Identification of single nucleotides in MoS2 nanopores

Ultrathin membranes have drawn much attention due to their unprecedented spatial resolution for DNA nanopore sequencing. However, the high translocation velocity (3000-50000 nt/ms) of DNA molecules moving across such membranes limits their usability. To this end, we have introduced a viscosity gradient system based on room-temperature ionic liquids (RTILs) to control the dynamics of DNA translocation through a nanometer-size pore fabricated in an atomically thin MoS2 membrane. This allows us for the first time to statistically identify all four types of nucleotides with solid state nanopores. Nucleotides are identified according to the current signatures recorded during their transient residence in the narrow orifice of the atomically thin MoS2 nanopore. In this novel architecture that exploits high viscosity of RTIL, we demonstrate single-nucleotide translocation velocity that is an optimal speed (1-50 nt/ms) for DNA sequencing, while keeping the signal to noise ratio (SNR) higher than 10. Our findings pave the way for future low-cost and rapid DNA sequencing using solid-state nanopores.Comment: Manuscript 24 pages, 4 Figures Supporting Information 24 pages, 12 Figures, 2 Tables Manuscript in review Nature Nanotechnology since May 27th 201

Relevance of the Drag Force during Controlled Translocation of a DNA–Protein Complex through a Glass Nanocapillary

Combination of glass nanocapillaries with optical tweezers allowed us to detect DNA-protein complexes in physiological conditions. In this system, a protein bound to DNA is characterized by a simultaneous change of the force and ionic current signals from the level observed for the bare DNA. Controlled displacement of the protein away from the nanocapillary opening revealed decay in the values of the force and ionic current. Negatively charged proteins EcoRI, RecA, and RNA polymerase formed complexes with DNA that experienced electrophoretic force lower than the bare DNA inside nanocapillaries. Force profiles obtained for DNA-RecA in our system were different than those in the system with nanopores in membranes and optical tweezers. We suggest that such behavior is due to the dominant impact of the drag force comparing to the electrostatic force acting on a DNA-protein complex inside nanocapillaries. We explained our results using a stochastic model taking into account the conical shape of glass nanocapillaries

Single Molecule Localization and Discrimination of DNA–Protein Complexes by Controlled Translocation Through Nanocapillaries

Through the use of optical tweezers we performed controlled translocations of DNA-protein complexes through nanocapillaries. We used RNA polymerase (RNAP) with two binding sites on a 7.2 kbp DNA fragment and a dCas9 protein tailored to have five binding sites on lambda-DNA (48.5 kbp). Measured localization of binding sites showed a shift from the expected positions on the DNA that we explained using both analytical fitting and a stochastic model. From the measured force versus stage curves we extracted the non equilibrium work done during the translocation of a DNA-protein complex and used it to obtain an estimate of the effective charge of the complex. In combination with conductivity measurements, we provided a proof of concept for discrimination between different DNA protein complexes simultaneous to the localization of their binding sites

Measurement of the Position-Dependent Electrophoretic Force on DNA in a Glass Nanocapillary

The electrophoretic force on a single DNA molecule inside a glass nanocapillary depends on the opening size and varies with the distance along the symmetrical axis of the nanocapillary. Using optical tweezers and DNA-coated beads, we measured the stalling forces and mapped the position-dependent force profiles acting on DNA inside nanocapillaries of different sizes. We showed that the stalling force is higher in nanocapillaries of smaller diameters. The position-dependent force profiles strongly depend on the size of the nanocapillary opening, and for openings smaller than 20 nm, the profiles resemble the behavior observed in solid-state nanopores. To characterize the position-dependent force profiles in nanocapillaries of different sizes, we used a model that combines information from both analytical approximations and numerical calculations

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNAbinding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and sitedirected modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria

DNA Translocation through Low-Noise Glass Nanopores

The effect of electron irradiation-induced shrinking on glass nanocapillaries with diameters ranging from 75 to 14 nm was analyzed by measuring the conductance characteristics with and without DNA translocation. We have investigated nanocapillary shrinking with a scanning electron microscope from several perspectives to understand the geometry of the shrunken nanocapillary. On the basis of this observation, the conductance was modeled with respect to the nanocapillary diameter, which allowed reproducing the experimental results. We then translocated DNA through the shrunken nanocapillaries and measured higher conductance drops for smaller diameters, reaching 1.7 nS for the 14 nm diameter nanocapillary. A model taking into account the conical shape of the shrunken nanocapillaries also supported this dependence. Next, we calculated the noise in the form of the standard deviation of the ionic conductance (between 0.04 and 0.15 nS) to calculate a signal-to-noise ratio (SNR) and compared it with nanopores embedded in 20 nm thick silicon nitride membranes. This shows that although nanocapillaries have smaller signal amplitudes due to their conical shape, they benefit from a lower noise. The glass nanocapillaries have a good SNR of about 25 compared with the SNR of 15 for smaller sized nanopores in silicon nitride membranes. The ability to use a modified model of nanopores to mimic the block conductance by DNA translocation provides a theoretical framework to support experimental results from translocating polymers such as DNA

Time-lapse microscopy series of bacteria exposed to exogenous DNA.

<p>The images were captured in the red channel to visualize ComEA-mCherry at intervals of 3 sec (A) or 2 min (B). Matlab-computed maximal fluorescence intensity-plots are shown in A (corresponding fluorescent image in inset). Heat-maps showing the fluorescence intensities of the mCherry signal are depicted in the lower row of panel B. (C) Time-lapse microscopy series as in (B), but in a <i>comEC</i> minus background. The corresponding movies are available online (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066.s012" target="_blank">movies S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066.s013" target="_blank">S3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066.s014" target="_blank">S4</a>).</p

ComEA binds to DNA <i>in vivo</i>.

<p>(A) Plasmid-encoded <i>gfp</i> (tat-GFP) or <i>comEA-gfp</i> (tat-ComEA-GFP), both preceded by a tat-signal sequence, were expressed in <i>E. coli</i>. The images shown correspond to the GFP channel, DAPI channel (to visualize DAPI-stained DNA), merged fluorescent images (merge), and phase contrast (Ph). The cells are outlined with dashed lines for tat-ComEA-GFP. Heat-maps showing the fluorescence intensities of the GFP and DAPI signals are depicted for the <i>tat-comEA-gfp</i> expressing cells below the images. (B) ComEA-mCherry aggregation and foci formation after the addition of external DNA. Competence-induced cells without (no DNA) or with external DNA were imaged in the red (mCherry; upper row) or the phase contrast channel (lower row) to visualize ComEA-mCherry localization. The DNA fragments differed in lengths (PCR fragment, 10.3 kb; λDNA, 48.5 kb; gDNA, various lengths). Transforming DNA did not lead to foci formation of periplasmic mCherry alone (preceded by the ComEA signal sequence; ss[ComEA]-mCherry). (C) Colocalization (merged image) of ComEA-mCherry (red channel) and YoYo-1-stained transforming DNA (green channel). The outline of the cells is shown in the phase contrast image (Ph). Scale bars in all images, 2 µm. (D) DNA uptake requires ComEA. DNA uptake of competent <i>V. cholerae</i> cells was tested using a whole-cell duplex PCR assay. All mutant strains were tested in a <i>comEC</i> positive (+) and negative (−) background. The lower PCR fragments indicate acceptor strain DNA (gDNA, acceptor); the upper band indicates internalized transforming DNA (tDNA). L, ladder.</p