The promotion of secondary structures in single-stranded DNA by drugs that bind to duplex DNA: an atomic force microscopy study

We study the behavior of single-stranded DNA (ssDNA) in the presence of well-known drugs with either an intercalating binding mode, such as daunorubicin, actinomycin D, and chloroquine, or a minor groove binding mode, such as netropsin and berenil, by atomic force microscopy (AFM). At very low salt conditions, ssDNA molecules adopt an unstructured conformation without secondary structures. We observe that under these conditions additions of?drugs that bind to double-stranded DNA (dsDNA) promote the formation of secondary structures in ssDNA. Furthermore, with an increase of concentration of the drugs, the extension as well as the thermal stabilization of these hairpins was observed.     

Sharp DNA bends as landmarks of protein-binding sites on straightened DNA

We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp). In the described method, protein-DNA complexes were straightened and immobilized on a flat surface using surface tension. A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex. The presence of DNA-binding proteins at the apex was verified by atomic force microscopy. The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy. We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity. The proposed protein-binding-site mapping technique is simple and easy to perform. Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA.     

Micro- and nano-scale deformation processes during scratch damage in high density polyethylene

Abstract

Atomic force microscopy and scanning electron microscopy techniques have been used to examine the surface deformation experienced by high density polyethylene during the scratch test. The scratch deformation process involves stretching of fibrils and microfibrils resulting in the formation of surface openings. At the molecular level the chains of molecules unfold and align in the direction of the moving indenter. In the scratch test, the scratch velocity may suggest that low strain rates are valid, but the local strain rates can be many orders of magnitiude higher as exemplified by atomic force microscopy. A number of modes of deformation are encountered during scratching. They include deformation bands, crazing, tearing, microcracking, regular cracking, and grooving. Crazing-tearing is the predominant mode of scratch deformation. It is envisaged that the sequence of tearing along the craze involves formation of deformation bands, development of craze, followed by tearing. Atomic force and scanning electron microscopy of scratch surface damage indicated that the nature and modes of scratch deformation are qualitatively similar to the case of uniaxial tensile deformation, implying similarities in the deformation behaviour between scratch and tensile deformation.     

Atomic force microscopy of the morphology and mechanical behaviour of barnacle cyprid footprint proteins at the nanoscale

Barnacles are a major biofouler of man-made underwater structures. Prior to settlement, cypris larvae explore surfaces by reversible attachment effected by a ‘temporary adhesive’. During this exploratory behaviour, cyprids deposit proteinaceous ‘footprints’ of a putatively adhesive material. In this study, footprints deposited by Balanus amphitrite cyprids were probed by atomic force microscopy (AFM) in artificial sea water (ASW) on silane-modified glass surfaces. AFM images obtained in air yielded better resolution than in ASW and revealed the fibrillar nature of the secretion, suggesting that the deposits were composed of single proteinaceous nanofibrils, or bundles of fibrils. The force curves generated in pull-off force experiments in sea water consisted of regions of gradually increasing force, separated by sharp drops in extension force manifesting a characteristic saw-tooth appearance. Following the relaxation of fibrils stretched to high strains, force–distance curves in reverse stretching experiments could be described by the entropic elasticity model of a polymer chain. When subjected to relaxation exceeding 500 ms, extended footprint proteins refolded, and again showed saw-tooth unfolding peaks in subsequent force cycles. Observed rupture and hysteresis behaviour were explained by the ‘sacrificial bond’ model. Longer durations of relaxation (>5 s) allowed more sacrificial bond reformation and contributed to enhanced energy dissipation (higher toughness). The persistence length for the protein chains (L_P) was obtained. At high elongation, following repeated stretching up to increasing upper strain limits, footprint proteins detached at total stretched length of 10 µm.     

Probing the kinetics of SYTOX Orange stain binding to double-stranded DNA with implications for DNA analysis

Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 10(8) M-1 s-1, suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry.     

Mechanical characterization of free-standing polypyrrole film

Free-standing polypyrrole films, neutral or doped with ClO₄⁻, have been mechanically characterized. Their elasticity of both dry and wet states was characterized by mean of the Young's modulus. In addition, the Young's modulus was calculated as a function of the film oxidation deep by “in situ” polarization at different potentials ranging from − 0.6 up to 0.8 V. The samples also were characterized under oxidation and reduction by reverse constant currents. When the films were submitted to a constant stretching force the length variations were obtained. At a constant length of the sample the electro-chemo-mechanical force developed by the redox processes was obtained. As predicted by the ESCR model lineal variations of both, length and force, as a function of the consumed charge were obtained.     

Screening the sequence selectivity of DNA-binding molecules using a gold nanoparticle-based colorimetric approach

We have developed a novel competition assay that uses a gold nanoparticle (Au NP)-based, high-throughput colorimetric approach to screen the sequence selectivity of DNA-binding molecules. This assay hinges on the observation that the melting behavior of DNA-functionalized Au NP aggregates is sensitive to the concentration of the DNA-binding molecule in solution. When short, oligomeric hairpin DNA sequences were added to a reaction solution consisting of DNA-functionalized Au NP aggregates and DNA-binding molecules, these molecules may either bind to the Au NP aggregate interconnects or the hairpin stems based on their relative affinity for each. This relative affinity can be measured as a change in the melting temperature (Tm) of the DNA-modified Au NP aggregates in solution. As a proof of concept, we evaluated the selectivity of 4',6-diamidino-2-phenylindone (an AT-specific binder), ethidium bromide (a nonspecific binder), and chromomycin A (a GC-specific binder) for six sequences of hairpin DNA having different numbers of AT pairs in a five-base pair variable stem region. Our assay accurately and easily confirmed the known trends in selectivity for the DNA binders in question without the use of complicated instrumentation. This novel assay will be useful in assessing large libraries of potential drug candidates that work by binding DNA to form a drug/DNA complex.     

Investigation of Surface Properties of Magnetorheological Elastomers by Atomic Force Microscopy

Magnetorheological elastomers generally consist of a natural or synthetic rubber matrix interspersed with micron-sized ferromagnetic particles. The magneto-elastic properties of such a composite are not merely a sum of the elasticity of the polymer and the stiffness and magnetic properties of the filler, but also the result of a complex synergy of several effects, relevant at different length scales and detectable by different techniques. In our present work we investigate the microstructures, the surface magnetic properties, and the elastic properties of new isotropic and anisotropic magnetorheological elastomers prepared using silicone rubber and soft magnetic carbonyl iron microspheres. Similar samples were previously investigated by means of small-angle neutron scattering, which proved to be a useful method in the investigation of their microscopic properties. We combined the data from the atomic force microscopy measurements with those from small-angle neutron scattering to better understand the complicated behaviour of the studied materials. The measurements were performed by atomic force microscopy in the following modes: standard imaging non-contact atomic force microscopy, magnetic force microscopy, and nanoindentation. A comparative study of samples with different particle concentrations and strength of magnetic field applied during the polymerization process is developed.     

Growth and characterization of Na3BaCl5·2H2O

Na₃BaCl₅·2H₂O crystals were prepared by the slow evaporation of an aqueous solution of a mixture of sodium chloride and barium chloride in stoichiometric ratio. Crystals were found to possess platelet habit. Crystals were analysed by infrared spectrophotometry, thermogravimetry and X-ray diffraction. The appearance of bands due to stretching and bending modes of water molecules in the IR spectra showed that the grown crystals were hydrated. X-ray oscillation and Weissenberg photographs were used to measure the dimensions of the unit cell.     

Investigation of quadruplex oligonucleotide-drug interactions by electrospray ionization mass spectrometry

Selectivity, binding stoichiometry, and mode of binding of Tel01, distamycin A, and diethylthiocarbocyanine iodide (DTC) to the parallel stranded G4-quadruplex [d(T2G5T)]4 were investigated by ESI-MS. The first drug/quadruplex complexes observed by ESI-MS are described. Tel01, distamycin A, and DTC all form complexes with quadruplex DNA, but only Tel01 is completely selective for quadruplex versus duplex oligonucleotide under the conditions employed. Previous solution determinations of the binding mode of Tel01 and distamycin A to quadruplex oligonucleotides indicate that Tel01 interacts through end-stacking with guanine tetrads of quadruplex DNA, while distamycin A interacts by binding to quadruplex grooves. When these two different drug/quadruplex complexes are subjected to collisionally activated dissociation in a mass spectrometer, the observed fragmentation patterns are distinct. Tel01/quadruplex complexes undergo facile loss of drug and dissociation to single-strand oligonucleotide ions, while distamycin/quadruplex complexes fragment into single-strand oligonucleotide ions in which the drug molecule is retained. Dissociation patterns for DTC/quadruplex complexes are similar to those of distamycin; therefore, it is concluded that DTC interacts with [d(T2G5T)]4 through groove-binding. These ESI-MS results are applicable to both the identification and characterization of G-quadruplex interactive agents and may also be useful in probing unusual DNA structures.     

Oxygen Phonon Branches in Detwinned YBa2Cu3O7

We report results of inelastic neutron scattering measurements of phonon dispersions on a detwinned sample of YBa₂Cu₃O₇ and compare them with model calculations. Plane oxygen bond stretching phonon branches disperse steeply downwards from the zone center in both the a and the b direction indicating a strong electron-phonon coupling. Half way to the zone boundary, the phonon peaks become ill-defined but we see no need to invoke unit cell doubling or charge stripe formation: lattice dynamical shell model calculations predict such behavior as a result of branch anticrossings. There were no observable superconductivity-related temperature effects on selected plane oxygen bond stretching modes measured on a twinned sample.     

Stochastic simulations of square aluminium tubes subjected to axial loading

Stochastic simulations of square aluminium tubes of 6060 T6 aluminium alloy subjected to axial crushing have been performed and compared to an experimental program carried out previously at SIMLab. The main variables during testing were the extrusion length, the wall thickness of the extrusions and the impact velocity of the impactor. Three different buckling modes were observed: progressive buckling, transition from progressive to global buckling and global buckling. Progressive buckling was primarily observed in the short specimens, while increasing the length a transition mode took place. However, for the thick-walled tubes a direct global buckling mode was found. In the present study, it has been investigated if geometric imperfections modelled by assumed Gaussian random fields could explain the experimentally observed behaviour. Variation of the random field parameters by use of a factorial design resulted in variations in especially the buckling modes and consequently the average force, and the same buckling modes found in the experiments were obtained in the simulations. For the chosen model, the shape of the geometric imperfections seemed to be more important than the amplitude. Further, simulations of profiles with three different lengths subjected to impacts at two different velocities were performed. The estimated probability for progressive buckling to appear decreased as the length increased, while the change in velocity had minor effects for the chosen parameter range.     

Forward and Backward Four-wave Mixing of Non-classical Light with Pump Depletion

Abstract

Photon statistics and non-classical behaviour of forward and backward four-wave mixing are examined including the coupling of modes in the approximation of small fluctuations around a stationary point, which is analytically determined and conditions for its stability are obtained. The depletion of pump modes is involved and effects of nonlinear dynamics, non-classical behaviour of input radiation, external noise and losses are included.     

Modelling of stretch forming behaviour in steel strip using finite element method

Abstract

Laboratory tests were carried out on CR1 grade steel strip involving punch stretching without lubrication and with PQ6 oil or polythene as lubricant. The finite element code was then used to simulate the forming behaviour during the test. The effects of normal anisotropy of the material and the contact friction between punch/die and the specimen on the forming behaviour were studied systematically. The predictability of the finite element modelling was confirmed by direct comparisons between the experimental measurements and the computer predictions. The aspects of further refinements required in the finite element models are discussed.

MST/1887     

Non-equilibrium all-atom molecular dynamics simulations of free and tethered DNA molecules in nanochannel shear flows

In order to gain insight into the mechanical and dynamical behaviour of free and tethered short chains of ss/ds DNA molecules in flow, and in parallel to investigate the properties of long chain molecules in flow fields, we have developed a series of quantum and molecular methods to extend the well developed equilibrium software CHARMM to handle non-equilibrium dynamics. These methods have been applied to cases of DNA molecules in shear flows in nanochannels. Biomolecules, both free and wall-tethered, have been simulated in the all-atom style in solvent-filled nanochannels. The new methods were demonstrated by carrying out NEMD simulations of free single-stranded DNA (ssDNA) molecules of 21 bases as well as double-stranded DNA (dsDNA) molecules of 21 base pairs tethered on gold surfaces in an ionic water shear flow. The tethering of the linker molecule (6-mercapto-1-hexanol) to perfect Au(111) surfaces was parametrized based on density functional theory (DFT) calculations. Force field parameters were incorporated into the CHARMM database. Gold surfaces are simulated in a Lennard-Jones style model that was fitted to the Morse potential model of bulk gold. The bonding force of attachment of the DNA molecules to the gold substrate linker molecule was computed to be up to a few nN when the DNA molecules are fully stretched at high shear rates. For the first time, we calculated the relaxation time of DNA molecules in picoseconds (ps) and the hydrodynamic force up to a few nanoNewtons (nN) per base pair in a nanochannel flow. The velocity profiles in the solvent due to the presence of the tethered DNA molecules were found to be nonlinear only at high shear flow rates. Free ssDNA molecules in a shear flow were observed to behave differently from each other depending upon their initial orientation in the flow field. Both free and tethered DNA molecules are clearly observed to be stretching, rotating and relaxing. Methods developed in this initial work can be incorporated into multiscale simulations including quantum mechanical, molecular and the microfluidic continuum regimes. The results may also be useful in extending existing macroscopic empirical models of DNA response dynamics in shear flows.     

MutS-mediated detection of DNA mismatches using atomic force microscopy

We have developed an atomic force microscopy-based method for detecting DNA base-pair mismatches using MutS protein isolated from E. coli. MutS is a biological sensor and a locator of DNA base-pair mismatches. It binds specifically to a mismatched DNA base pair and initiates a process of DNA repair. To test the possibility of visually detecting mismatched base pairs by atomic force microscopy, we prepared DNA templates approximately 500 bp in length consisting of a single or multiple base-pair mismatches. We demonstrate that MutS binding sites on individual DNA molecules were readily detectable by atomic force microscopy and that the observed positions were in good agreement with the predicted sites of base-pair mismatches at a few-nanometer resolution. The technique described here is rapid and sensitive and is expected to be useful in screening mutations and DNA polymorphisms.     

High flexibility of DNA on short length scales probed by atomic force microscopy

The mechanics of DNA bending on intermediate length scales (5-100 nm) plays a key role in many cellular processes, and is also important in the fabrication of artificial DNA structures, but previous experimental studies of DNA mechanics have focused on longer length scales than these. We use high-resolution atomic force microscopy on individual DNA molecules to obtain a direct measurement of the bending energy function appropriate for scales down to 5 nm. Our measurements imply that the elastic energy of highly bent DNA conformations is lower than predicted by classical elasticity models such as the worm-like chain (WLC) model. For example, we found that on short length scales, spontaneous large-angle bends are many times more prevalent than predicted by the WLC model. We test our data and model with an interlocking set of consistency checks. Our analysis also shows how our model is compatible with previous experiments, which have sometimes been viewed as confirming the WLC.     

pH- and salt-dependent molecular combing of DNA: experiments and phenomenological model

λ-DNA as well as plasmids can be successfully deposited by molecular combing on hydrophobic surfaces, for pH values ranging from 4 to 10. On polydimethylsiloxane (PDMS) substrates, the deposited DNA molecules are overstretched by about 60-100%. There is a significant influence of sodium ions (NaCl) on the surface density of the deposited DNA, with a maximum near to 100 mM NaCl for a DNA solution (28 ng μl(-1)) at pH 8. The combing process can be described by a micromechanical model including: (i) the adsorption of free moving coiled DNA at the substrate; (ii) the stretching of the coiled DNA by the preceding meniscus; (iii) the relaxation of the deposited DNA to the final length. The sticky ends of λ-DNA cause an adhesion force in the range of about 400 pN which allows a stable overstretching of the DNA by the preceding meniscus. The exposing of hidden hydrophobic bonds of the overstretched DNA leads to a stable deposition on the hydrophobic substrate. The pH-dependent density of deposited DNA as well as the observed influence of sodium ions can be explained by their screening of the negatively charged DNA backbone and sticky ends, respectively. The final DNA length can be derived from a balance of the stored elastic energy of the overstretched molecules and the energy of adhesion.     

Direct visualization of RecQ helicase–DNA interaction with fluorescence microscopy and atomic force microscopy

RecQ helicase–DNA interactions were directly visualized with fluorescence microscopy. DNA–RecQ complexes formed in binding and unwinding reaction were stretched onto the hydrophobic surface by molecular combing method. The complexes can be observed with fluorescence microscope because the DNA molecules were labeled with dye molecules of YOYO-1. The DNA binding and unwinding activity of RecQ helicase leads to reduced lengths of the observed DNA molecules. More direct observations with atomic force microscopy were also made. It was seen that RecQ is mainly monomeric both in solution and after binding to DNA.