Effect of base methylation on binding and mobility of bacterial protein Hfq on double-stranded DNA.

Regulation of protein mobility is a fundamental aspect of cellular processes. In this study, we examined the impact of DNA methylation on the diffusion of nucleoid associated protein Hfq. This protein is one of the most abundant proteins that shapes the bacterial chromosome and is involved in several aspects of nucleic acid metabolism.

Form factor of helical structures and twisted fibres.

A general formalism is presented for the isotropically averaged single-chain scattering function (form factor) of single, double, triple and higher-order helices, as well as twisted fibres consisting of concentric layers of strands. Form factors for double and triple helices with differently sized grooves have also been derived. The formulas include the longitudinal and transverse interference over the pitch and radius of the helices, respectively.

Amyloid-like Hfq interaction with single-stranded DNA: involvement in recombination and replication in .

Interactions between proteins and single-stranded DNA (ssDNA) are crucial for many fundamental biological processes, including DNA replication and genetic recombination. Thus, understanding detailed mechanisms of these interactions is necessary to uncover regulatory rules occurring in all living cells. The RNA-binding Hfq is a pleiotropic bacterial regulator that mediates many aspects of nucleic acid metabolism.

Reconstituted TAD-size chromatin fibers feature heterogeneous nucleosome clusters.

Large topologically associated domains (TADs) contain irregularly spaced nucleosome clutches, and interactions between such clutches are thought to aid the compaction of these domains. Here, we reconstituted TAD-sized chromatin fibers containing hundreds of nucleosomes on native source human and lambda-phage DNA and compared their mechanical properties at the single-molecule level with shorter '601' arrays with various nucleosome repeat lengths. Fluorescent imaging showed increased compaction upon saturation of the DNA with histones and increasing magnesium concentration.

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.

SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by and .

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by and that commonly cause secondary bacterial pneumonia.

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.

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.

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.

Interactions between DNA and the Hfq Amyloid-like Region Trigger a Viscoelastic Response.

Molecular transport of biomolecules plays a pivotal role in the machinery of life. Yet, this role is poorly understood due the lack of quantitative information. Here, the role and properties of the C-terminal region of Hfq is reported, involved in controlling the flow of a DNA solution.

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.

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.

Single-molecule compaction of megabase-long chromatin molecules by multivalent cations.

To gain insight into the conformational properties and compaction of megabase-long chromatin molecules, we reconstituted chromatin from T4 phage DNA (165 kb) and recombinant human histone octamers (HO). The unimolecular compaction, induced by divalent Mg2+ or tetravalent spermine4+ cations, studied by single-molecule fluorescence microscopy (FM) and dynamic light scattering (DLS) techniques, resulted in the formation of 250-400 nm chromatin condensates. The compaction on this scale of DNA size is comparable to that of chromatin topologically associated domains (TAD) in vivo.

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.

Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain.

Emerging DNA-based nanotechnologies would benefit from the ability to modulate the properties (e.g., solubility, melting temperature, chemical stability) of diverse DNA templates (single molecules or origami nanostructures) through controlled, self-assembling coatings.

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.

Structure of the H-NS-DNA nucleoprotein complex.

Nucleoid associated proteins (NAPs) play a key role in the compaction and expression of the prokaryotic genome. Here we report the organisation of a major NAP, the protein H-NS on a double stranded DNA fragment. For this purpose we have carried out a small angle neutron scattering study in conjunction with contrast variation to obtain the contributions to the scattering (structure factors) from DNA and H-NS.

Revisiting the Anomalous Bending Elasticity of Sharply Bent DNA.

Several recent experiments suggest that sharply bent DNA has a surprisingly high bending flexibility, but the cause of this flexibility is poorly understood. Although excitation of flexible defects can explain these results, whether such excitation can occur with the level of DNA bending in these experiments remains unclear. Intriguingly, the DNA contained preexisting nicks in most of these experiments but whether nicks might play a role in flexibility has never been considered in the interpretation of experimental results.

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.

Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides.

Biofilms are densely populated communities of microbial cells protected and held together by a matrix of extracellular polymeric substances. The structure and rheological properties of the matrix at the microscale influence the retention and transport of molecules and cells in the biofilm, thereby dictating population and community behavior. Despite its importance, quantitative descriptions of the matrix microstructure and microrheology are limited.

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.

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.

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.

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.