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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.

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5 nm diameter pore we find that, in a threshold electric field regime of ∼30-100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5 nm and 2.0 nm diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens new avenues for exploring protein folding structures, internal contacts, and electric field-induced deformability. Significance Statement Can localized electric fields drive the complete unfolding of a protein molecule? Protein unfolding prior to its translocation through a nanopore constriction is an important step in protein transport across biological membranes and also an important step in nanopore-based protein sequencing. We studied here the electric-field-driven translocation behavior of a model protein (cyt c) through nanopores of diameters ranging from 1.5 to 5.5 nm. These single molecule measurements show that electric fields at the nanopore constriction can select both partially and fully unfolded protein conformations. Zero-field free energy gaps between these conformations, found using a simple thermodynamic model, are in remarkable agreement with previously reported studies of cyt c unfolding energetics.

High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 μs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D0) by a factor of ∼50, and are voltage-independent in the regime tested. We reason that deviations of D from D0 are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of μ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved.

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.

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.

In recent years, nanopores have emerged as exceptionally promising single-molecule sensors due to their ability to detect biomolecules at subfemtomole levels in a label-free manner. Development of a high-throughput nanopore-based biosensor requires multiplexing of nanopore measurements. Electrical detection, however, poses a challenge, as each nanopore circuit must be electrically independent, which requires complex nanofluidics and embedded electrodes. Here, we present an optical method for simultaneous measurements of the ionic current across an array of solid-state nanopores, requiring no additional fabrication steps. Proof-of-principle experiments are conducted that show simultaneous optical detection and characterization of ssDNA and dsDNA using an array of pores. Through a comparison with electrical measurements, we show that optical measurements are capable of accessing equivalent transmembrane current information.

Nanopores have garnered much scientific interest due for their applications in modeling biological systems and as use for biosensors. Their size creates a system in which transported ions must interact with the pore walls. These nanoscale effects have led to the devolvement of ion controlling devices such as diodes and transistors with a broad range of applications. Past studies of ionic diodes have mainly focused on lateral dimension dependencies on rectification while keeping the length in the micrometer range. The ability to shorten nanopore diodes while retaining their rectification abilities has important applications to the production of ionic circuits, artificial cells, and lab-on-a-chip systems. We look at the rectification abilities of 30 nm long nanopores drilled by the transmission electron microscope (TEM) into a Silicon Nitride membrane between ±1V at 10 mM, 100 mM, and 1 M KCl. We perform current-voltage (I-V) measurements on single nanopores in 3 states: as-prepared by TEM, one side evaporated with 8 nm thick layer of gold, and after modifying the gold layer with thiols. In the two modified states, the pores show rectification at all concentrations. The rectification direction indicates that the pores rectify as if conical rather than cylindrical indicating the double conical nature of TEM drilled nanopores plays a significant role at the nanometer scale. Additionally, with only gold, the rectification and current magnitudes are enhanced due to nonlinear kinetic flow near one of the pore openings. The recorded current-voltage curves exhibit three regimes with ohmic, limiting and overlimiting currents, previously observed in nanofluidic large-aspect ratio channels. Rectification in nanometer long pores would be of significant importance to the future development and miniaturization of ionic nanoscale systems.

Intracellular RNA molecules adopt a wide variety of 3D structures, many of which have been studied using high-resolution structural techniques such as X-ray crystallography and NMR. However, while canonical structural features for a class of RNA molecules are assumed based on such studies, structural variations within a class are extremely difficult to detect, as only a limited set of structures are available. For example, the number of unique transfer-RNA (tRNA) molecules in eukaryotes can be ∼100, not including variant products of post-transcriptional modification. It has been hypothesized that tRNA accommodation in the ribosome during translation is modulated by tRNA stiffness, which in turn affects the translation kinetics. Despite this, no experimental studies that probe tRNA stiffness are available. In this work, we electrophoretically drive individual tRNA molecules through ∼3nm diameter pores in order to study their deformation kinetics during translocation. Surprisingly, we find that all six tRNAs studied here exhibit unique translocation kinetics, which we believe correspond to variations in their mechanical properties. Implementation of machine learning algorithms reveal a multidimensional set of parameters that can be used to ascertain the identity of a tRNA from a single ionic current pulse with 70-90% accuracy. Our results pave the way for a deeper understanding of RNA tertiary structure and its dependence on post-transcriptional modifications.

Solid-state nanopores have emerged as a useful tool for studying biopolymers. The ability to map regions of interest along stretched biopolymers, as well as to detect subtle changes in conformation, makes nanopores attractive single-molecule sensors. Using solid-state nanopores with diameters in the range of 3-10 nm, DNA transport has been too fast but seems to be more regulated. In contrast, transport through smaller pores in the 3-5 nm range is noticeably slower, yet sticking and other interactions complicate the transport dynamics. Therefore, a full understanding of the translocation process requires exploring regimes where polymer linearization is coupled to regulated transport. In this study, we report on an interesting nanopore geometry regime that allows for regulated DNA transport process, while slowing down DNA in order to achieve true detection from individual base pairs. We will show that transport matches theoretical prediction in this geometric regime, and that these results allow us to obtain quantitative information on polymer length with unprecedented detail.

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.

Solid-state nanopore has been proved as a versatile method for label-free single molecule detection with fairly high resolution. Protein detection using solid-state nanopores is generally challenging because its globular shape, compared with worm-shape DNA, leads to fast transit times which are confounded with limited bandwidth. One strategy is to use a comparable size solid-state nanopore to the size of protein; the confinement effect of a smaller nanopore achieves slow protein protein transit times and better resolution. However, certain proteins such as adenylate kinase (AdK) and dihydrofolate reductase (DHFR) stick to pores that are of similar diameters to the proteins, making it challenging to acquire sufficient translocation events prior to clogging. With larger pores, however, many events are missed due to fast protein translocation times. In this report, we overcome this issue by developing a setup for driving proteins by employing two driving forces, namely pressure-driven flow and an electrical potential across the pore. Using this method, we can tune protein transit times and maintain a high signal-to-noise, even for pores that are 1.5 times larger than the protein size. Control is highlighted by detecting the large conformational change of AdK upon binding a lock-substrate, as well as by detecting a smaller conformational change in DHFR upon binding the anti-cancer drug methotrexate.

Nanopore-based detection of macromolecules has gained interest in recent years, because of the enormous potential of single-molecule analysis at ultra-low analyte concentrations. In this work, we describe how nanopores can be used to trap, manipulate, and study individual bimolecular complexes with control over the applied force and other experimental parameters, such as temperature and pressure. Key to our results is the combination of ultrathin nanopores and high-bandwidth electronics, which together enable high-resolution measurements. In addition, characterizing the pore's surface charge enables more quantitative assessment of the role of electroosmosis on molecular capture and transport. We will discuss specific examples of molecular transport on well-characterized nanopores in different materials. Our results represent a key advance towards more quantitative macromolecular characterization at the single-molecule level.

Accompanying the diverse palette of functions exhibited by proteins is a rich set of structural properties. For example, whereas a particular protein function such as electron transfer requires well-positioned residues in space to provide precise interatomic distances, many enzymes require conformational flexibility to facilitate substrate recognition and catalysis. While crystallography provides atomic-level structural information, there are no rival techniques that can analyze the size and extent of structural diversity in proteins in their solution environment.

We present a study of designed self-assembly of 2D peptide monolayer crystals on the surface of graphene and graphitic interfaces and their properties in various biologically significant conditions. Atomic force microscopy imaging of dried peptides adsorbed on graphitic surfaces reveals an amorphous monolayer structure that contains voids due to drying. After rehydration, the peptide monolayer reorganizes into highly ordered domains comprised by parallel arranged peptides that are oriented on the graphitic structure with C3 symmetry, in close agreement with computational predictions.

Carbon nanotube porins (CNTPs), ultra-short carbon nanotubes (5-20nm) that can self-insert into lipid bilayers, allow single-channel planar lipid bilayer measurements of ionic transport through carbon nanotube (CNT) pores. CNTs with sub-nanometer diameters have been predicted to display ultra-fast water transport and high levels of ionic exclusion resulting from one-dimensional water wire transport through their hydrophobic cores. Here, we show that ultra-short 0.8-nm-diameter CNTPs, can spontaneously insert into lipid bilayers to form highly cation selective channels that exhibit low ionic conductance. Using reversal potential in various asymmetric salt conditions, we observe that these CNTPs exhibit large permselectivity values, corresponding to a high selectivity for both potassium and sodium over chloride. We also show evidence that neutralization of charges on the CNTs enhances ion selectivity. We also show that by using a solid-state nanopore to act as a support and form a completely solvent-free bilayer system, we can observe extremely stable ionic currents through these nanotubes, in contrast to previous reports with solvent containing bilayers. These results establish CNTPs as a promising biomimetic platform for developing cell interfaces, creating stochastic sensors, and water desalination.

Carbon nanotubes (CNTs) have narrow hydrophobic inner pores, highly reminiscent of protein channels, that enable fast transport of water and ions. Their rigid structure and chemical robustness allow for diverse ex vivo applications. We have recently reported biomimetic membrane channels cased on carbon nanotubes—carbon nanotube porins (CNTPs). We have shown that CNTPs self-insert into lipid membranes and form stable pores. In this study, we used electrophysiological single-channel measurements to quantify ion conductance and selectivity of 0.8 nm diameter CNTPs. We report the conductance of single CNTP channels, as well as the scaling of the conductance with the ion concentration. We also used the reversal potential measurements to characterize the selectivity of the ion transport and found that CNTPs are highly selective to cations over anions. We will discuss the mechanism of this selectivity, and demonstrate ways to control it. These findings are important and relevant for developing CNTPs into a potential candidate nanopore for water treatment applications. Our studies on intrinsic characterization of CNTP transport properties will also enable researchers to consider this new material for a variety of biophysical applications.

Nanopores are attractive electrochemical sensors for their single-molecule sensitivity and simple experimental setup. Many of the electronic systems used for nanopore measurements are inherited from electrophysiology applications, and as a result they are primarily optimized for current levels of 50pA or less, at bandwidths below 10kHz. However, state-of-the-art solid-state nanopore sensors now commonly produce signals in the nanoampere range, for which common patch clamp amplifiers are no longer ideal. We present a new experimental platform which integrates a custom CMOS preamplifier with nanopores in thin silicon nitride membranes. The low parasitic capacitance of the integrated platform reduces noise at high frequencies, allowing nanopore measurements at finer temporal resolution than supported by popular instruments. We demonstrate the viability of this system with high-bandwidth electrical recordings of the passage of short DNA molecules through nanopores. This new platform will improve the signal-to-noise ratios of nanopore sensors, enabling further single-molecule studies of fast biophysical processes.

Molecular-sized holes through ultrathin membranes are simple yet powerful reporters of biomolecular structure. In the regime where the nanopore size approaches the critical dimension of a biomolecule, nanopores are extremely useful as sensors with discrimination capabilities. In this presentation, I will show how synthetic nanopores with a length comparable with lipid membranes enables a precise discrimination among small biomolecules. I will demonstrate how these pores can detect a specific microRNA extracted from a biological sample, as well as examples in which discrimination among biomolecules was achieved. Also, I will describe the use of nanopores as discrimination among biomolecules that have slight chemical alterations.View Large Image | View Hi-Res Image | Download PowerPoint Slide

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.