Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology.

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA structure would also stabilize its pathogenic variants is unknown. Here we show that the N 1 -methylation of guanosine at position 9 (m 1 G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has an opposite and destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated by the observation that demethylation of m 1 G9, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving its structure and activity. These results have new therapeutic implications, suggesting that the N 1 -methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.

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.

Response to Comment on "Enhanced water permeability and tunable ion selectivity in subnanometer carbon nanotube porins".

Horner and Pohl argue that high water transport rates reported for carbon nanotube porins (CNTPs) originate from leakage at the nanotube-bilayer interface. Our results and new experimental evidence are consistent with transport through the nanotube pores and rule out a defect-mediated transport mechanism. Mechanistic origins of the high Arrhenius factor that we reported for narrow CNTPs at pH 8 require further investigation.

Label-Free Single-Molecule Thermoscopy Using a Laser-Heated Nanopore.

When light is used to excite electronic transitions in a material, nonradiative energy during relaxation is often released in the form of heat. In this work, we show that photoexcitation of a silicon nitride nanopore using a focused visible laser results in efficient localized photothermal heating, which reduces the nearby electrolyte viscosity and increases the ionic conductance. In addition, a strong localized thermal gradient in the pore vicinity is produced, evidenced by finite-element simulations and experimental observation of both ion and DNA thermophoresis. After correcting for thermophoresis, the nanopore current can be used as a nanoscale thermometer, enabling rapid force thermoscopy. We utilize this to probe thermal melting transitions in synthetic and native biomolecules that are heated at the nanopore. Our results on single molecules are validated by correspondence to bulk measurements, which paves the way to various biophysical experiments that require rapid temperature and force control on individual molecules.

Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing.

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.

Enhanced water permeability and tunable ion selectivity in subnanometer carbon nanotube porins.

Fast water transport through carbon nanotube pores has raised the possibility to use them in the next generation of water treatment technologies. We report that water permeability in 0.8-nanometer-diameter carbon nanotube porins (CNTPs), which confine water down to a single-file chain, exceeds that of biological water transporters and of wider CNT pores by an order of magnitude. Intermolecular hydrogen-bond rearrangement, required for entry into the nanotube, dominates the energy barrier and can be manipulated to enhance water transport rates. CNTPs block anion transport, even at salinities that exceed seawater levels, and their ion selectivity can be tuned to configure them into switchable ionic diodes. These properties make CNTPs a promising material for developing membrane separation technologies.

Studies of RNA Sequence and Structure Using Nanopores.

Nanopores are powerful single-molecule sensors with nanometer scale dimensions suitable for detection, quantification, and characterization of nucleic acids and proteins. Beyond sequencing applications, both biological and solid-state nanopores hold great promise as tools for studying the biophysical properties of RNA. In this review, we highlight selected landmark nanopore studies with regards to RNA sequencing, microRNA detection, RNA/ligand interactions, and RNA structural/conformational analysis.

Electrophoretic Deformation of Individual Transfer RNA Molecules Reveals Their Identity.

It has been hypothesized that the ribosome gains additional fidelity during protein translation by probing structural differences in tRNA species. We measure the translocation kinetics of different tRNA species through ∼3 nm diameter synthetic nanopores. Each tRNA species varies in the time scale with which it is deformed from equilibrium, as in the translocation step of protein translation. Using machine-learning algorithms, we can differentiate among five tRNA species, analyze the ratios of tRNA binary mixtures, and distinguish tRNA isoacceptors.

Osmium-Based Pyrimidine Contrast Tags for Enhanced Nanopore-Based DNA Base Discrimination.

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.

Direct and Scalable Deposition of Atomically Thin Low-Noise MoS2 Membranes on Apertures.

Molybdenum disulfide (MoS2) flakes can grow beyond the edge of an underlying substrate into a planar freestanding crystal. When the substrate edge is in the form of an aperture, reagent-limited nucleation followed by edge growth facilitate direct and selective growth of freestanding MoS2 membranes. We have found conditions under which MoS2 grows preferentially across micrometer-scale prefabricated solid-state apertures in silicon nitride membranes, resulting in sealed membranes that are one to a few atomic layers thick. We have investigated the structure and purity of our membranes by a combination of atomic-resolution transmission electron microscopy, elemental analysis, Raman spectroscopy, photoluminescence spectroscopy, and low-noise ion-current recordings through nanopores fabricated in such membranes. Finally, we demonstrate the utility of fabricated ultrathin nanopores in such membranes for single-stranded DNA translocation detection.

Label-free optical detection of biomolecular translocation through nanopore arrays.

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.

Nanopore-based conformational analysis of a viral RNA drug target.

Nanopores are single-molecule sensors that show exceptional promise as a biomolecular analysis tool by enabling label-free detection of small amounts of sample. In this paper, we demonstrate that nanopores are capable of detecting the conformation of an antiviral RNA drug target. The hepatitis C virus uses an internal ribosome entry site (IRES) motif in order to initiate translation by docking to ribosomes in its host cell. The IRES is therefore a viable and important drug target. Drug-induced changes to the conformation of the HCV IRES motif, from a bent to a straight conformation, have been shown to inhibit HCV replication. However, there is presently no straightforward method to analyze the effect of candidate small-molecule drugs on the RNA conformation. In this paper, we show that RNA translocation dynamics through a 3 nm diameter nanopore is conformation-sensitive by demonstrating a difference in transport times between bent and straight conformations of a short viral RNA motif. Detection is possible because bent RNA is stalled in the 3 nm pore, resulting in longer molecular dwell times than straight RNA. Control experiments show that binding of a weaker drug does not produce a conformational change, as consistent with independent fluorescence measurements. Nanopore measurements of RNA conformation can thus be useful for probing the structure of various RNA motifs, as well as structural changes to the RNA upon small-molecule binding.

High-bandwidth protein analysis using solid-state nanopores.

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.