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.

We have developed a method to analyze in detail, translocation events providing a novel and flexible tool for data analysis of nanopore experiments. Our program, called OpenNanopore, is based on the cumulative sums algorithm (CUSUM algorithm). This algorithm is an abrupt change detection algorithm that provides fitting of current blockages, allowing the user to easily identify the different levels in each event. Our method detects events using adaptive thresholds that adapt to low-frequency variations in the baseline. After event identification, our method uses the CUSUM algorithm to fit the levels inside every event and automatically extracts their time and amplitude information. This facilitates the statistical analysis of an event population with a given number of levels. The obtained information improves the interpretation of interactions between the molecule and nanopore. Since our program does not require any prior information about the analyzed molecules, novel moleculenanopore interactions can be characterized. In addition our program is very fast and stable. With the progress in fabrication and control of the translocation speed, in the near future, our program could be useful in identification of the different bases of DNA.

No abstract

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.