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 DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed 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.

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.

Lipid-coated nanocapillaries for DNA sensing.

We report a simple and efficient way to accomplish the chemical modification of glass nanopores by means of lipid self-assembly. Lipid coating improves the success rate of these glass nanopores as biosensors to detect λ-DNA.

Voltage-driven transport of ions and DNA through nanocapillaries.

We study the effect of salt concentration on the ionic conductance and translocation of single DNA molecules through nanocapillaries made out of quartz glass. DNA translocation experiments were performed in aqueous solution for concentrations of KCl between 10 mM and 2 M while ion conductance was characterized from 1 mM to 2 M KCl concentration. Here, we develop a model for the conductance of conical nanocapillaries taking into consideration the surface charge of the quartz glass. We demonstrate that the conductance of our nanocapillaries shows similar behavior to silicon oxide nanopores at low and high KCl concentrations. Finally, we show that DNA translocations in high KCl concentrations (400 mM-2 M) cause a reduction in the ionic current. In contrast, DNA translocations at low KCl concentrations (10-300 mM) lead to increases in the ionic current. Our new results, which until now have not been shown for nanocapillaries, can be well understood with an adapted model.

Simple reconstitution of protein pores in nano lipid bilayers.

We developed a new, simple and robust approach for rapid screening of single molecule interactions with protein channels. Our glass nanopipets can be fabricated simply by drawing glass capillaries in a standard pipet puller, in a matter of minutes, and do not require further modification before use. Giant unilamellar vesicles break when in contact with the tip of the glass pipet and form a supported bilayer with typical seal resistances of ∼140 GΩ, which is stable for hours and at applied potentials up to 900 mV. Bilayers can be formed, broken, and re-formed more than 50 times using the same pipet enabling rapid screening of bilayers for single protein channels. The stability of the lipid bilayer is significantly superior to that of traditionally built bilayers supported by Teflon membranes, particularly against perturbation by electrical and mechanical forces. We demonstrate the functional reconstitution of the E. coli porin OmpF and α-hemolysin in a glass nanopipet supported bilayer. Interactions of the antibiotic enrofloxacin with the OmpF channel have been studied at the single-molecule level, demonstrating the ability of this method to detect single molecule interactions with protein channels. High-resolution conductance measurements of protein channels can be performed with low sample and buffer consumption. Glass nanopipet supported bilayers are uniquely suited for single-molecule studies as they are more rigid and the lifetime of a stable membrane is on the scale of hours, closer to that of natural cell membranes.

Detecting DNA folding with nanocapillaries.

We demonstrate for the first time the detection of the folding state of double-stranded DNA in nanocapillaries with the resistive pulse technique. We show that glass capillaries can be pulled into nanocapillaries with diameters down to 45 nm. We study translocation of lambda -DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single lambda -DNA molecules. Our experiments prove that nanocapillaries are suitable for label-free analysis of DNA in aqueous solutions and viable alternatives to solid-state nanopores made by silicon nanotechnology.

Wavelength Dependence of Photoinduced Microcantilever Bending in the UV-VIS Range.

Micromechanical devices such as microcantilevers (MC) respond to irradiationwith light by at least two different, photon-mediated processes, which induce MC bendingas a consequence of differential surface stress. The first and slow bending is due to theabsorption of photons, whose energy is transformed into heat and causes bending ofbimetallic microcantilevers due to thermal expansion. The second type of deflection is fastand caused by photons of sufficient energy to promote electrons across the Schottky barrierand thus create charge carriers, resulting in photoinduced stress that causes MC bending. Inthis study, the MC bending response to irradiation with light of wavelengths ranging from250 to 700 nm was investigated. Measurements of the immediate mechanical response tophotoinduced stress as a function of the wavelength of incident light provide an avenue tothe determination of the cut-off wavelength/energy of the Schottky barrier in the MCdevices under investigation. For a gold coated Si3Ni4 microcantilever we measured a cutoffwavelength of 1206 nm, which lies in the range of the literature value of 1100 nm.

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.

Analyzing single DNA molecules by nanopore translocation.

Small holes in membranes or nanocapillaries can be employed to detect single molecules in solution. In fact, the resistive-pulse technique based on nanopores allows for determination of length, charge, and folding state of deoxyribonucleic acid (DNA). Here, we describe the experimental procedures necessary for measuring single DNA molecules in nanocapillaries. We also discuss the measures for data analysis and how to determine that only single molecule events are observed.