Background:The intrinsically disordered protein ␣-synuclein, a hallmark of Parkinson disease, is involved in mitochondrial dysfunction in neurodegeneration and directly interacts with mitochondria. Results: ␣-Synuclein regulates VDAC permeability; ␣-synuclein toxicity in yeast depends on VDAC. Conclusion: ␣-Synuclein both blocks VDAC and translocates via this channel across the mitochondrial outer membrane. Significance: (Patho)physiological roles of monomeric ␣-synuclein may originate from its interaction with VDAC.

We identify a contribution to the ionic current noise spectrum in solid-state nanopores that exceeds all other noise sources in the frequency band 0.1-10 kHz. Experimental studies of the dependence of this excess noise on pH and electrolyte concentration indicate that the noise arises from surface charge fluctuations. A quantitative model based on surface functional group protonization predicts the observed behaviors and allows us to locally measure protonization reaction rates. This noise can be minimized by operating the nanopore at a deliberately chosen pH.As nanodevices are increasingly used for single-molecule detection and manipulation in the time domain, their noise characteristics at kHz frequencies gain additional significance. In the case of protein nanopores, noise has elucidated both pore and analyte dynamics [1][2][3]; in solidstate nanopores to date, noise has simply interfered with detection of DNA and proteins [4][5][6][7][8][9]. Previous work has described a variety of noise sources in solid-state nanopores and typical measuring apparatus [10][11][12].In this Letter, we identify a new source of current noise in solid-state nanopores that is dominant in the frequency range from 0.1-10 kHz. A simple analysis of the noise dependence on pH and the electrolyte ionic strength, based on a few general assumptions, suggests that the noise originates from surface charge fluctuations. Finally, we develop a quantitative model based on protonization of surface functional groups [1] and compare it to the experimental results. Our technique is sensitive to a few tens of active surface groups in the nanopore, allowing us to observe variations in local surface properties from nanopore to nanopore and to guide optimization of single-molecule detectors.Nanopores were fabricated with a transmission electron microscope [13] in a thin film of lowstress, amorphous silicon nitride, resulting in single, roughly hourglass-shaped [14] channels 55 nm long and ~4 nm in diameter at the smallest point. The silicon nitride film containing the nanopore separates two reservoirs of potassium chloride electrolyte in a sealed fluidic cell; current measurements were made between Ag/AgCl electrodes in each reservoir (Fig. 1). Noise measurements on different nanopores show the same general features and pH spectral shapes, while the sensitivity and local nature of the technique reveals sample-to-sample variability [15]. The noise sources we observe appear to be intrinsic to the silicon nitride nanopore surface and are not as variable as 1/f noise [12].The power spectral densities (PSDs) of typical current recordings (Fig. 1, inset) demonstrate various expected noise sources: with no applied voltage bias (lower spectrum), only thermal (Johnson) noise (thick dashed line) and high-frequency capacitive noise driven by the preamplifier input are present. With applied voltage bias across the nanopore device (upper spectrum), conductance fluctuations such as 1/f noise (dotted line) become apparent. We also observe the appearance of an...

We describe experiments and modeling results that reveal and explain the distribution of times that identical double-stranded DNA (dsDNA) molecules take to pass through a voltage-biased solid-state nanopore. We show that the observed spread in this distribution is caused by viscous-drag-induced velocity fluctuations that are correlated with the initial conformation of nanopore-captured molecules. This contribution exceeds that due to diffusional Brownian motion during the passage. Nevertheless, and somewhat counterintuitively, the diffusional Brownian motion determines the fundamental limitations of rapid DNA strand sequencing with a nanopore. We model both diffusional and conformational fluctuations in a Langevin description. It accounts well for passage time variations for DNA molecules of different lengths, and predicts conditions required for low-error-rate nanopore-strand DNA sequencing with nanopores.

Voltage-biased solid-state nanopores are well established in their ability to detect and characterize single polymers, such as DNA, in electrolytes. The addition of a pressure gradient across the nanopore yields a second molecular driving force that provides new freedom for studying molecules in nanopores. In this work, we show that opposing pressure and voltage bias enables nanopores to detect and resolve very short DNA molecules, as well as to detect near-neutral polymers.

A detailed understanding of the origin of the electrophoretic force on DNA molecules in a solid-state nanopore is important for the development of nanopore-based sequencing technologies. Because of the discrepancies between recent attempts to predict this force and both direct and indirect experimental measurements, this topic has been the focus of much recent discussion. We show that the force is predictable to very good accuracy if all of the experimental conditions are accounted for properly. To resolve this issue, we compare the calculation efficiency and accuracy of numerical solutions of Poisson-Boltzmann and Poisson-Nernst-Planck descriptions of electrolyte behavior in the nanopore in the presence of DNA molecules. Two geometries--axially symmetric and cross-sectional--are compared and shown to be compatible. Numerical solutions are carried out on a sufficiently fine mesh to evaluate the viscous drag force acting on DNA inside a silicon nitride nanopore. By assuming the DNA is immobilized in the axial center of the nanopore, the calculation result of this viscous drag force is found to be rather larger than the experimental result. Because the viscous drag force decreases if DNA is closer to the surface of the nanopore, however, the relevant effective driving force is the average over all possible positions of the DNA in the nanopore. When this positional uncertainty is taken into account, the effective driving force acting on DNA inside the nanopore is found to agree very well with the experimental results.

We report the formation of a tunable single DNA molecule trap near a solid-state nanopore in an electrolyte solution under conditions where an electric force and a pressure-induced viscous flow force on the molecule are nearly balanced. Trapped molecules can enter the pore multiple times before escaping the trap by passing through the pore or by diffusing away. Statistical analysis of many individually trapped molecules yields a detailed picture of the fluctuation phenomena involved, which are successfully modeled by a one-dimensional first passage approach.

A growing number of new technologies are supported by a single- or multi-nanopore architecture for capture, sensing, and delivery of polymeric biomolecules. Nanopore-based single-molecule DNA sequencing is the premier example. This method relies on the uniform linear charge density of DNA, so that each DNA strand is overwhelmingly likely to pass through the nanopore and across the separating membrane. For disordered peptides, folded proteins, or block copolymers with heterogeneous charge densities, by contrast, translocation is not assured, and additional strategies to monitor the progress of the polymer molecule through a nanopore are required. Here, we demonstrate a single-molecule method for direct, model-free, real-time monitoring of the translocation of a disordered, heterogeneously charged polypeptide through a nanopore. The crucial elements are two "selectivity tags"-regions of different but uniform charge density-at the ends of the polypeptide. These affect the selectivity of the nanopore differently and enable discrimination between polypeptide translocation and retraction. Our results demonstrate exquisite sensitivity of polypeptide translocation to applied transmembrane potential and prove the principle that nanopore selectivity reports on biopolymer substructure. We anticipate that the selectivity tag technique will be broadly applicable to nanopore-based protein detection, analysis, and separation technologies, and to the elucidation of protein translocation processes in normal cellular function and in disease.

We probe the energy landscape governing the passage of α-synuclein, a natural "diblock copolymer"-like polypeptide, through a nanoscale pore. α-Synuclein is an intrinsically disordered neuronal protein associated with Parkinson's pathology. The motion of this electrically heterogeneous polymer in the β-barrel voltage-dependent anion channel (VDAC) of the mitochondrial outer membrane strongly depends on the properties of both the charged and uncharged regions of the α-synuclein polymer. We model this motion in two ways. First, a simple Markov model accounts for the transitions of the channel between the states of different occupancy by α-synuclein. Second, the detailed energy landscape of this motion can be accounted for using a drift-diffusion framework that incorporates the α-synuclein binding energy and the free energy cost of its confinement in the VDAC pore. The models directly predict the probability of α-synuclein translocation across the mitochondrial outer membrane, with immediate implications for the physiological role of α-synuclein in regulation of mitochondrial bioenergetics. Time-resolved measurements of the electrical properties of VDAC occupied by α-synuclein reveal distinct effects of the motion of the junction separating the differently charged regions of the polymer.