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Force fluctuations assist nanopore unzipping of DNA
V Viasnoff, N Chiaruttini, J Muzard, and U Bockelmann
Journal of Physics : Condensed Matter - 22(45) :454122 - DOI:10.1088/0953-8984/22/45/454122 - 2010
We experimentally study the statistical distributions and the voltage dependence of the unzipping time of 45 base-pair-long double-stranded DNA through a nanopore. We then propose a quantitative theoretical description considering the nanopore unzipping process as a random walk of the opening fork through the DNA sequence energy landscape biased by a time-fluctuating force. To achieve quantitative agreement fluctuations need to be correlated over the millisecond range and have an amplitude of order kBT/bp. Significantly slower or faster fluctuations are not appropriate, suggesting that the unzipping process is efficiently enhanced by noise in the kHz range. We further show that the unzipping time of short 15 base-pair hairpins does not always increase with the global stability of the double helix and we theoretically study the role of DNA elasticity on the conversion of the electrical bias into a mechanical unzipping force.
T7 RNA polymerase studied by force measurements varying cofactor concentration
P. Thomen, P. J. Lopez, U. Bockelmann, J. Guillerez, M. Dreyfus, and F. Heslot
Biophys. J. - 95(5) :2423–33 - DOI:10.1529/biophysj.107.125096 - 2009
RNA polymerases carry out the synthesis of an RNA copy from a DNA template. They move along DNA, incorporate nucleotide triphosphate (NTP) at the end of the growing RNA chain, and consume chemical energy. In a single-molecule assay using the T7 RNA polymerase, we study how a mechanical force opposing the forward motion of the enzyme along DNA affects the translocation rate. We also study the influence of nucleotide and magnesium concentration on this process. The experiment shows that the opposing mechanical force is a competitive inhibitor of nucleotide binding. Also, the single-molecule data suggest that magnesium ions are involved in a step that does not depend on the external load force. These kinetic results associated with known biochemical and mutagenic data, along with the static information obtained from crystallographic structures, shape a very coherent view of the catalytic cycle of the enzyme: translocation does not take place upon NTP binding nor upon NTP cleavage, but rather occurs after PPi release and before the next nucleotide binding event. Furthermore, the energetic bias associated with the forward motion of the enzyme is close to kT and represents only a small fraction of the free energy of nucleotide incorporation and pyrophosphate hydrolysis.
Probing DNA base pairing energy profiles using a nanopore
Virgile Viasnoff, Nicolas Chiaruttini, and Ulrich Bockelmann
European Biophysics Journal - 38(2) :263–9 - PMID:18836709 - 2009
We experimentally show that the voltage driven unzipping of long DNA duplexes by an a-hemolysin pore is sensitive to the shape of the base pairing energy landscape. Two sequences of equal global stability were investigated. The sequence with an homogeneous base pairing profile translocates faster than the one with alternative weak and strong regions. We could qualitatively account for theses observations by theoretically describing the voltage driven translocation as a biased random walk of the unzipping fork in the sequence dependent energy landscape.
A simple and universal tool to remove on-line impurities in mono- or two-dimensional liquid chromatography–mass spectrometry analysis
Hesse A-M, Marcelo P, Rossier J and Vinh J.
J. Chrom. A - 1189(1-2) :175-82 - DOI:10.1016/j.chroma.2007.12.060 - 2008
Several recurrent problems have always hindered mono-dimensional liquid chromatography-mass spectrometry proteomic analyses. Polymer contamination is a major problem because polymers could co-elute with compounds of interest (peptides). In this case spectral suppression degrades dynamic range and sensitivity. Polyethylene glycol derivatives count among the major contaminants. They are targeted in this work. They are eluted at 35-40% acetonitrile from C18 phase in every single reversed-phase run. Moreover, they are also observed in two-dimensional liquid chromatography in every salt fraction. A simple and robust method is presented here for rapid and efficient on-line removal of these impurities using self-regenerating purification microdevices.
Chiral Colloidal Clusters
D. Zerrouki, J. Baudry, D. Pine, P. Chaikin, J. Bibette
Nature - 455 :380-2 - DOI:10.1038/nature07237 - 2008
Chirality is an important element of biology, chemistry and physics. Once symmetry is broken and a handedness is established, biochemical pathways are set. In DNA, the double helix arises from the existence of two competing length scales, one set by the distance between monomers in the sugar backbone, and the other set by the stacking of the base pairs1. Here we use a colloidal system to explore a simple forcing route to chiral structures. To do so we have designed magnetic colloids that, depending on both their shape and induced magnetization, self-assemble with controlled helicity. We model the two length scales with asymmetric colloidal dumbbells linked by a magnetic belt at their waist. In the presence of a magnetic field the belts assemble into a chain and the steric constraints imposed by the asymmetric spheres force the chain to coil. We show that if the size ratio between the spheres is large enough, a single helicity is adopted, right or left. The realization of chiral colloidal clusters opens up a new link between colloidal science and chemistry. These colloidal clusters may also find use as mesopolymers, as optical and light-activated structures2, and as models for enantiomeric separation.
Decompressing Emulsion Droplets Favors Coalescence
N. Brémond, A.R. Thiam, J. Bibette
Phys. Rev. Lett. - 100(2) :024501 - DOI:10.1103/PhysRevLett.100.024501 - 2008
The destabilization process of an emulsion under flow is investigated in a microfluidic device. The experimental approach enables us to generate a periodic train of droplet pairs, and thus to isolate and analyze the basic step of the destabilization, namely, the coalescence of two droplets which collide. We demonstrate a counterintuitive phenomenon: coalescence occurs during the separation phase and not during the impact. Separation induces the formation of two facing nipples in the contact area that hastens the connection of the interfaces prior to fusion. Moreover, droplet pairs initially stabilized by surfactants can be destabilized by forcing the separation. Finally, we note that the fusion mechanism is responsible for a cascade of coalescence events in a compact system of droplets where the separation is driven by surface tension.
Measuring the kinetics of biomolecular recognition with magnetic colloids
L. Cohen-Tannoudji, E. Bertrand, J. Baudry, C. Robic, C. Goubault, M. Pellissier, A. Johner, F. Thalmann, N. K. Lee, C. M. Marques, J. Bibette
Phys. Rev. Lett. - 100(10) :108301 - DOI:10.1103/PhysRevLett.100.108301 - 2008
We introduce a general methodology based on magnetic colloids to study the recognition kinetics of tethered biomolecules. Access to the full kinetics of the reaction is provided by an explicit measure of the time evolution of the reactant densities. Binding between a single ligand and its complementary receptor is here limited by the colloidal rotational diffusion. It occurs within a binding distance that can be extracted by a reaction-diffusion theory that properly accounts for the rotational Brownian dynamics. Our reaction geometry allows us to probe a large diversity of bioadhesive molecules and tethers, thus providing a quantitative guidance for designing more efficient reactive biomimetic surfaces, as required for diagnostic, therapeutic, and tissue engineering techniques.
Theoretical study of sequence-dependent nanopore unzipping of DNA
U. Bockelmann and V. Viasnoff
Biophys. J. - 94(7) :2716–24 - DOI:10.1529/biophysj.107.111732 - 2008
We theoretically investigate the unzipping of DNA electrically driven through a nanometer-size pore. Taking the DNA base sequence explicitly into account, the unpairing and translocation process is described by a biased random walk in a one-dimensional energy landscape determined by the sequential basepair opening. Distributions of translocation times are numerically calculated as a function of applied voltage and temperature. We show that varying these two parameters changes the dynamics from a predominantly diffusive behavior to a dynamics governed by jumps over local energy barriers. The work suggests experimentally studying sequence effects, by comparing the average value and standard deviation of the statistical distribution of translocation times.
Interference and crosstalk in double optical tweezers using a single laser source
P. Mangeol and U. Bockelmann
Rev Sci Instrum - 79(8) :083103 - DOI:10.1063/1.2957652 - 2008
Experimental studies of single molecule mechanics require high force sensitivity and low drift, which can be achieved with optical tweezers. We built an optical tweezer setup for force measurements in a two bead assay. A cw infrared laser beam is split by polarization and focused by a high numerical aperture objective to create two traps. The same laser is used to form both traps and to measure the force by back focal plane interferometry. We show that although the two beams entering the microscope are designed to exhibit orthogonal polarization, interference and a significant parasitic force signal occur. Comparing the experimental results with a ray optics model, we show that the interference patterns are caused by the rotation of polarization on microscope lens surfaces and slides. The model qualitatively describes the pattern and the dependence of the parasitic force signal on the experimental parameters. We present two different approaches to experimentally reduce the crosstalk, namely, polarization rectification and frequency shifting.
Time-course analysis of mouse serum proteome changes following exposure of the skin to ionizing radiation
Guipaud O, Holler V, Buard V, Tarlet G, Royer N, Vinh J, Benderitter M.
Proteomics - 7(21) :3992-4002 - PMID:17960731 - 2007
Radiation-induced lesion outcomes of normal tissues are difficult to predict. In particular, radiotherapy or local exposure to a radioactive source by accident can trigger strong injury to the skin. The finding of biomarkers is of fundamental relevance for the prediction of lesion apparition and its evolution, and for the settlement of therapeutic strategies. In order to study radiation-induced cutaneous lesions, we developed a mouse model in which the dorsal skin was selectively exposed to ionizing radiation (IR). 2-D difference gel electrophoresis (2-D DIGE) coupled with MS was used to investigate proteins altered in expression and/or PTM in serum. Proteome changes were monitored from 1 day to 1 month postirradiation, at a dose of 40 Gy, in this specific model developing reproducible clinical symptoms ranging from erythema to skin ulceration with wound healing. About 60 proteins (including some isoforms and likely post-translational variants), representing 20 different proteins, that exhibited significant and reproducible kinetic expression changes, were identified using MS and database searches. Several proteins, down- or up-regulated from day one, could prove to be good candidates to prognosticate the evolution of a skin lesion such as necrosis. In addition, we observed shifts in pI of several spot trains, revealing potential PTM changes, which could also serve as indicators of irradiation or as predictors of lesion severity.


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579 publications.