The re-engineering of biological protein pore scaffolds is a successful approach 12, 13 which has led, for example, to components for label-free biosensing [14][15][16][17] and portable genome sequencing 18, 19 . Creating completely new architectures with synthetic materials can offer greater design freedom and translate into more functions and applications [20][21][22][23][24][25][26] . A key challenge in the de-novo design of membrane channels is, however, to achieve an atomistically defined structure of predictable nanomechanical properties 3 because the traditional building blocks of polypeptides and organic polymers are highly flexible 2, 4 . DNA, by contrast, is known to fold into predetermined structures and is able to meet most the criteria required for creating synthetic channels [27][28][29][30][31][32][33] . Indeed, membrane-spanning DNA nanopores have been very 3" "recently built to feature a central hollow barrel which is open at both ends [5][6][7][8][9] . The barrel is composed of six hexagonally arranged, interconnected DNA duplexes that enclose a 2 nm-wide lumen with a length ranging from 17 to 42 nm. The innovative step was the inclusion of hydrophobic anchors 5, 7, 9 to insert the negatively charged pores into the hydrophobic bilayer membrane. While of novelty and considerable interest 34, 35 , the barrels do not exploit the full design flexibility offered by DNA nanotechnology and do not exhibit the higher-order functions of ion channels which can bind ligands, respond by nanomechanical opening, and select cargo for transport.We used the simple geometric shape of an open barrel as a starting point to rationally design a nanodevice that can regulate the flux of matter across a bilayer membrane.The aim of the first design step was reduce the pore height to approximate the bilayer thickness 36 and thereby avoid structural flexibility and potential leakiness 37 . A pore height of 7 nm (Fig. 1a, NP) was achieved using a six-helix-bundle architecture with six concatenated DNA strands, each of which connects two neighboring duplexes at their termini (Fig. 1b). This connectivity is drastically simpler than classical origami 38 based on cadnano software where oligonucleotides run through multiple duplexes and cause a minimum height of approx. 15 nm 5, 38 . Our design with connections at the duplex ends also avoids traditional internal cross-overs that cause structural deviations from parallel aligned duplexes 39 .The second step was to design a molecular gate that closes one barrel entrance but reopens the channel upon binding of a ligand. A origami plate has been previously used as a controllable lid for a DNA origami box 32 . But our molecular models supported by biophysical studies 37 suggest that the plate might be structurally too flexible and leaky 4" "to form a tight seal. As a solution, we designed a nanodevice that features in its closed state, NP-C (Fig. 1c), a simple "lock" strand which is bound closely to the entrance by hybridization to two docking sites. The sites are formed by th...

Membrane-spanning nanopores from folded DNA are a recent example of biomimetic man-made nanostructures that can open up applications in biosensing, drug delivery, and nanofluidics. In this report, we generate a DNA nanopore based on the archetypal six-helix-bundle architecture and systematically characterize it via single-channel current recordings to address several fundamental scientific questions in this emerging field. We establish that the DNA pores exhibit two voltage-dependent conductance states. Low transmembrane voltages favor a stable high-conductance level, which corresponds to an unobstructed DNA pore. The expected inner width of the open channel is confirmed by measuring the conductance change as a function of poly(ethylene glycol) (PEG) size, whereby smaller PEGs are assumed to enter the pore. PEG sizing also clarifies that the main ion-conducting path runs through the membrane-spanning channel lumen as opposed to any proposed gap between the outer pore wall and the lipid bilayer. At higher voltages, the channel shows a main low-conductance state probably caused by electric-field-induced changes of the DNA pore in its conformation or orientation. This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes. These findings settle a discrepancy between two previously published conductances. By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.

The temperature-sensitive gating of human Connexin 26 (hCx26) was analyzed with confocal Raman microscopy. High-resolution Raman spectra covering the spectral range between 400 and 1500 rel. cm(-1) with a spectral resolution of 1 cm(-1) were fully annotated, revealing notable differences between the spectrum recorded from solubilized hCx26 in Ca(2+)-buffered POPC at 10°C and any other set of protein conditions (temperature, Ca(2+) presence, POPC presence). Spectral components originating from specific amino acids show that the TM1/EL1 parahelix and probably the TM4 trans-membrane helix and the plug domain are involved in the gating process responsible for fully closing the hemichannel.

No abstract

No abstract

Planar lipid bilayers can be automatically formed by remotely actuated painting in parallel on Micro-Electrode-Cavity-Arrays (MECA)-Chips. Voltage-clamp recording of ionic current through bacterial channels in bilayers is a powerful technique, yielding information on both the physicochemical characteristics and the physiological role of membrane proteins. Antibiotics penetrate bacterial membranes through general diffusion protein channels, like OmpF in E. coli. The MECA chip approach supports the analysis of translocation events in high resolution by detection of the transient block of ionic currents when a single molecule resides in the pore. Here, reconstituting the porins CarO from A. Baumannii, OmpC and OmpF from E. coli and orthologs in K. pneumoniae/E. cloacae, we resolved the transport of antibiotics and thereby study kinetics of antibiotics permeation through porins. This information can be useful in optimizing the design of new antibacterial drugs effective against resistant bacteria.