Johan R. C. van der Maarel

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.

Molecular dynamics simulation of multivalent-ion mediated attraction between DNA molecules.

All atom molecular dynamics simulations with explicit water were done to study the interaction between two parallel double-stranded DNA molecules in the presence of the multivalent counterions putrescine (2+), spermidine (3+), spermine (4+) and cobalt hexamine (3+). The inter-DNA interaction potential is obtained with the umbrella sampling technique. The attractive force is rationalized in terms of the formation of ion bridges, i.e., multivalent ions which are simultaneously bound to the two opposing DNA molecules. The lifetime of the ion bridges is short on the order of a few nanoseconds.

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.

Dynamic Remodeling of Microbial Biofilms by Functionally Distinct Exopolysaccharides

ABSTRACT 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. Here, particle-tracking microrheology in combination with genetic approaches was used to spatially and temporally study the rheological contributions of the major exopolysaccharides Pel and Psl in Pseudomonas aeruginosa biofilms. Psl increased the elasticity and effective cross-linking within the matrix, which strengthened its scaffold and appeared to facilitate the formation of microcolonies. Conversely, Pel reduced effective cross-linking within the matrix. Without Psl, the matrix becomes more viscous, which facilitates biofilm spreading. The wild-type biofilm decreased in effective cross-linking over time, which would be advantageous for the spreading and colonization of new surfaces. This suggests that there are regulatory mechanisms to control production of the exopolysaccharides that serve to remodel the matrix of developing biofilms. The exopolysaccharides were also found to have profound effects on the spatial organization and integration of P. aeruginosa in a mixed-species biofilm model of P. aeruginosa-Staphylococcus aureus. Pel was required for close association of the two species in mixed-species microcolonies. In contrast, Psl was important for P. aeruginosa to form single-species biofilms on top of S. aureus biofilms. Our results demonstrate that Pel and Psl have distinct physical properties and functional roles during biofilm formation. IMPORTANCE Most bacteria grow as biofilms in the environment or in association with eukaryotic hosts. Removal of biofilms that form on surfaces is a challenge in clinical and industrial settings. One of the defining features of a biofilm is its extracellular matrix. The matrix has a heterogeneous structure and is formed from a secretion of various biopolymers, including proteins, extracellular DNA, and polysaccharides. It is generally known to interact with biofilm cells, thus affecting cell physiology and cell-cell communication. Despite the fact that the matrix may comprise up to 90% of the biofilm dry weight, how the matrix properties affect biofilm structure, maturation, and interspecies interactions remain largely unexplored. This study reveals that bacteria can use specific extracellular polymers to modulate the physical properties of their microenvironment. This in turn impacts biofilm structure, differentiation, and interspecies interactions. Most bacteria grow as biofilms in the environment or in association with eukaryotic hosts. Removal of biofilms that form on surfaces is a challenge in clinical and industrial settings. One of the defining features of a biofilm is its extracellular matrix. The matrix has a heterogeneous structure and is formed from a secretion of various biopolymers, including proteins, extracellular DNA, and polysaccharides. It is generally known to interact with biofilm cells, thus affecting cell physiology and cell-cell communication. Despite the fact that the matrix may comprise up to 90% of the biofilm dry weight, how the matrix properties affect biofilm structure, maturation, and interspecies interactions remain largely unexplored. This study reveals that bacteria can use specific extracellular polymers to modulate the physical properties of their microenvironment. This in turn impacts biofilm structure, differentiation, and interspecies interactions.

Effect of Nanochannel Geometry on DNA Structure in the Presence of Macromolecular Crowding Agent

We experimentally and numerically study the effects of macromolecular crowding agents on DNA structure when confined to a nanochannel. Curiously, DNA response to crowding is significantly different between bulk phase, nanoslit confinement, and nanotube confinement. Coarse grained Brownian dynamics simulations reproduce trends seen in the experiments and allow us to develop a deeper understanding of the key physics at play in these systems. It is proposed that the occupancy of free volume next to the channel wall by crowders causes an effective reduction in confining dimensions of the channel that initially swells DNA in nanoconfinement.

Introduction to biopolymer physics

Biopolymers Polymer Conformation Polyelectrolytes Polymer Dynamics Higher-Order Structures and Their Transitions Mesoscopic Structures.

A systematic study of DNA conformation in slitlike confinement

DNA conformation in slitlike confinement is studied using Monte Carlo simulation and scaling theory. We focus on analysing the in-plane DNA extension as a function of slit height and DNA properties, such as contour length, persistence length and width. Similar to tube confinement, we identify an extended de Gennes regime which is prevalent in many experimental studies. However, unlike previous studies in tubes, we find two highly confined regimes (so called Odijk regimes) that depend on chain crossing. Our results support the majority of experiments which display a gradual transition into the Odijk regime and give new insight into the importance of chain width for DNA in nanoslits.

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.

Viscoelasticity of entangled λ-phage DNA solutions

The viscoelastic moduli of lambda-phage DNA through the entanglement transition were obtained with particle tracking microrheology. With increasing frequency, the viscous loss modulus first increases, then levels off, and eventually increases again. Concurrently, the elastic storage modulus monotonously increases and eventually levels off to a constant high frequency plateau value. Once the DNA molecules become entangled at about ten times the overlap concentration, the elastic storage modulus becomes larger than the viscous loss modulus in an intermediate frequency range. The number of entanglements per chain is obtained from the plateau value of the elasticity modulus. The longest, global relaxation time pertaining to the motion of the DNA molecules is obtained from the low shear viscosity as well as from the lowest crossover frequency of the viscous loss and elastic storage moduli. The concentration dependencies of the low shear viscosity, the number of entanglements per chain, and the relaxation time agree with the relevant scaling laws for reptation dynamics of entangled polyelectrolytes in an excess of simple, low molecular weight salt with screened electrostatic interactions.

MRP14 (S100A9) protein interacts with alzheimer beta-amyloid peptide and induces its fibrillization

Increasing evidence supports the contribution of local inflammation to the development of Alzheimers disease (AD) pathology, although the precise mechanisms are not clear. In this study, we demonstrate that the pro-inflammatory protein S100A9 interacts with the A1–40 peptide and promotes the formation of fibrillar -amyloid structures. This interaction also results in reduced S100A9 cytotoxicity by the binding of S100A9 toxic species to A1–40 amyloid structures. These results suggest that secretion of S100A9 during inflammation promotes the formation of amyloid plaques. By acting as a sink for toxic species, plaque formation may be the result of a protective response within the brain of AD patients, in part mediated by S100A9.