Université PSL



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A nanostructure made of a small non-coding RNA
Laboratoire Physico-biologie aux méso-échelles - Cayrol B., Nogues C., Dawid A., Sagi I., Silberzan P., Isambert H.
JACS - 131(47) :17270-6 - DOI:10.1021/ja906076e - 2009
Natural RNAs, unlike many proteins, have never been reported to form extended nanostructures, despite their wide variety of cellular functions. This is all the more striking, as synthetic DNA and RNA forming large nanostructures have long been successfully designed. Here, we show that DsrA, a 87-nt noncoding RNA of Escherishia coli, self-assembles into a hierarchy of nanostructures through antisense interactions of three contiguous self-complementary regions. Yet, the extended nanostructures, observed using atomic force microscopy (AFM) and fluorescence microscopy, are easily disrupted into >100 nm long helical bundles of DsrA filaments, including hundreds of DsrA monomers, and are surprisingly resistant to heat and urea denaturation. Molecular modeling demonstrates that this structural switch of DsrA nanostructures into filament bundles results from the relaxation of stored torsional constraints and suggests possible implications for DsrA regulatory function.
Mechanisms of chiral discrimination by topoisomerase IV
Laboratoire Physique des biomolécules - K. C. Neuman, G. Charvin, D. Bensimon and V. Croquette
Proc. Nat. Acad. Sci. USA - 106(17) :6986–91 - DOI:10.1073/pnas.0900574106 - 2009
Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase, transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results.
Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers
Laboratoire Physique des biomolécules - F. Mosconi, J.-F. Allemand, D. Bensimon and V. Croquette
Phys. Rev. Lett. - 102(7) :78301 - PMID:19257716 - 2009
We deduced the torque applied on a single stretched and twisted DNA by integrating the change in the molecule's extension with respect to force as it is coiled. While consistent with previous direct measurements of the torque at high forces (F > 1 pN), this method, which is simple and does not require a sophisticated setup, allows for lower force estimates. We used this approach to deduce the effective torsional modulus of DNA, which decreases with force, and to estimate the buckling torque of DNA as a function of force in various salt conditions.
Single-cell response to stiffness exhibits muscle-like behavior
Laboratoire Physique des biomolécules - D. Mitrossilis, J. Fouchard, A. Guiroy, N. Desprat, N. Rodriguez, B. Fabry and A. Asnacios
Proc. Nat. Acad. Sci. USA - 106(43) :18243–8 - DOI:10.1073/pnas.0903994106 - 2009
Living cells sense the rigidity of their environment and adapt their activity to it. In particular, cells cultured on elastic substrates align their shape and their traction forces along the direction of highest stiffness and preferably migrate towards stiffer regions. Although numerous studies investigated the role of adhesion complexes in rigidity sensing, less is known about the specific contribution of acto-myosin based contractility. Here we used a custom-made single-cell technique to measure the traction force as well as the speed of shortening of isolated myoblasts deflecting microplates of variable stiffness. The rate of force generation increased with increasing stiffness and followed a Hill force–velocity relationship. Hence, cell response to stiffness was similar to muscle adaptation to load, reflecting the force-dependent kinetics of myosin binding to actin. These results reveal an unexpected mechanism of rigidity sensing, whereby the contractile acto-myosin units themselves can act as sensors. This mechanism may translate anisotropy in substrate rigidity into anisotropy in cytoskeletal tension, and could thus coordinate local activity of adhesion complexes and guide cell migration along rigidity gradients.
Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome
Laboratoire Physique des biomolécules - M. Manosas, M. M. Spiering, Z. Zhuang, S. J. Benkovic and V. Croquette
Nature Chemical Biology - 5(12) :904–12 - DOI:10.1038/nchembio.236 - 2009
The unwinding and priming activities of the bacteriophage T4 primosome, which consists of a hexameric helicase (gp41) translocating 5' to 3' and an oligomeric primase (gp61) synthesizing primers 5' to 3', have been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a primosome disassembly mechanism or a new DNA looping mechanism. A fused gp61-gp41 primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms by disfavoring primosome disassembly, thereby increasing primase processivity. In contrast to T4, priming in bacteriophage T7 and Escherichia coli involves discrete pausing of the primosome and dissociation of the primase from the helicase, respectively. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.
Photomanipulation of a Droplet by the Chromocapillary Effect
Laboratoire Pôle Microfluidique - A. Diguet, R. M. Guillermic, N. Magome, A. Saint-Jalmes, Y. Chen, K. Yoshikawa and D. Baigl
Angew Chem Int Ed Engl. - 48(49) :9281-4 - DOI:10.1002/anie.200904868 - 2009
Ace of hearts: Liquid droplets can be manipulated in a controlled fashion along trajectories of any desired shape (such as a heart, see picture) by using light to create a wavelength-dependent interfacial tension gradient at a liquid/liquid interface. In this new phenomenon, the “chromocapillary effect”, an interfacial flow generates droplet motion in the direction opposite to the gradient.
Sequence-Independent and Reversible Photocontrol of Transcription/Expression Systems Using a Photosensitive Nucleic Acid Binder
Laboratoire Pôle Microfluidique - A. Estevez-Torres, C. Crozatier, A. Diguet, T. Hara, H. Saito, K. Yoshikawa and D. Baigl
Proc. Nat. Acad. Sci. USA - 106(30) :12219-23 - DOI:10.1073/pnas.0904382106 - 2009
To understand non-trivial biological functions, it is crucial to develop minimal synthetic models that capture their basic features. Here, we demonstrate a sequence-independent, reversible control of transcription and gene expression using a photosensitive nucleic acid binder (pNAB). By introducing a pNAB whose affinity for nucleic acids is tuned by light, in vitro RNA production, EGFP translation, and GFP expression (a set of reactions including both transcription and translation) were successfully inhibited in the dark and recovered after a short illumination at 365 nm. Our results indicate that the accessibility of the protein machinery to one or several nucleic acid binding sites can be efficiently regulated by changing the conformational/condensation state of the nucleic acid (DNA conformation or mRNA aggregation), thus regulating gene activity in an efficient, reversible, and sequence-independent manner. The possibility offered by our approach to use light to trigger various gene expression systems in a system-independent way opens interesting perspectives to study gene expression dynamics as well as to develop photocontrolled biotechnological procedures.
Wetting and spreading
Laboratoire Procédés, Plasmas, Microsystèmes - Bonn D., Eggers J., Iindekeu J., Meunier J., Rolley E.
REVIEWS OF MODERN PHYSICS - 81(2) :739-805 - DOI:10.1103/RevModPhys.81.739 - 2009
Wetting phenomena are ubiquitous in nature and technology. A solid substrate exposed to the environment is almost invariably covered by a layer of fluid material. In this review, the surface forces that lead to wetting are considered, and the equilibrium surface coverage of a substrate in contact with a drop of liquid. Depending on the nature of the surface forces involved, different scenarios for wetting phase transitions are possible; recent progress allows us to relate the critical exponents directly to the nature of the surface forces which lead to the different wetting scenarios. Thermal fluctuation effects, which can be greatly enhanced for wetting of geometrically or chemically structured substrates, and are much stronger in colloidal suspensions, modify the adsorption singularities. Macroscopic descriptions and microscopic theories have been developed to understand and predict wetting behavior relevant to microfluidics and nanofluidics applications. Then the dynamics of wetting is examined. A drop, placed on a substrate which it wets, spreads out to form a film. Conversely, a nonwetted substrate previously covered by a film dewets upon an appropriate change of system parameters. The hydrodynamics of both wetting and dewetting is influenced by the presence of the three-phase contact line separating ``wet'' regions from those that are either dry or covered by a microscopic film only. Recent theoretical, experimental, and numerical progress in the description of moving contact line dynamics are reviewed, and its relation to the thermodynamics of wetting is explored. In addition, recent progress on rough surfaces is surveyed. The anchoring of contact lines and contact angle hysteresis are explored resulting from surface inhomogeneities. Further, new ways to mold wetting characteristics according to technological constraints are discussed, for example, the use of patterned surfaces, surfactants, or complex fluids.
Development of oligonucleotide microarray involving plasma polymerized acrylic acid” Thin Solid Films
Laboratoire Procédés, Plasmas, Microsystèmes - R. Jafari , M. Tatoulian, D. Le Clerre, L. Talini, F. Richard, F. Arefi-Khonsari
Thin Solid Films - 517(19) :5763-8 - DOI:10.1016/j.tsf.2009.03.217 - 2009
This paper presents the manufacturing of biochips by using the COOH– derived polymer coating deposited by plasma polymerization of acrylic acid. This technology is based on depositing a thin layer obtained by plasma polymerization of acrylic acid which allows a further covalent immobilization of biomolecules on glass substrates. The plasma power value was optimized to maximize the stability of plasma polymerized acrylic acid (PPAA) coatings in water, which has a very important role for such applications. In order to obtain a covalent immobilization of DNA probes on the PPAA coated surface, the activation protocol of carboxylic function was carried out with the help of N-Hydroxy Succinimide and 1-Ethyl-3-(3-DimethylAminopropyl) Carbodiimide. The efficiency of PPAA coated in microarray applications was compared with two types of commercial slides. Such surfaces have shown very interesting results in terms of relative density of attached DNA probe molecules and signal-to-background ratio measured for target DNA hybridization. Nonspecific DNA bonding measurements showed only a small amount of nonspecific physisorption between the DNA probe and the PPAA-activated surfaces. This work shows that the plasma polymerization technique can be successfully applied to produce a high-quality glass surface for the manufacturing of DNA arrays.
Determination of Cocaine in Human Plasma by Selective Solid-Phase Extraction Using an Aptamer-Based Sorbent
Laboratoire Sciences Analytiques, Bioanalytiques et Miniaturisation - B. Madru, F. Chapuis-Hugon, E. Peyrin, V. Pichon
Anal. Chem. - 81(16) :7081-6 - DOI:10.1021/ac9006667 - 2009
A complete characterization is presented of a highly selective solid-phase extraction (SPE) sorbent which exploits the properties of aptamers. An oligosorbent based on aptamers immobilized on a solid support was synthesized and tested for the selective extraction of cocaine from human plasma. Anticocaine aptamers were immobilized to CNBr-activated Sepharose, and an extraction procedure was developed in pure media. Specific retention of cocaine on the oligosorbent was demonstrated, and the capacity of the support was determined. This oligosorbent was then applied to the selective extraction of cocaine from plasma at a concentration of 0.4 mg L(-1), i.e., corresponding to the plasma concentration reached after an intake of a single dose of cocaine. Extraction recovery close to 90% was obtained. Moreover, interfering compounds that perturbed cocaine quantification when using a standard SPE sorbent were not retained on the oligosorbent, thus allowing fast and reliable analyses of plasma samples with an estimated limit of detection of 0.1 microg mL(-1).

410 publications.