Tunable corrugated patterns in an active nematic sheet

International audienc

Microfluidic patterning of miniaturized DNA arrays on plastic substrates

This paper describes the patterning of DNA arrays on plastic surfaces using an elastomeric, two dimensional (2D) microcapillary system (\u3bcCS). Fluidic structures were realized through hot embossing lithography using Versaflex\uae CL30. Like elastomers based on poly(dimethylsiloxane) (PDMS), this thermoplastic block co-polymer is able to seal a surface in a reversible manner, making it possible to confine DNA probes with a level of control that is unparalleled using standard microspotting techniques. We focus on \u3bcCSs that support arrays comprising up to 48 spots each being 45 \u3bcm in diameter. Substrates were fabricated from two hard termoplastic materials - poly(methylmethacrylate) (PMMA) and a polycyclic olefin (e.g., Zeonor\uae 1060R) - which were both activated with N-hydroxysuccinimide (NHS) ester to mediate covalent attachment of DNA molecules. The approach was exemplified by using 0.25 to 32 \u3bcM solutions of amino-modified oligonucleotides labeled with either Cy3 or Cy5 fluorescent dye in phosphate buffered saline (PBS), allowing for a direct and sensitive characterization of the printed arrays. Solutions were incubated for durations of 1 to >48 h at22, 30 and 40\u2103 to probe the conditions for obtaining uniform spots of high fluorescence intensity. The length (l) and depth (d) of microfluidic supply channels were both important with respect to depletion as well as evaporation of the solvent. While selective activation of the substrate proved helpful to limit unproductive loss of oligonucleotides along trajectories, incubation of solution in a humid environment was necessary to prevent uncontrolled drying of liquid, keeping the immobilization process intact over extended periods of time. When combined, these strategies effectively promoted the formation of high-quality DNA arrays, making it possible to arrange multiple probes in parallel with a high degree of uniformity. Moreover, we show that resultant arrays are compatible with standard hybridization protocols, which allowed for reliable discrimination of individual stands when exposed to a specific ssDNA target molecule.Dans le pr\ue9sent article, on d\ue9crit la structuration de puces d\u2019ADN sur des surfaces en mati\ue8re plastique au moyen d\u2019un syst\ue8me \ue9lastom\ue8re microcapillaire \ue0 deux dimensions (\u3bcCS). Les structures des microcanaux ont \ue9t\ue9 r\ue9alis\ue9es par gaufrage lithographique \ue0 chaud avec du Versaflex CL30. Comme les \ue9lastom\ue8res \ue0 base de poly(dim\ue9thylsiloxane), ce copolym\ue8re thermoplastique s\ue9quenc\ue9 permet de sceller une surface de mani\ue8re r\ue9versible, rendant possible le confinement de sondes d\u2019ADN avec un niveau de contr\uf4le jamais atteint avec des techniques classiques de micropositionnement. On s\u2019est concentr\ue9 sur les \u3bcCS permettant l\u2019obtention de micropuces ayant jusqu\u2019\ue0 2 7 48 points, chacun de 45 \u3bcm de diam\ue8tre. Les substrats ont \ue9t\ue9 fabriqu\ue9s \ue0 partir de deux mati\ue8res thermoplastiques dures, du poly(m\ue9thacrylate de m\ue9thyle) et une poly(cyclool\ue9fine) (p. ex. Zeonor 1060R), qui ont toutes deux \ue9t\ue9 activ\ue9es avec du chlorhydrate de 1-\ue9thyl-3-[3-(dim\ue9thylamino)propyl]carbodiimide et du N hydroxysuccinimide pour m\ue9dier la liaison covalente aux mol\ue9cules d\u2019ADN. L\u2019approche a \ue9t\ue9 authentifi\ue9e au moyen de solutions 0,25 1232 \u3bcM d\u2019oligonucl\ue9otides modifi\ue9s avec un groupe amino et marqu\ue9s avec du Cy3 ou du Cy5 fluorescent dans un tampon phosphate salin, permettant une caract\ue9risation directe et sensible des puces imprim\ue9es. Les solutions ont \ue9t\ue9 incub\ue9es pendant des dur\ue9es allant de 1 \ue0 plus de 48 h \ue0 22, 30 et 40 \ub0C pour tester les conditions permettant d\u2019obtenir des points ayant une intensit\ue9 de fluorescence \ue9lev\ue9e et uniforme. La longueur (l) et la profondeur (d) des microcanaux d\u2019alimentation \ue9taient toutes deux importantes sur le plan de la diminution ainsi que de l\u2019\ue9vaporation du solvant. Bien qu\u2019une activation s\ue9lective du substrat se soit av\ue9r\ue9e utile pour limiter les pertes improductives d\u2019oligonucl\ue9otides le long des trajectoires, l\u2019incubation de la solution dans un environnement humide \ue9tait n\ue9cessaire pour pr\ue9venir le s\ue9chage incontr\uf4l\ue9 du liquide et ainsi conserver le processus d\u2019immobilisation intact pendant de longues p\ue9riodes. Lorsqu\u2019elles \ue9taient combin\ue9es, ces strat\ue9gies favorisaient efficacement la formation de micropuces d\u2019ADN de haute qualit\ue9, ce qui rend possible le placement de plusieurs sondes en parall\ue8le avec un haut degr\ue9 d\u2019uniformit\ue9. De plus, on a montr\ue9 que les puces obtenues \ue9taient compatibles avec les protocoles classiques d\u2019hybridation, qui permettent la discrimination fiable de brins individuels quand ils sont expos\ue9s \ue0 une mol\ue9cule cible sp\ue9cifique d\u2019ADN simple brin.Peer reviewed: YesNRC publication: Ye

Systems medicine and integrated care to combat chronic noncommunicable diseases

We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems

Programmed mechano-chemical coupling in reaction-diffusion active matter

Embryo morphogenesis involves a complex combination of pattern-forming mechanisms. However, classical in vitro patterning experiments explore only one mechanism at a time, thus missing coupling effects. Here, we conjugate two major pattern-forming mechanisms —reaction-diffusion and active matter— by integrating dissipative DNA/enzyme reaction networks within an active gel composed of cytoskeletal motors and filaments. We show that the strength of the flow generated by the active gel controls the mechano-chemical coupling between the two subsystems. We use this property to engineer the mechanical activation of chemical reaction networks both in time and space, thus mimicking key aspects of the polarization mechanism observed in C. elegans oocytes. We anticipate that reaction-diffusion active matter may be useful to investigate mechano-chemical transduction and to design new materials with life-like properties

DNA-Controlled Spatiotemporal Patterning of a Cytoskeletal Active Gel

International audienceLiving cells move and change their shape because signaling chemical reactions modify the state of their cyto-skeleton; an active gel that converts chemical energy into mechanical forces. To create lifelike materials, it is thus key to engineer chemical pathways that drive active gels. Here, we describe the preparation of DNA-responsive surfaces that control the activity of a cytoskeletal active gel comprised of microtubules: a DNA signal triggers the release of molecular motors from the surface into the gel bulk, generating forces that structure the gel. Depending on the DNA sequence and concentration , the gel forms a periodic band pattern or contracts globally. Finally, we show that the structuration of the active gel can be spatially controlled in the presence of a gradient of DNA concentration. We anticipate that such DNA-controlled active matter will contribute to the development of lifelike materials with self-shaping properties

DNA-based long-lived reaction-diffusion patterning in a host hydrogel

International audienc

Long-Lasting and Responsive DNA/Enzyme-Based Programs in Serum-Supplemented Extracellular Media

International audienceDNA molecular programs are emerging as promising pharmaceutical approaches due to their versatility for biomolecular sensing and actuation. However, the implementation of DNA programs has been mainly limited to serum-deprived in vitro assays due to the fast deterioration of the DNA reaction networks by the nucleases present in the serum. Here, we show that DNA/enzyme programs are functional in serum for 24 h but are later disrupted by nucleases that give rise to parasitic amplification. To overcome this, we implement three-letter code networks that suppress autocatalytic parasites while still conserving the functionality of DNA/enzyme programs for at least 3 days in the presence of 10% serum. In addition, we define a new buffer that further increases the biocompatibility and conserves responsiveness to changes in molecular composition across time. Finally, we demonstrate how serum-supplemented extracellular DNA molecular programs remain responsive to molecular inputs in the presence of living cells, having responses 6-fold faster than the cellular division rate, and are sustainable for at least three cellular divisions. This demonstrates the possibility of implementing in situ biomolecular characterization tools for serum-demanding in vitro models. We foresee that the coupling of chemical reactivity to our DNA programs by aptamers or oligonucleotide conjugations will allow the implementation of extracellular synthetic biology tools, which will offer new biomolecular pharmaceutical approaches and the emergence of complex and autonomous in vitro models

Synthesis of Programmable Reaction-Diffusion Fronts Using DNA Catalyzers

International audienceWe introduce a DNA-based reaction-diffusion (RD) system in which reaction and diffusion terms can be precisely and independently controlled. The effective diffusion coefficient of an individual reaction component, as we demonstrate on a traveling wave, can be reduced up to 2.7-fold using a self-assembled hydrodynamic drag. The intrinsic programmability of this RD system allows us to engineer, for the first time, orthogonal autocatalysts that counter-propagate with minimal interaction. Our results are in excellent quantitative agreement with predictions of the Fisher-Kolmogorov-Petrovskii-Piscunov model. These advances open the way for the rational engineering of pattern formation in pure chemical RD systems. Reaction-diffusion (RD) models are a rich source of spatiotemporal pattern formation phenomena. Not only is this mechanism relevant to biological morphogene-sis [1], but it is one of the few conceptualizations that physics can offer for the spontaneous emergence of order in molecular systems [2]. Traveling waves [3], spirals [4] and Turing patterns [5], among other structures [6, 7], have been observed experimentally. However, in contrast to pattern formation in hydrodynamics, few of these studies are quantitative [8, 9]. The reason is that we lack a fully controllable and easily modeled experimental RD system. In addition, to generate arbitrary spatiotemporal patterns the following properties need to be programmable: i) the topology of the chemical reaction network (CRN), ii) the reaction rates, and iii) the diffusion coefficients of individual species D i. The majority of attempts to achieve these goals concern redox or acid-base reactions related to the Belousov-Zhabotinsky (BZ) reaction [10–12]. Our current understanding does not allow to engineer CRNs with such chemistries in a rational way. Although semi-heuristic methods have been developed [13–15], they are neither general nor modular. Particular solutions to control diffusion have been devised for BZ-related reactions [5, 16] but no general strategy is available. DNA-based chemical reaction networks provide an interesting solution to the issues mentioned above. Due to base complementarity, the kinetics of the DNA hybridiza-tion reaction can be predicted from the sequence [17, 18]. Recent advances in DNA nanotechnology allow us to program the topology of quite complex CRNs. Enzyme-free DNA circuits have been used for producing tunable cascading reactions [19] and encoding edge detection algorithms [20]. In combination with enzymatic reactions, non-equilibrium dissipative behaviors with DNA circuits have been obtained, such as non-linear oscillators [21– 23], memory switches [24], and propagating waves and spirals [25]. Here we introduce a general method to control specifically the reaction and diffusion rates of DNA species involved in such programmable reaction networks. We demonstrate this on the minimal reaction capable of self-organization in space: an autocatalytic front propagating in a 1-dimensional reactor. As such, we used an auto-catalytic node of the DNA polymerase exonuclease nicking enzyme (PEN) toolbox, that works as follows [21]. Species A, an 11-mer single-stranded DNA (ssDNA), cat-alyzes its own growth in the presence of a template strand T, a 22-mer that carries two contiguous domains complementary to A: species A reversibly hybridizes with T on either of these domains and one of the resulting complexes can be extended by a polymerase (pol), which is the rate-limiting step in our conditions. The resulting double-stranded DNA (dsDNA) complex carries a recognition site for a nicking enzyme (nick) such that the upper strand is cut at its midpoint, releasing two molecules of A and the intact T. The kinetics of this process is captured by the simplified mechanism sketched in (FIG. 1a, for details refer to [21, 25]). The total concentration of each species, free or bound, is noted in italics in the following. In a one dimensional reactor the evolution of A is described by the reaction-diffusion equation ∂A ∂t = r(A) + ∂ ∂x D eff (A) ∂A ∂x , (1) where r(A) is the reaction term, and we have made explicit that the effective diffusion coefficient D eff (A) depends on A. This reflects the existence of A in states with different diffusion coefficients (free and bound to T). When D eff (A) = D, equation (1) together with reasonable assumptions about r(A) [26], form the Fisher-Kolmogorov-Petrovskii-Piscunov (Fisher-KPP) case: there exists a single stable asymp-totic traveling wave solution A(x, t) = A(x − v m t), where v m = 2 r (0)D depends neither on other details of the growth function r(A) nor on the shape of the initial condition [27, 28]. In our case, if the front propagation is controlled by the growth at the leading edge, where A 0

Crosslinking and depletion determine spatial instabilities in cytoskeletal active matter

International audienceActive gels made of cytoskeletal proteins are valuable materials with attractive non-equilibrium properties such as spatial self-organization and self-propulsion. At least four typical routes to spatial patterning have been reported to date in different types of cytoskeletal active gels: bending and buckling instabilities in extensile systems, and global and local contraction instabilities in contractile gels. Here we report the observation of these four instabilities in a single type of active gel and we show that they are controlled by two parameters: the concentrations of ATP and depletion agent. We demonstrate that as the ATP concentration decreases, the concentration of passive motors increases until the gel undergoes a gelation transition. At this point, buckling is selected against bending, while global contraction is favored over local ones. Our observations are coherent with a hydrodynamic model of a viscoelastic active gel where the filaments are crosslinked with a characteristic time that diverges as the ATP concentration decreases. Our work thus provides a unified view of spatial instabilities in cytoskeletal active matter