Single T4-DNA molecules were confined in rectangular-shaped channels with a depth of 300 nm and a width in the range 150-300 nm casted in a poly(dimethylsiloxane) nanofluidic chip. The extensions of the DNA molecules were measured with fluorescence microscopy as a function of the ionic strength and composition of the buffer as well as the DNA intercalation level by the YOYO-1 dye. The data were interpreted with scaling theory for a wormlike polymer in good solvent, including the effects of confinement, charge, and self-avoidance. It was found that the elongation of the DNA molecules with decreasing ionic strength can be interpreted in terms of an increase of the persistence length. Self-avoidance effects on the extension are moderate, due to the small correlation length imposed by the channel cross-sectional diameter.Intercalation of the dye results in an increase of the DNA contour length and a partial neutralization of the DNA charge, but besides effects of electrostatic origin it has no significant effect on the bare bending rigidity. In the presence of divalent cations, the DNA molecules were observed to contract, but they do not collapse into a condensed structure. It is proposed that this contraction results from a divalent counterion mediated attractive force between the segments of the DNA molecule.

To overcome the diffraction constraints of traditional optical lithography, the next generation lithographies (NGLs) will utilize any one or more of EUV (extreme ultraviolet), X-ray, electron or ion beam technologies to produce sub-100 nm features. Perhaps the most under-developed and under-rated is the utilization of ions for lithographic purposes. All three ion beam techniques, FIB (Focused Ion Beam), Proton Beam Writing (p-beam writing) and Ion Projection Lithography (IPL) have now breached the technologically difficult 100 nm barrier, and are now capable of fabricating structures at the nanoscale. FIB, p-beam writing and IPL have the flexibility and potential to become leading contenders as NGLs. The three ion beam techniques have widely different attributes, and as such have their own strengths, niche areas and application areas. The physical principles underlying ion beam interactions with materials are described, together with a comparison with other lithographic techniques (electron beam writing and EUV/X-ray lithography). IPL follows the traditional lines of lithography, utilizing large area masks through which a pattern is replicated in resist material which can be used to modify the near-surface properties. In IPL, the complete absence of diffraction effects coupled with ability to tailor the depth of ion penetration to suit the resist thickness or the depth of modification are prime characteristics of this technique, as is the ability to pattern a large area in a single brief irradiation exposure without any wet processing steps. p-beam writing and FIB are direct write (maskless) processes, which for a long time have been considered too slow for mass production. However, these two techniques may have some distinct advantages when used in combination with nanoimprinting and pattern transfer. FIB can produce master stamps in any material, and p-beam writing is ideal for producing three-dimensional high-aspect ratio metallic stamps of precise geometry. The transfer of large scale patterns using nanoimprinting represents a technique of high potential for the mass production of a new generation of high area, high density, low dimensional structures. Finally a cross section of applications are chosen to demonstrate the potential of these new generation ion beam nanolithographies.

The effect of dextran nanoparticles on the conformation and compaction of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. It was observed that the DNA molecules elongate and eventually condense into a compact form with increasing volume fraction of the crowding agent. Under crowded conditions, the channel diameter is effectively reduced, which is interpreted in terms of depletion in DNA segment density in the interfacial region next to the channel wall. Confinement in a nanochannel also facilitates compaction with a neutral crowding agent at low ionic strength. The threshold volume fraction for condensation is proportional to the size of the nanoparticle, due to depletion induced attraction between DNA segments. We found that the effect of crowding is not only related to the colligative properties of the agent and that confinement is also important. It is the interplay between anisotropic confinement and osmotic pressure which gives the elongated conformation and the possibility for condensation at low ionic strength.A substantial fraction of the total volume of biological media is occupied by macromolecules, which do not directly participate in biochemical reactions. Nevertheless, it is now well established that these background species have an important effect on molecular transport, reaction rates, and chemical equilibrium (1). The steric repulsion between impenetrable macromolecules in a crowded medium is a major factor in determining the thermodynamic activities of the reactants. Crowding by an inert osmotic agent can also affect macromolecular structure. A well known example is the transition of DNA to a compact form (condensation) in the presence of overthreshold concentrations of simple neutral polymers and simple salts (2, 3, 4). It has been proposed that macromolecular crowding is the basis for phase separation in the cytoplasm (5) and condensation of DNA into the nucleoid of bacterial cells (6). The latter hypothesis is supported by the observation that DNA can be condensed by cytoplasmic extracts from Escherichia coli at extract concentrations corresponding to about 1 ⁄2 the cellular concentration (7). Besides background species, the cytoplasm of most eukaryotic cells contains stationary elements such as fiber lattices and membranes. These structures affect macromolecular conformation through confinement in 1D or 2D. Accordingly, macromolecular crowding and confinement are intimately related and deserve an integrated approach to understand their modes of operation and how they couple.DNA condensation can be assisted and directed by a surface. In surface directed condensation, DNA is first adsorbed onto an interface, after which it is condensed with an agent. Examples that have been reported in the literature are the condensation of single molecules into rods and toroidal structures with protamine or ethanol (8, 9). Single DNA molecules can be confined and visualized with f luorescence microscopy in quasi 1D nanochannels. The extension in the longitudinal ...

We report the utilization of a focused mega-electron-volt (MeV) proton beam to write accurate high-aspect-ratio structures at sub-100 nm dimensions. Typically, a MeV proton beam is focused to a sub-100 nm spot size and scanned over a suitable resist material. When the proton beam interacts with matter it follows an almost straight path. The secondary electrons induced by the primary proton beam have low energy and therefore limited range, resulting in minimal proximity effects. These features enable smooth three-dimensional structures to be direct written into resist materials. Initial tests have shown this technique capable of writing high aspect ratio walls of 30 nm width with sub-3 nm edge smoothness.

No abstract

Hfq is a bacterial protein that is involved in several aspects of nucleic acids metabolism. It has been described as one of the nucleoid associated proteins shaping the bacterial chromosome, although it is better known to influence translation and turnover of cellular RNAs. Here, we explore the role of Escherichia coli Hfq's C-terminal domain in the compaction of double stranded DNA. Various experimental methodologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy, isothermal titration microcalorimetry and electrophoretic mobility assays have been used to follow the assembly of the C-terminal and N-terminal regions of Hfq on DNA. Results highlight the role of Hfq's C-terminal arms in DNA binding, change in mechanical properties of the double helix and compaction of DNA into a condensed form. The propensity for bridging and compaction of DNA by the C-terminal domain might be related to aggregation of bound protein and may have implications for protein binding related gene regulation.

Hfq is a bacterial pleiotropic regulator that mediates several aspects of nucleic acids metabolism. The protein notably influences translation and turnover of cellular RNAs. Although most previous contributions concentrated on Hfq's interaction with RNA, its association to DNA has also been observed in vitro and in vivo. Here, we focus on DNA-compacting properties of Hfq. Various experimental technologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy and small angle neutron scattering have been used to follow the assembly of Hfq on DNA. Our results show that Hfq forms a nucleoprotein complex, changes the mechanical properties of the double helix and compacts DNA into a condensed form. We propose a compaction mechanism based on protein-mediated bridging of DNA segments. The propensity for bridging is presumably related to multi-arm functionality of the Hfq hexamer, resulting from binding of the C-terminal domains to the duplex. Results are discussed in regard to previous results obtained for H-NS, with important implications for protein binding related gene regulation.

We report a way of fabricating high-quality void-free high-aspect-ratio metallic stamps of 100nm width and 2μm depth, using the technique of proton beam writing coupled with electroplating using a nickel sulfamate solution. Proton beam writing is a one-step direct-write process with the ability to fabricate nanostructures with high-aspect-ratio vertical walls and smooth sides, and as such has ideal characteristics for three-dimensional (3D) stamp fabrication. Nanoindentation and atomic force microscopy measurements of the nickel surfaces of the fabricated stamp show a hardness and side-wall roughness of 5GPa and 7nm, respectively. The fabricated 100nm 3D stamps have been used to transfer test patterns into poly(methylmethacrylate) films, spin coated onto a silicon substrate. Proton beam writing coupled with electroplating offers a process of high potential for the fabrication of high quality metallic 3D nanostamps.