Internal Motion of Chromatin Fibers Is Governed by Dynamics of Uncompressed Linker Strands.

Chromatin compaction and internal motion are fundamental aspects of gene expression regulation. Here, we have investigated chromatin fibers comprising recombinant histone octamers reconstituted with double-stranded bacteriophage T4-DNA. The size of the fibers approaches the typical size of genomic topologically associated domains. Atomic force and fluorescence (correlation) microscopy have been used to assess the structural organization, histone-induced compaction, and internal motion. In particular, the fibers are stretched on arrays of nanochannels, each channel with a diameter of 60 or 125 nm. Major intrafiber segregation and fast internal fluctuations are observed. Full compaction was only achieved by triggering an attractive nucleosome interaction through the addition of magnesium cations. Besides compaction, histone complexation results in a dramatic decrease in the fiber's relaxation time. The relaxation times are similar to those of naked DNA with a comparable stretch, which indicates that internal motion is governed by the dynamics of uncompressed linker strands. Furthermore, the main reorganization process is association-dissociation of individually compacted regions. We surmise that the modulation of chromatin's internal motion by histone complexation might have implications for transcriptional bursting.

Compaction and condensation of DNA mediated by the C-terminal domain of Hfq.

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.

Effect of HU protein on the conformation and compaction of DNA in a nanochannel.

The effect of the heat unstable nucleoid structuring protein HU on the conformation of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. Pre-incubated DNA molecules contract in the longitudinal direction of the channel with increasing concentration of HU. This contraction is mainly due to HU-mediated bridging of distal DNA segments and is controlled by channel diameter as well as ionic composition and strength of the buffer. For over-threshold concentrations of HU, the DNA molecules compact into an condensed form. Divalent magnesium ions facilitate, but are not required for bridging nor condensation. The conformational response following exposure to HU was investigated with a nanofluidic device that allows an in situ change in environmental solution conditions. The stretch of the nucleoprotein complex first increases, reaches an apex in ∼20 min, and subsequently decreases to an equilibrium value pertaining to pre-incubated DNA molecules after ∼2 h. This observation is rationalised in terms of a time-dependent bending rigidity by structural rearrangement of bound HU protein followed by compaction through bridging interaction. Results are discussed in regard to previous results obtained for nucleoid associated proteins H-NS and Hfq, with important implications for protein binding related gene regulation.

Effects of Hfq on the conformation and compaction of DNA.

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.

Amplified stretch of bottlebrush-coated DNA in nanofluidic channels.

The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.

Effect of H-NS on the elongation and compaction of single DNA molecules in a nanospace.

The effect of the bacterial heat-stable nucleoid-structuring protein (H-NS) on the conformation of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. With increasing concentration of H-NS, the DNA molecules either elongate or contract. The conformational response is related to filamentation of H-NS on DNA through oligomerization and H-NS mediated bridging of distal DNA segments and is controlled by the concentration and ionic composition of the buffer. Confinement in a nanochannel also facilitates compaction of DNA into a condensed form for over-threshold concentrations of H-NS. Divalent ions such as magnesium facilitate but are not required for bridging nor condensation. The time scale of the collapse after exposure to H-NS was determined to be on the order of minutes, which is much shorter than the measured time required for filamentation of around one hour. We found that the effect of H-NS is not only related to its binding properties but also the confinement is of paramount importance. The interplay between confinement, H-NS-mediated attraction, and filamentation controls the conformation and compaction of DNA. This finding might have implications for gene silencing and chromosome organisation, because the cross-sectional dimensions of the channels are comparable to those of the bacterial nucleoid.

Macromolecular crowding induced elongation and compaction of single DNA molecules confined in a nanochannel.

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.

Probing Amyloid-DNA Interaction with Nanofluidics.

Nanofluidics is an emerging methodology to investigate single biomacromolecules without functionalization and/or attachment of the molecules to a substrate. In conjunction with fluorescence microscopy, it can be used to investigate structural and dynamical aspects of amyloid-DNA interaction. Here, we summarize the methodology for fabricating lab-on-chip devices in relatively cheap polymer resins and featuring quasi one-dimensional nanochannels with a cross-sectional diameter of tens to a few hundred nanometers. Site-specific staining of amyloid-forming protein Hfq with a fluorescence dye is also described. The methodology is illustrated with two application studies. The first study involves assembling bacterial amyloid proteins such as Hfq on double-stranded DNA and monitoring the folding and compaction of DNA in a condensed state. The second study is about the concerted motion of Hfq on DNA and how this is related to DNA's internal motion. Explicit details of procedures and workflows are given throughout.

Mobility of Bacterial Protein Hfq on dsDNA: Role of C-Terminus-Mediated Transient Binding.

The mobility of protein is fundamental in the machinery of life. Here, we have investigated the effect of DNA binding in conjunction with DNA segmental fluctuation (internal motion) of the bacterial Hfq master regulator devoid of its amyloid C-terminus domain. Hfq is one of the most abundant nucleoid associated proteins that shape the bacterial chromosome and is involved in several aspects of nucleic acid metabolism. Fluorescence microscopy has been used to track a C-terminus domain lacking mutant form of Hfq on double-stranded DNA, which is stretched by confinement to a rectangular nanofluidic channel. The mobility of the mutant is strongly accelerated with respect to the wild-type variant. Furthermore, it shows a reverse dependence on the internal motion of DNA, in that slower motion results in slower protein diffusion. The results demonstrate the subtle role of DNA internal motion in controlling the mobility of a nucleoid associated protein, and, in particular, the importance of transient binding and moving DNA strands out of the way.

Role of Internal DNA Motion on the Mobility of a Nucleoid-Associated Protein.

Protein transport on DNA is at the core of the machinery of life. Here we investigated the influence of DNA internal motion on the mobility of Hfq, which is involved in several aspects of nucleic acid metabolism and is one of the nucleoid-associated proteins that shape the bacterial chromosome. Fluorescence microscopy was used to follow Hfq on double-stranded DNA that was stretched by confinement to a channel with a diameter of 125 nm. The protein mobility shows a strong dependence on the internal motion of DNA in that slower motion results in faster protein diffusion. A model of released diffusion is proposed that is based on three-dimensional diffusion through the interior of the DNA coil interspersed by periods in which the protein is immobilized in a bound state. We surmise that the coupling between DNA internal motion and protein mobility has important implications for DNA metabolism and protein-binding-related regulation of gene expression.

Considerations for the nano aperture ion source: Geometrical design and electrical control.

A new type of ion source is being developed for proton beam writing and other focused ion beam applications. The potential of this source as well as achieved performance of the nano aperture ion source will be evaluated. Based on the ideal source parameters, critical geometrical parameters constraining chromatic aberrations and a possible pathway to achieve this performance will be presented. Finally, an electronic control system to minimize chromatic and spherical aberrations to an acceptable level will be demonstrated.

Linearization and Labeling of Single-Stranded DNA for Optical Sequence Analysis.

Genetic profiling would benefit from linearization of ssDNA through the exposure of the unpaired bases to gene-targeting probes. This is compromised by ssDNA's high flexibility and tendency to form self-annealed structures. Here, we demonstrate that self-annealing can be avoided through controlled coating with a cationic-neutral diblock polypeptide copolymer. Coating does not preclude site-specific binding of fluorescence labeled oligonucleotides. Bottlebrush-coated ssDNA can be linearized by confinement inside a nanochannel or molecular combing. A stretch of 0.32 nm per nucleotide is achieved inside a channel with a cross-section of 100 nm and a 2-fold excess of polypeptide with respect to DNA charge. With combing, the complexes are stretched to a similar extent. Atomic force microscopy of dried complexes on silica revealed that the contour and persistence lengths are close to those of dsDNA in the B-form. Labeling is based on hybridization and not limited by restriction enzymes. Enzyme-free labeling offers new opportunities for the detection of specific sequences.

Effects of electrostatic screening on the conformation of single DNA molecules confined in a nanochannel.

Single T4-DNA molecules were confined in rectangular-shaped channels with a depth of 300 nm and a width in the range of 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 the 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.

Separation of superparamagnetic particles through ratcheted Brownian motion and periodically switching magnetic fields.

Brownian ratchet based particle separation systems for application in lab on chip devices have drawn interest and are subject to ongoing theoretical and experimental investigations. We demonstrate a compact microfluidic particle separation chip, which implements an extended on-off Brownian ratchet scheme that actively separates and sorts particles using periodically switching magnetic fields, asymmetric sawtooth channel sidewalls, and Brownian motion. The microfluidic chip was made with Polydimethylsiloxane (PDMS) soft lithography of SU-8 molds, which in turn was fabricated using Proton Beam Writing. After bonding of the PDMS chip to a glass substrate through surface activation by oxygen plasma treatment, embedded electromagnets were cofabricated by the injection of InSn metal into electrode channels. This fabrication process enables rapid production of high resolution and high aspect ratio features, which results in parallel electrodes accurately aligned with respect to the separation channel. The PDMS devices were tested with mixtures of 1.51 µm, 2.47 µm, and 2.60 µm superparamagnetic particles suspended in water. Experimental results show that the current device design has potential for separating particles with a size difference around 130 nm. Based on the promising results, we will be working towards extending this design for the separation of cells or biomolecules.

Buried centimeter-long micro- and nanochannel arrays in porous silicon and glass.

We developed a simple process to fabricate deeply buried micro- and nanoscale channels in glass and porous silicon from bulk silicon using a combination of ion beam irradiation, electrochemical anodization and high temperature oxidation. The depth, width and length of these structures can be controllably varied and we successfully fabricated an array of centimeter-long buried micro- and nanochannels. This process allows densely packed, arbitrary-shaped channel geometries with micro- to nanoscale dimensions to be produced in a three-dimensional multilevel architecture, providing a route to fabricate complex devices for use in nanofluidics and lab-on-a-chip systems. We demonstrate the integration of these channels with large reservoirs for DNA linearization in high aspect ratio nanochannels.

Highly sensitive and multispectral responsive phototransistor using tungsten-doped VO2 nanowires.

In this work, we report a novel and feasible strategy for the practical applications of one-dimensional ultrasensitive phototransistors made of tungsten-doped VO2 single nanowires. The photoconductive response of the single nanowire device was investigated under different visible light excitations (405 nm, 532 nm, and 660 nm). The phototransistor device exhibited ultrafast photoresponse, high responsivity, broad multispectral response, and rapid saturation characteristic curves. These promising results help to promote the applications of this material in nano-scale optoelectronic devices such as efficient multispectral phototransistors and optical switches.

A nanofluidic device for single molecule studies with in situ control of environmental solution conditions.

We report an approach to study the in situ conformational response of single biomolecules such as DNA to a change in environmental solution conditions. These conditions are, for example, the composition of the buffer or the presence of protein. For this purpose, we designed and fabricated a nanofluidic device featuring two arrays of parallel nanochannels in a perpendicular configuration. The cross-sections of the channels are rectangular with a diameter down to 175 nm. These lab-on-a-chip devices were made of polydimethylsiloxane (PDMS) cast on a high quality master stamp, obtained by proton beam writing and UV lithography. Biomolecules can be inserted into the device through the array of channels in one direction, whereas the buffer can be exchanged through the intersecting array of channels in the other direction. A buffer exchange time inside the grid of nanochannels of less than one second was measured by monitoring the conductivity of salt solutions. The exchange time of a protein was typically a few seconds, as determined by imaging the influx of fluorescence labelled protamine. We demonstrate the functionality of the device by investigating the compaction of DNA by protamine and the unpacking of pre-compacted DNA through an increase in the concentration of salt.

High throughput fabrication of disposable nanofluidic lab-on-chip devices for single molecule studies.

An easy method is introduced allowing fast polydimethylsiloxane (PDMS) replication of nanofluidic lab-on-chip devices using accurately fabricated molds featuring cross-sections down to 60 nm. A high quality master is obtained through proton beam writing and UV lithography. This master can be used more than 200 times to replicate nanofluidic devices capable of handling single DNA molecules. This method allows to fabricate nanofluidic devices through simple PDMS casting. The extensions of YOYO-1 stained bacteriophage T4 and λ-DNA inside these nanochannels have been investigated using fluorescence microscopy and follow the scaling prediction of a large, locally coiled polymer chain confined in nanochannels.

Nanouidic compaction of DNA by like-charged protein.

The effects of the like-charged proteins bovine serum albumin and hemoglobin on the conformation and compaction of single DNA molecules confined in rectangular nanochannels were investigated with fluorescence microscopy. The channels have lengths of 50 µm and cross-sectional diameters in the range of 80-300 nm. In the wider channels, the DNA molecules are compressed and eventually condense into a compact form with increasing concentration of protein. In the narrow channels, no condensation was observed. The threshold concentration for condensation depends on the channel cross-sectional diameter as well as the ionic strength of the supporting medium. The critical values for full compaction are typically less than one-tenth of a millimolar. In the bulk phase and in the same environmental conditions, no condensation was observed. Anisotropic nanoconfinement hence facilitates compaction of DNA by negatively charged protein. We tentatively interpret this behavior in terms of enhanced depletion interaction between segments of the DNA molecule due to orientation order imposed by the channel walls.

A single-channel microparticle sieve based on Brownian ratchets.

We present a novel device for the separation of microparticles in a single channel, which is made of inversely asymmetric Brownian ratchets. It enables separation into two different fractions with an adjustable threshold and can be modeled with good agreement. This device serves as proof of concept for an extremely compact class of microsieves.