opens the door to a new class of nanopore devices in which electronic sensing and control are performed directly at the pore.