Using techniques of molecular biology, we have designed a molecular construction which allows to attach the two complementary strands of one end of a single molecule of bacteriophage λ DNA separately to a glass microscope slide and a microscopic bead. A soft microneedle acting as a force sensor is chemically attached to the bead and its deflection is measured by an optical microscope. Keeping the base of the force lever fixed, the glass slide is displaced slowly, leading to a progressive opening of the double helix. The force measured during the opening process shows a characteristic variation which is related to the sequence of the bases along the DNA molecule. We present a brief summary of the present state of our work.

We investigate the statistical mechanics of a torsionally constrained polymer. The polymer is modelled as an inextensible chain with bend rigidity A, twist rigidity C, and twist-stretch coupling D. In such a model, thermal bend fluctuations couple geometrically to an applied torque through the relation Lk = Tw + Wr. We explore this coupling and find excellent agreement between the predictions of our model and the single λ-DNA molecule stretching experiments of Strick et al. [Science 271 (1996) 1835]. This analysis affords an experimental determination of the microscopic twist rigidity C. Quantitative agreement between theory and experiment is obtained using C = 120 nm and D = 50 nm. The theory further predicts a thermal reduction of the effective twist rigidity induced by bend fluctuations.

We study fluctuations of DNA-cationic lipid complexes in their lamellar membrane phases with DNA intercalated between lipid membranes. We theoretically elucidate this novel state of matter by characterizing it as the very first realization of a decoupled (unregistered) phase of strongly fluctuating 2-d smectic manifolds weakly interacting across membranes. Due to couplings between adjacent 2-d smectic Lx, × Ly planes, the experimentally observed ordinary 2-d smectic behavior [Salditt et al., Phys. Rev. Lett. 79, 2582 (1997)] of DNA in-plane undulations, with , must cross over, at the longest scales, to a novel fluctuation behavior, with < u2 > ˜ (logLy )2 ˜ (logLx.)2.

Experiments have been performed on the cationic bilayer and liposome-forming surfactant DODAB, a successful DNA transfection agent. The surface forces apparatus (SFA) was utilized to measure the long-range colloidal and short-range adhesion forces between Langmuir-Blodgett bilayers exposing DODAB in the outer monolayers. Forces were measured in aqueous solutions of varying salt concentrations between 1 and 10 mM, with and without single-stranded DNA added to the solution. These represent the first measurements of the forces between surfactant/DNA assemblies. At low salt concentrations without DNA, the interbilayer forces are repulsive due to their electrostatic interaction, in agreement with DLVO theory. In contrast, in the presence of DNA which adsorbs to the bilayer surfaces, the forces between the bilayers changed to an enhanced and longer ranged repulsive steric-electrostatic interaction. At shorter range. the DNA can be excluded from the gap leading to an enhanced adhesion at contact accompanied by lipid rearrangement.

The thermally driven motions of fluorescently labeled microtubules embedded in a network of filamentous actin polymers are analysed as the diffusion of a rod-like polymer within a virtual tube formed by the surrounding semiflexible actin filaments. The apparent diffusion constant parallel to the tube scales with the inverse of the microtubule length and the magnitude is consistant with diffusion through a medium with a viscosity of approximately 10 centipoise. Introduction of crosslinks between the actin filaments does not alter the diffusion of the microtubules in the actin network.

We report here the first observation and microscopic characterization of tactoidal granules of actin in concentrated gels of actin filaments (F-actin). Phase contrast microscopy shows these stable tactoids of densely packed F-actin to be of various sizes on the order of 10 μm. The background gel of F-actin is optically birefringent, indicating orientational order of the filaments consistent with theoretical predictions. In contrast, no birefringence is detected through the tactoids, suggesting very distinct yet undetermined packing of the filaments inside. The tactoids demonstrate elastic response upon micromanipulation. The microscopic segregation of F-actin into two states of different protein concentrations is consistent with a first order phase transition between a nematic gel of lower concentration on the order of ten mg/nl and a highly packed, possibly columnar state of protein filaments. In addition to the excluded volume effects which are known to drive thermodynamic phase transitions as a function of particle concentration, additional molecular forces must also play important roles since the formation was found to be irreversible upon dilution.

Plant and animal cells contain a complex polymeric network known as the cytoskeleton. A principal component of this is the actin cortex, a gel-like network of F-actin protein filaments. Recently, solutions of reconstituted F-actin have provided in vitro models of the actin cortex, as well as excellent model systems in which to study semiflexible polymers. We describe models of viscoelasticity in semifexible polymers, and report theoretical and experimental results for thermal fluctuations of embedded particles, which act as local viscoelastic probes of soft materials such as biopolymer solutions. Specifically, we report high-frequency scaling behavior of the shear modulus, as the 3/4 power of frequency, in contrast with the behavior of flexible polymer systems.

We discuss a dynamical model for the frequency-dependent shear modulus of an entangled solution of semifexible polymers, based on longitudinal fluctuations in filaments between entanglement points or crosslinks. The goal is to explain non-Rouse, power-law scaling of the bulk shear modulus that is found via microscopic rheology of highly entangled F-actin solutions. This generalizes a previous model for the static modulus. Hydrodynamic effects, and the validity of a local drag approximation below the scale of the mesh size, are discussed. We test aspects of our model via a molecular dynamics simulation, and also present for comparison experimental results from microrheology on F-actin.

The behaviour of polymer chains grafted to a surface has attracted considerable interest due to its broad range of applications in both colloid science and biological systems.

This work compares the electrostatic bundling of two Inovirus particles: fd and M13, that are structurally identical except that the effective axial charge density of M 13 is approximately 30% lower than that of fd. The electrostatic force (or osmotic pressure) between these ordered biopolyelectrolytes, as a function of inter-rod separation, has been calculated with Monte Carlo simulations. Comparison is made with experiments on the bundling of fd and M 13 caused by divalent ions, as detected by light scattering. In the theoretical calculations, the bundling results from electrostatic attraction between the neighbouring polyelectrolytes, caused by the correlated interactions between the ion clouds. The importance of this effect (i. e. capacity to induce bundling) is governed by surface charge density of the polyelectrolyte, amount of multivalent ion present, charge and size of the hydrated multivalent ion. These predictions are in very good agreement with the presented experimental results on bundling of fd and M 13 caused by Ca2+ and Mg2+

We present an application of first principles molecular dynamics simulations, based on the Car-Parrinello method, to the study of the properties of glucose in aqueous solution. By treating both solute and solvent quantum-mechanically, we provide a reliable description of the hydrogen bonds, which determine the water structure around the glucose. These results show the power of our technique in describing biologically relevant problems, where an accurate microscopic description is essential.

The yeast S. cereviseae represents the first eukaryotic organism whose genome has been entirely sequenced as a result of the Human Genome Project(1). In this report we demonstrate the good agreement between an experimental high resolution melting curve of total nuclear S. cereviseae DNA and the theoretical melting calculated for the complete yeast DNA genome (12,067,277 bp: Saccharomyces Genome Database) by the statistical thermodynamics program MELTSIM, parameterized for long DNA sequences(2,3). The experimental and theoretical melting curves are both fairly symmetrical and possess nearly identical Tm values. Calculated melting of coding and flanking DNA regions indicates that flanking DNAs are more (A+T)-rich than coding sequences and account for the earliest melting DNA. Calculated melting curves of the 16 individual yeast chromosomes are very similar and with few exceptions exhibit symmetric melting curves. MELTSIM was also used to calculate a theoretical denaturation map of Chromosome III DNA. The agreement between MELTSIM calculated and experimental melting data demonstrates our ability to accurately simulate long DNA sequence melting in complex eukaryotic genomes, whose sequences are becoming increasingly available for study in public databases. This has important consequences for the understanding of sequence dependent energetic properties of DNA in their biological sequence context and also for their potential use in biomaterials applications.

The general Lifshitz theory of van der Waals interaction was used to formulate and compute energies between the nucleotides situated on the opposite ends of a broken DNA helix. Our calculation show that infrared and ultraviolet resonances in the dielectric functions of DNA and the intracellular liquid account for less then 10 percent of the forces of interaction, at a range of 5-15 angstroms, between nucleotides. Thus the fundamental contribution to the interaction is presented by the group of resonances with frequencies of X - ray range. It was shown that during the interaction between thymine - guanine, adenine - guanine and cytosine - guanine there exists a potential barrier which prevents DNA selfrepairing after a mutanous, over a distance of about 7 - 20 angstroms, at the room temperature and with reference to the viscosity factor for pure water. All the remaining pairs of nucleotides have no such barrier. In addition the barrier vanishes and DNA undergoes complete selfrepairing with the decreace in viscosity of intracellular medium.

The ATP-dependent motility of the kinesin-related non claret disjunctional (ncd) mechanoenzyme was observed in an in vitro bead motility assay using optical tweezers in combination with a new two-dimensional displacement detection method. The detection technique is based on observing the far-field interference pattern formed in the back focal plane (BFP) of the microscope condenser by the illuminating laser focus and the light scattered from the trapped dielectric bead. The ability to observe the two-dimensional motion, with high temporal and spatial resolution, and in a manner largely independent of position in the microscope field-of-view, is the particular advantage of this detection method. In the assay, a fusion protein (GST-N195) of truncated ncd and glutathione-Stransferase was adsorbed to silica beads and the axial and lateral motions of the beads along the microtubule surface were observed. The average axial velocity of the ncd coated beads was 230 ± 30 nm/s (± std. dev.). Spectral analysis of bead motion showed an increase in viscous drag near the surface. Furthermore, we also found that any elastic constraints of the moving motors are much smaller than the constraints due to binding in the presence of the non-hydrolyzable nucleotide adenylylimido-diphosphate (AMP-PNP).

It is becoming clear that quantal behavior is a central feature of contractile systems. Steplike behavior has been demonstrated in the kinesin - microtubule system and in the myosin - actin filament system-both on molecular scale. We show here that step-like features appear also in the single intact sarcomere. We studied single sarcomeres of single bumblebee myofibrils. Motorimposed ramp length changes on activated myofibrils resulted in sarcomere-length changes that were stepwise. Computer analysis of the stepwise shortening patterns revealed a step-size distribution containing multiple peaks. The peaks were separated by 2.7 nm per half-sarcomere which is the linear actin-subunit spacing. Thus, translation steps are an integer multiple of the actin-subunit spacing. This result parallels the one observed in the kinesin-tubulin spacing where step size is a multiple of the tubulin-subunit spacing. In the muscle system, however, the steps are preserved on a macroscopic scale, implying high synchrony. The quantal steps are easily explained by a model in which the actin filament propels itself over stationary cross-bridges: if actin binds to the cross-bridges between steps, then the observed quantal result is inevitable.

We monitor the effect of transversal membrane asymmetry on the morphology of giant uni-lamellar vesicles in sugar and polymer solutions. The shapes of fluid lipid vesicles are governed by the bending elasticity of their membrane which is characterized by the bending modulus and the spontaneous curvature of the bilayer. We present a recently developed technique for the measurement of the spontaneous curvature using quantitative phase contrast microscopy. Different mechanisms for elastic membrane asymmetry and the role of the bending energy concept for the morphology of cellular organelles are discussed.

We used the micropipet aspiration technique for a study of biomembrane adhesion. Adhesion was caused by contact site A, a highly specific cell adhesion molecule, reconstituted in lipid vesicles of DOPC with 5 %(mol/mol) DOPE-PEG2000. We found adhesion and subsequent receptor aggregation in the contact zone. Additionally, electrostatic modulation of membrane adhesion was studied. Whereas addition of the negatively charged lipid SOPS to the lecithin (SOPC) host membrane suppressed adhesion due to electrostatic repulsion, a positively charged lipid (DOTAP) was surprisingly ineffective. This might be due to either phase separation of the mixture or DOTAP changing other membrane properties as bending stiffness and the Hamaker constant.

Single and multicomponent membrane-mimetic surfaces of DPPC and synthetic lipid-peptide conjugates were formed on an alkylated glass surface by a process of vesicle fusion. Correlative atomic force microscopy and radiochemical titration techniques confirmed the generation of a single substrate supported monolayer and predictable deposition of defined concentrations of lipopeptide. Mixed systems were stable for periods exceeding 1 month if stored at room temperature in phosphate buffered saline.

Electric field-induced transient birefringence and light scattering are reported for aqueous suspensions of synthetic unilamellar bilayer vesicles, prepared from the lipid dioleoylphosphatidylcholine (DOPC). The multiexponential birefringence relaxations observed on the microsecond and millisecond timescales are interpreted in terms of elongation and reorientation of induced dipolar vesicles, their linear chain formation, and electrofusion (and possibly electroporation) of the vesicles. Above certain threshold values of vesicle concentration, field-induced light scattering occurs concomitant with the birefringence. The corresponding transient signals corroborate the linear chain formation and subsequent fusion of the induced dipolar vesicles.

An equimolar mixture of palmitic acid (PA) and 1-lyso-palmitoyl-phosphatidylcholine (Lyso-PPC) has been studied by time-resolved small angle and wide angle X-ray scattering during temperature cycles between room temperature and 53.5 °C. In addition to the X-ray experiments, differential scanning calorimetry (DSC) with a similar temperature vs. time course was performed. At room temperature we observe a coexistence of two different lamellar phases. Our results indicate that in one of these lamellar phases, Lyso-PPC and PA associate in lamellar structures resembling di-palmitoyl-phosphatidylcholine (DPPC) bilayers, whereas the other phase is an interdigitated lamellar phase, where the acyl chains of both Lyso-PPC and PA extend across the entire hydrocarbon width of the bilayer. Upon heating, the latter phase disappears at 42 °C, corresponding to the chain-melting temperature for DPPC bilayers. At a higher temperature the remaining lamellar phase undergoes a phase transition into an isotropic micellar phase. In the cooling scans a particular slow kinetics of the regeneration of the two lamellar phases is observed. Our experiments provide new information about this system where only the DPPC-like mesophase has been reported before.