Tunable 2D diffusion of DNA nanostructures on lipid membranes.

DNA nanotechnology facilitates the synthesis of biomimetic models for studying biological systems. This work uses lipid bilayers as platforms for two-dimensional single-particle tracking of the dynamics of DNA nanostructures. Three different DNA origami structures adhere to the membrane through hybridization with cholesterol-modified strands. Their two-dimensional diffusion coefficient is modulated by changing the concentration of monovalent and divalent salts and the number of anchors. In addition, the diffusion coefficient is tuned by targeting cholesterol-modified anchor strands with strand-displacement reactions. We demonstrate a responsive system with changing diffusivity by selectively displacing membrane-bound anchor strands. We also show the programmed release of origami structures from the lipid membranes.

Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System.

Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.

Preparative refolding of small monomeric outer membrane proteins.

The outer membrane of gram-negative bacteria constitutes an important hurdle for the transport of hydrophobic molecules into the cell. Mass flux is often facilitated by various outer membrane proteins. These proteins are of biotechnological importance because they could help to improve the performance of whole-cell biocatalysts or be incorporated into artificial cell-like systems. The characterization and understanding of their transport properties greatly benefits from the possibility to express and purify these proteins. We investigated folding parameters for the refolding of four small monomeric outer membrane proteins from Escherichia coli (OmpW) and different pseudomonads (AlkL, OprG and TodX). To this aim we screened a number of inexpensive detergents and detergent concentrations, folding additives as well as protein concentrations. Interestingly, detergents with a C12 chain were most effective in promoting the folding reaction, particularly the negatively charged N-Lauroylsarcosine for OmpW, OprG and TodX as well as the zwitterionic N,N-Dimethyl-n-dodecylamine N-oxide (LDAO) for AlkL. The addition of 1 M urea (AlkL, OmpW), 0.1 M glutamate (OprG) or 0.1 M glycine (TodX) could further improve the folding efficiency. In order to be able to reproducibly produce larger amounts of the proteins, we then established the folding in a miniaturized stirred-tank reactor system combined with a liquid handler. This approach led to a near-complete refolding of OprG (96%), a very good folding of AlkL (84%) and OmpW (71%), only TodX folding was more variable with a final folding efficiency of 52%, all obtained at a final protein concentration of 0.5 g/L.

Electrotransfection of Polyamine Folded DNA Origami Structures.

DNA origami structures are artificial molecular nanostructures in which DNA double helices are forced into a closely packed configuration by a multitude of DNA strand crossovers. We show that three different types of origami structures (a flat sheet, a hollow tube, and a compact origami block) can be formed in magnesium-free buffer solutions containing low (<1 mM) concentrations of the condensing agent spermidine. Much like in DNA condensation, the amount of spermidine required for origami folding is proportional to the DNA concentration. At excessive amounts, the structures aggregate and precipitate. In contrast to origami structures formed in conventional buffers, the resulting structures are stable in the presence of high electric field pulses, such as those commonly used for electrotransfection experiments. We demonstrate that spermidine-stabilized structures are stable in cell lysate and can be delivered into mammalian cells via electroporation.

Noncovalent hydrogel beads as microcarriers for cell culture.

Hydrogel beads as microcarriers could have many applications in biotechnology. However, bead formation by noncovalent cross-linking to achieve high cell compatibility by avoiding chemical reactions remains challenging because of rapid gelation rates and/or low stability. Here we report the preparation of homogeneous, tunable, and robust hydrogel beads from peptide-polyethylene glycol conjugates and oligosaccharides under mild, cell-compatible conditions using a noncovalent crosslinking mechanism. Large proteins can be released from beads easily. Further noncovalent modification allows for bead labeling and functionalization with various compounds. High survival rates of embedded cells were achieved under standard cell culture conditions and after freezing the beads, demonstrating its suitability for encapsulating and conserving cells. Hydrogel beads as functional system have been realized by generating protein-producing microcarriers with embedded eGFP-secreting insect cells.

A large size-selective DNA nanopore with sensing applications.

Transmembrane nanostructures like ion channels and transporters perform key biological functions by controlling flow of molecules across lipid bilayers. Much work has gone into engineering artificial nanopores and applications in selective gating of molecules, label-free detection/sensing of biomolecules and DNA sequencing have shown promise. Here, we use DNA origami to create a synthetic 9 nm wide DNA nanopore, controlled by programmable, lipidated flaps and equipped with a size-selective gating system for the translocation of macromolecules. Successful assembly and insertion of the nanopore into lipid bilayers are validated by transmission electron microscopy (TEM), while selective translocation of cargo and the pore mechanosensitivity are studied using optical methods, including single-molecule, total internal reflection fluorescence (TIRF) microscopy. Size-specific cargo translocation and oligonucleotide-triggered opening of the pore are demonstrated showing that the DNA nanopore can function as a real-time detection system for external signals, offering potential for a variety of highly parallelized sensing applications.

Molecular transport through large-diameter DNA nanopores.

DNA-based nanopores are synthetic biomolecular membrane pores, whose geometry and chemical functionality can be tuned using the tools of DNA nanotechnology, making them promising molecular devices for applications in single-molecule biosensing and synthetic biology. Here we introduce a large DNA membrane channel with an ≈4 nm diameter pore, which has stable electrical properties and spontaneously inserts into flat lipid bilayer membranes. Membrane incorporation is facilitated by a large number of hydrophobic functionalizations or, alternatively, streptavidin linkages between biotinylated channels and lipids. The channel displays an Ohmic conductance of ≈3 nS, consistent with its size, and allows electrically driven translocation of single-stranded and double-stranded DNA analytes. Using confocal microscopy and a dye influx assay, we demonstrate the spontaneous formation of membrane pores in giant unilamellar vesicles. Pores can be created both in an outside-in and an inside-out configuration.

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.