Single polypeptide detection using a translocon EXP2 nanopore

DNA sequencing using nanopores has already been achieved and commercialized; the next step in advancing nanopore technology is towards protein sequencing. Although trials have been reported for discriminating the 20 amino acids using biological nanopores and short peptide carriers, it remains challenging. The size compatibility between nanopores and peptides is one of the issues to be addressed. Therefore, exploring biological nanopores that are suitable for peptide sensing is key in achieving amino acid sequence determination. Here, we focus on EXP2, the transmembrane protein of a translocon from malaria parasites, and describe its pore-forming properties in the lipid bilayer. EXP2 mainly formed a nanopore with a diameter of 2.5 nm assembled from 7 monomers. Using the EXP2 nanopore allowed us to detect poly-L-lysine (PLL) at a single-molecule level. Furthermore, the EXP2 nanopore has sufficient resolution to distinguish the difference in molecular weight between two individual PLL, long PLL (Mw: 30,000–70,000) and short PLL (Mw: 10,000). Our results contribute to the accumulation of information for peptide-detectable nanopores.     

Mechanisms of pressure-induced water infiltration process through graphene nanopores

Understanding the mechanism of water infiltration through nanopores is essential for wide applications ranging from membrane separation to gene therapy. In this paper, the molecular dynamics simulation method is used to investigate the pressure-assisted water transport process through graphene nanopores. Various factors including the hydrophobicity of nanopore surface, nanopore dimension, temperature as well as external electric field that affect water in permeation into graphene nanopores are discussed. It is found that classic Laplace-Young equation fails and the relationship between pressure and diameter (D) does not follow the 1/D dependence as the characteristic dimension of a nanopore is sufficiently small (smaller than 1?nm). The critical pressure significantly depends on both the pore length and electric field as D is smaller than 5?nm. Besides, enhancing temperature and electric field intensity are obviously beneficial for water infiltration through those nanopores with a diameter smaller than 5?nm.     

On possible trypsin-induced biases in peptides analysis with aerolysin nanopore

Nanopore-based single-molecule analysis technique is a promising approach in the field of proteomics. In this Technical Brief, the interaction between the biological nanopore of Aerolysin (AeL) and peptides is investigated, focusing on potential biases depending on the AeL activation protocol. Our results reveal that residual trypsin, which may be unintentionally introduced in analyte solution when using a classical AeL activation protocol, can induce a significant formation of shorter peptides by enzymatic degradation of longer ones, which may lead to unwanted effects and/or misinterpretations. AeL free-trypsin activation protocol eliminates this bias and appears more appropriate for peptide/proteins analysis, specifically in the perspective of nanopore-based molecular fingerprinting or of low-abundance species characterization.     

Enhanced identification of Tau acetylation and phosphorylation with an engineered aerolysin nanopore

Posttranslational modifications (PTMs) affect protein function/dysfunction, playing important roles in the occurrence and development of tauopathies including Alzheimer's disease. PTM detection is significant and still challenging due to the requirements of high sensitivity to identify the subtle structural differences between modifications. Herein, in terms of the unique geometry of the aerolysin (AeL) nanopore, we elaborately engineered a T232K AeL nanopore to detect the acetylation and phosphorylation of Tau segment (Pep). By replacing neutral threonine (T) with positively charged lysine (K) at the 232 sites, the T232K and K238 rings of this engineered T232K AeL nanopore corporately work together to enhance electrostatic trapping of the acetylated and phosphorylated Tau peptides. Translocation speed of the monophosphorylated Pep-P was decelerated by up to 46 folds compared to the wild-type (WT) AeL nanopore. The prolonged residences within the T232K AeL nanopore enabled to simultaneously identify the monoacetylated Pep-Ac, monophosphorylated Pep-P, di-modified Pep-P-Ac and non-modified Pep. The tremendous potential is demonstrated for PTM sensing by manipulating non-covalent interactions between nanopores and single analytes.     

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins¹. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low^2,3 and size control is nontrivial^4,5. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.     

Developing synthetic conical nanopores for biosensing applications

In this review we bring together recent results from our group focused towards the development of biosensors from single conically-shaped artificial nanopores. The nanopores, used in the work presented here, were prepared using the track-etch process. The fabrication of track-etched conical nanopores has been optimized to allow for single nanopores with reproducible dimensions to be prepared. We have also demonstrated techniques that allow for easy and controllable manipulation of nanopore geometry (e.g., cone angle). We will consider the ion transport properties of the conical nanopores and factors that affect these properties. Methods for introducing functions that mimic biological ion channels, such as voltage-gating, into these nanopores will also be addressed. Three prototype sensors developed from single conical nanopores will be presented. In the first two sensors, the single conical nanopores function as resistive-pulse sensors and detect the presence of analytes as current-blockade events in the ion current. The third sensor functions in an on/off mode, much like a ligand-gated ion channel. In the presence of a target analyte, the ion current permanently shuts off.     

Genomic Pathogen Typing Using Solid-State Nanopores

In clinical settings, rapid and accurate characterization of pathogens is essential for effective treatment of patients; however, subtle genetic changes in pathogens which elude traditional phenotypic typing may confer dangerous pathogenic properties such as toxicity, antibiotic resistance, or virulence. Existing options for molecular typing techniques characterize the critical genomic changes that distinguish harmful and benign strains, yet the well-established approaches, in particular those that rely on electrophoretic separation of nucleic acid fragments on a gel, have room for only incremental future improvements in speed, cost, and complexity. Solid-state nanopores are an emerging class of single-molecule sensors that can electrophoretically characterize charged biopolymers, and which offer significant advantages in terms of sample and reagent requirements, readout speed, parallelization, and automation. We present here the first application of nanopores for single-molecule molecular typing using length based “fingerprints” of critical sites in bacterial genomes. This technique is highly adaptable for detection of different types of genetic variation; as we illustrate using prototypical examples including Mycobacterium tuberculosis and methicillin-resistant Streptococcus aureus, the solid-state nanopore diagnostic platform may be used to detect large insertions or deletions, small insertions or deletions, and even single-nucleotide variations in bacterial DNA. We further show that Bayesian classification of test samples can provide highly confident pathogen typing results based on only a few tens of independent single-molecule events, making this method extremely sensitive and statistically robust.     

How Nanopore Translocation Experiments Can Measure RNA Unfolding

Electrokinetic translocation of biomolecules through solid-state nanopores represents a label-free single-molecule technique that may be used to measure biomolecular structure and dynamics. Recent investigations have attempted to distinguish individual transfer RNA (tRNA) species based on the associated pore translocation times, ion-current noise, and blockage currents. By manufacturing sufficiently smaller pores, each tRNA is required to undergo a deformation to translocate. Accordingly, differences in nanopore translocation times and distributions may be used to infer the mechanical properties of individual tRNA molecules. To bridge our understanding of tRNA structural dynamics and nanopore measurements, we apply molecular dynamics simulations using a simplified “structure-based” energetic model. Calculating the free-energy landscape for distinct tRNA species implicates transient unfolding of the terminal RNA helix during nanopore translocation. This provides a structural and energetic framework for interpreting current experiments, which can aid the design of methods for identifying macromolecules using nanopores.     

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

Solid-state nanopores have been used extensively in biomolecular studies involving DNA and proteins. However, the interpretation of signals generated by the translocation of proteins or protein-DNA complexes remains challenging. Here, we investigate the behavior of monovalent streptavidin and the complex it forms with short biotinylated DNA over a range of nanopore sizes, salts, and voltages. We describe a simple geometric model that is broadly applicable and employ it to explain observed variations in conductance blockage and dwell time with experimental conditions. The general approach developed here underscores the value of nanopore-based protein analysis and represents progress toward the interpretation of complex translocation signals.     

Distinguishable populations report on the interactions of single DNA molecules with solid-state nanopores

Solid-state nanopores have received increasing interest over recent years because of their potential for genomic screening and sequencing. In particular, small nanopores (2-5 nm in diameter) allow the detection of local structure along biological molecules, such as proteins bound to DNA or possibly the secondary structure of RNA molecules. In a typical experiment, individual molecules are translocated through a single nanopore, thereby causing a small deviation in the ionic conductance. A correct interpretation of these conductance changes is essential for our understanding of the process of translocation, and for further sophistication of this technique. Here, we present translocation measurements of double-stranded DNA through nanopores down to the diameter of the DNA itself (1.8-7 nm at the narrowest constriction). In contrast to previous findings on such small nanopores, we find that single molecules interacting with these pores can cause three distinct levels of conductance blockades. We attribute the smallest conductance blockades to molecules that briefly skim the nanopore entrance without translocating, the intermediate level of conductance blockade to regular head-to-tail translocations, and the largest conductance blockades to obstruction of the nanopore entrance by one or multiple (duplex) DNA strands. Our measurements are an important step toward understanding the conductance blockade of biomolecules in such small nanopores, which will be essential for future applications involving solid-state nanopores.     

Ion Current Rectification,Limiting and Overlimiting Conductances in Nanopores

Previous reports on Poisson-Nernst-Planck (PNP) simulations of solid-state nanopores have focused on steady state behaviour under simplified boundary conditions. These are Neumann boundary conditions for the voltage at the pore walls, and in some cases also Donnan equilibrium boundary conditions for concentrations and voltages at both entrances of the nanopore. In this paper, we report time-dependent and steady state PNP simulations under less restrictive boundary conditions, including Neumann boundary conditions applied throughout the membrane relatively far away from the nanopore. We simulated ion currents through cylindrical and conical nanopores with several surface charge configurations, studying the spatial and temporal dependence of the currents contributed by each ion species. This revealed that, due to slow co-diffusion of oppositely charged ions, steady state is generally not reached in simulations or in practice. Furthermore, it is shown that ion concentration polarization is responsible for the observed limiting conductances and ion current rectification in nanopores with asymmetric surface charges or shapes. Hence, after more than a decade of collective research attempting to understand the nature of ion current rectification in solid-state nanopores, a relatively intuitive model is retrieved. Moreover, we measured and simulated current-voltage characteristics of rectifying silicon nitride nanopores presenting overlimiting conductances. The similarity between measurement and simulation shows that overlimiting conductances can result from the increased conductance of the electric double-layer at the membrane surface at the depletion side due to voltage-induced polarization charges. The MATLAB source code of the simulation software is available via the website http://micr.vub.ac.be.     

Peptide Nanopores and Lipid Bilayers: Interactions by Coarse-Grained Molecular-Dynamics Simulations

A set of 49 protein nanopore-lipid bilayer systems was explored by means of coarse-grained molecular-dynamics simulations to study the interactions between nanopores and the lipid bilayers in which they are embedded. The seven nanopore species investigated represent the two main structural classes of membrane proteins (α-helical and β-barrel), and the seven different bilayer systems range in thickness from ∼28 to ∼43 Å. The study focuses on the local effects of hydrophobic mismatch between the nanopore and the lipid bilayer. The effects of nanopore insertion on lipid bilayer thickness, the dependence between hydrophobic thickness and the observed nanopore tilt angle, and the local distribution of lipid types around a nanopore in mixed-lipid bilayers are all analyzed. Different behavior for nanopores of similar hydrophobic length but different geometry is observed. The local lipid bilayer perturbation caused by the inserted nanopores suggests possible mechanisms for both lipid bilayer-induced protein sorting and protein-induced lipid sorting. A correlation between smaller lipid bilayer thickness (larger hydrophobic mismatch) and larger nanopore tilt angle is observed and, in the case of larger hydrophobic mismatches, the simulated tilt angle distribution seems to broaden. Furthermore, both nanopore size and key residue types (e.g., tryptophan) seem to influence the level of protein tilt, emphasizing the reciprocal nature of nanopore-lipid bilayer interactions.     

Direct Sensing and Discrimination among Ubiquitin and Ubiquitin Chains Using Solid-State Nanopores

Nanopore sensing involves an electrophoretic transport of analytes through a nanoscale pore, permitting label-free sensing at the single-molecule level. However, to date, the detection of individual small proteins has been challenging, primarily due to the poor signal/noise ratio that these molecules produce during passage through the pore. Here, we show that fine adjustment of the buffer pH, close to the isoelectric point, can be used to slow down the translocation speed of the analytes, hence permitting sensing and characterization of small globular proteins. Ubiquitin (Ub) is a small protein of 8.5 kDa, which is well conserved in all eukaryotes. Ub conjugates to proteins as a posttranslational modification called ubiquitination. The immense diversity of Ub substrates, as well as the complexity of Ub modification types and the numerous physiological consequences of these modifications, make Ub and Ub chains an interesting and challenging subject of study. The ability to detect Ub and to identify Ub linkage type at the single-molecule level may provide a novel tool for investigation in the Ub field. This is especially adequate because, for most ubiquitinated substrates, Ub modifies only a few molecules in the cell at a given time. Applying our method to the detection of mono- and poly-Ub molecules, we show that we can analyze their characteristics using nanopores. Of particular importance is that two Ub dimers that are equal in molecular weight but differ in 3D structure due to their different linkage types can be readily discriminated. Thus, to our knowledge, our method offers a novel approach for analyzing proteins in unprecedented detail using solid-state nanopores. Specifically, it provides the basis for development of single-molecule sensing of differently ubiquitinated substrates with different biological significance. Finally, our study serves as a proof of concept for approaching nanopore detection of sub-10-kDa proteins and demonstrates the ability of this method to differentiate among native and untethered proteins of the same mass.     

Voltage-Driven Translocation of DNA through a High Throughput Conical Solid-State Nanopore

Nanopores have become an important tool for molecule detection at single molecular level. With the development of fabrication technology, synthesized solid-state membranes are promising candidate substrates in respect of their exceptional robustness and controllable size and shape. Here, a 30–60 (tip-base) nm conical nanopore fabricated in 100 nm thick silicon nitride (Si₃N₄) membrane by focused ion beam (FIB) has been employed for the analysis of λ-DNA translocations at different voltage biases from 200 to 450 mV. The distributions of translocation time and current blockage, as well as the events frequencies as a function of voltage are investigated. Similar to previously published work, the presence and configurations of λ-DNA molecules are characterized, also, we find that greater applied voltages markedly increase the events rate, and stretch the coiled λ-DNA molecules into linear form. However, compared to 6–30 nm ultrathin solid-state nanopores, a threshold voltage of 181 mV is found to be necessary to drive DNA molecules through the nanopore due to conical shape and length of the pore. The speed is slowed down ∼5 times, while the capture radius is ∼2 fold larger. The results show that the large nanopore in thick membrane with an improved stability and throughput also has the ability to detect the molecules at a single molecular level, as well as slows down the velocity of molecules passing through the pore. This work will provide more motivations for the development of nanopores as a Multi-functional sensor for a wide range of biopolymers and nano materials.     

The nanopore connection to cell membrane unitary permeability

Artificial nanopores have recently emerged as versatile tools for analyzing and sorting single molecules at high speed. However, the biological cell has already developed a large set of sophisticated protein nanopores that are able to selectively translocate all types of molecules through membranes. Therefore, hybrid devices combining artifical solid-state with biomimetic protein nanopores appear to us as a particularly promising approach to the creation of powerful diagnostic, preparative and therapeutic devices. Here, we discuss a technique, optical single-transporter recording (OSTR), in which arrays of artificial micropores and nanopores are employed to analyze protein nanopores of cellular membranes. After briefly summarizing some salient features of OSTR, the technique is compared with the electrical patch clamp method and the first results of our efforts to amalgamate optical and electrical recording are described. Finally, prospects for combining OSTR with 4Pi microscopy, single-molecule fluorescence spectroscopy and fluorescence correlation spectroscopy are discussed.     

Nanopore analysis reveals differences in structural stability of ovine PrPC proteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrP^C). Species barriers to transmission also reflect PrP^C sequence differences. Defining the structure-activity relationship of PrP^C/PrP^Sc with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrP^C corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrP^Sc-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrP^Sc-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrP^Sc-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.     

Integrated nanopore sensing platform with sub-microsecond temporal resolution

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.     

209 Designing stiff protein nanopores for challenging tasks in biosensing

Protein nanopore-based sensing elements represent a pressing need in molecular biomedical diagnosis (Bayley & Cremer, 2001). However, the integration of protein nanopores with other nanofluidic devices is a challenging task. This is especially true, if we consider that isolated single proteins are in general fragile and unstable under harsh conditions of detection. Here, I will present a strategy for improving the stability of the open-state current of a redesigned nanopore using ferric hydroxamate uptake component A (FhuA), a beta-barrel membrane protein channel of E. coli (Mohammad et al., 2012). The primary function of FhuA is to facilitate the energy-driven, high-affinity Fe³⁺ uptake complexed by the siderophore ferrichrome (Paweleket al., 2006). The key ingredient of this strategy was the coupling of direct genetic engineering of FhuA with a fast-dilution refolding approach to obtain an unusually stable protein nanopore under a broad range of experimentation. These advantageous characteristics were recently demonstrated by examining proteolytic activity of an enzyme at a highly acidic pH, a condition at which majority of beta-barrel protein nanopores are normally gated or unfolded. Future membrane protein design work will not only reveal a better understanding of the processes employed in membrane protein folding and stability, but will also serve as a platform for the integration of robust protein components into devices (Astier, Bayley, & Howorka, 2005).     

Single-Stranded DNA within Nanopores: Conformational Dynamics and Implications for Sequencing; a Molecular Dynamics Simulation Study

Engineered protein nanopores, such as those based on α-hemolysin from Staphylococcus aureus have shown great promise as components of next-generation DNA sequencing devices. However, before such protein nanopores can be used to their full potential, the conformational dynamics and translocation pathway of the DNA within them must be characterized at the individual molecule level. Here, we employ atomistic molecular dynamics simulations of single-stranded DNA movement through a model α-hemolysin pore under an applied electric field. The simulations enable characterization of the conformations adopted by single-stranded DNA, and allow exploration of how the conformations may impact on translocation within the wild-type model pore and a number of mutants. Our results show that specific interactions between the protein nanopore and the DNA can have a significant impact on the DNA conformation often leading to localized coiling, which in turn, can alter the order in which the DNA bases exit the nanopore. Thus, our simulations show that strategies to control the conformation of DNA within a protein nanopore would be a distinct advantage for the purposes of DNA sequencing.