Lorenz J. Steinbock

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.

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.

Sensing DNA-coatings of microparticles using micropipettes

The resistive pulse technique is widely used to detect the size of small particles in aqueous solutions. This work demonstrates that a few tens of DNA molecules and thus the charges on a particle can be simply detected by pressure-driven translocation through a microcapillary based Coulter counter. The typical opening of the capillaries ranges from 2 to 6 microm. The custom-built system gives optical access using a high numerical aperture objective allowing to observe colloids passing the sensing volume by optical means. We show the feasibility of our setup by distinguishing colloids with one and two micron diameters. Our measurements prove that a few ten strands of DNA bound to the colloids can be detected. This can be achieved by simple comparison of current amplitudes for blank and coated colloids at low salt concentrations (2-40 mmol [NaCl]). Our results clearly demonstrate that the Coulter counter can be used to detect the surface charges on colloids. Moreover, the results are in good agreement with a dynamical computer model taking into account the full geometry of the capillary.

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.

The emergence of nanopores in next-generation sequencing

Next-generation sequencing methods based on nanopore technology have recently gained considerable attention, mainly because they promise affordable and fast genome sequencing by providing long read lengths (5 kbp) and do not require additional DNA amplification or enzymatic incorporation of modified nucleotides. This permits health care providers and research facilities to decode a genome within hours for less than

Probing DNA with micro-?and nanocapillaries and optical tweezers

1000. This review summarizes past, present, and future DNA sequencing techniques, which are realized by nanopore approaches such as those pursued by Oxford Nanopore Technologies.

Voltage-driven transport of ions and DNA through nanocapillaries

We combine for the first time optical tweezer experiments with the resistive pulse technique based on capillaries. Quartz glass capillaries are pulled into a conical shape with tip diameters as small as 27 nm. Here, we discuss the translocation of λ-phage DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single λ-phage DNA molecules. Our flow cell design allows for the straightforward incorporation of optical tweezers. We show that a DNA molecule attached to an optically trapped colloid is pulled into a capillary by electrophoretic forces. The detected electrophoretic force is in good agreement with measurements in solid-state nanopores.

Modeling of colloidal transport in capillaries

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.

Controllable Shrinking and Shaping of Glass Nanocapillaries under Electron Irradiation

We dynamically model the full ionic current signature of micron-sized colloids passing through microcapillaries in silico for the first time. Our novel computer simulation allows free adjustment of all relevant experimental parameters such as the geometry of the used orifice, noise sources, external applied pressure or voltage, and the charge of the particles passing through the channel. We demonstrate that our algorithm correctly describes the experimentally observed signals in our recently introduced microcapillary based Coulter counters. Finally, we quantitatively investigate the influence of DNA-functionalized particles on the signal amplitude as a function of salt concentration and particle size.

Simple Reconstitution of Protein Pores in Nano Lipid Bilayers

The ability to reshape nanopores and observe their shrinkage under an electron microscope is a powerful and novel technique. It increases the sensitivity of the resistive pulse sensing and enables to detect very short and small molecules. However, this has not yet been shown for glass nanocapillaries. In contrast to their solid-state nanopore counterparts, nanocapillaries are cheap, easily fabricated and in the production do not necessitate clean room facilities. We show for the first time that quartz nanocapillaries can be shrunken under a scanning electron microscope beam. Since the shrinking is caused by the thermal heating of the electrons, increasing the beam current increases the shrink rate. Higher acceleration voltage on the contrary increases the electron penetration depth and reduces the electron density causing slower shrinkage. This allows us to fine control the shrink rate and to stop the shrinking process at any desired diameter. We show that a shrunken nanocapillary detects DNA translocation with six times higher signal amplitudes than an unmodified nanocapillary. This will open a new path to detect small and short molecules such as proteins or RNA with nanocapillaries.