Crosslinking and depletion determine spatial instabilities in cytoskeletal active matter.

Active 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.

Front speed and pattern selection of a propagating chemical front in an active fluid.

Spontaneous pattern formation in living systems is driven by reaction-diffusion chemistry and active mechanics. The feedback between chemical and mechanical forces is often essential to robust pattern formation, yet it remains poorly understood in general. In this analytical and numerical paper, we study an experimentally motivated minimal model of coupling between reaction-diffusion and active matter: a propagating front of an autocatalytic and stress-generating species.

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

DNA 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 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.

Programmed mechano-chemical coupling in reaction-diffusion active matter.

Embryo morphogenesis involves a complex combination of self-organization mechanisms that generate a great diversity of patterns. However, classical in vitro patterning experiments explore only one self-organization mechanism at a time, thus missing coupling effects. Here, we conjugate two major out-of-equilibrium patterning mechanisms—reaction-diffusion and active matter—by integrating dissipative DNA/enzyme reaction networks within an active gel composed of cytoskeletal motors and filaments.

DNA-Controlled Spatiotemporal Patterning of a Cytoskeletal Active Gel.

Living cells move and change their shape because signaling chemical reactions modify the state of their cytoskeleton, an active gel that converts chemical energy into mechanical forces. To create life-like 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 composed of microtubules: A DNA signal triggers the release of molecular motors from the surface into the gel bulk, generating forces that structure the gel.

Spatiotemporal Patterning of Living Cells with Extracellular DNA Programs.

Reactive extracellular media focus on engineering reaction networks outside the cell to control intracellular chemical composition across time and space. However, current implementations lack the feedback loops and out-of-equilibrium molecular dynamics for encoding spatiotemporal control. Here, we demonstrate that enzyme-DNA molecular programs combining these qualities are functional in an extracellular medium where human cells can grow.

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

The development of living organisms is a source of inspiration for the creation of synthetic life-like materials. Embryo development is divided into three stages that are inextricably linked: patterning, differentiation and growth. During patterning, sustained out-of-equilibrium molecular programs interpret underlying molecular cues to create well-defined concentration profiles.

Tunable corrugated patterns in an active nematic sheet.

Active matter locally converts chemical energy into mechanical work and, for this reason, it provides new mechanisms of pattern formation. In particular, active nematic fluids made of protein motors and filaments are far-from-equilibrium systems that may exhibit spontaneous motion, leading to actively driven spatiotemporally chaotic states in 2 and 3 dimensions and coherent flows in 3 dimensions (3D). Although these dynamic flows reveal a characteristic length scale resulting from the interplay between active forcing and passive restoring forces, the observation of static and large-scale spatial patterns in active nematic fluids has remained elusive.

rEXPAR: An Isothermal Amplification Scheme That Is Robust to Autocatalytic Parasites.

In the absence of DNA, a solution containing the four deoxynucleotidetriphosphates (dNTPs), a DNA polymerase, and a nicking enzyme generates a self-replicating mixture of DNA species called parasite. Parasites are problematic in template-based isothermal amplification schemes such as EXPAR as well as in related molecular programming approaches, such as the PEN DNA toolbox. Here we show that using a nicking enzyme with only three letters (C, G, T) in the top strand of its recognition site, such as Nb.

Synthesis and materialization of a reaction-diffusion French flag pattern.

During embryo development, patterns of protein concentration appear in response to morphogen gradients. These patterns provide spatial and chemical information that directs the fate of the underlying cells. Here, we emulate this process within non-living matter and demonstrate the autonomous structuration of a synthetic material.

Observing and Controlling the Folding Pathway of DNA Origami at the Nanoscale.

DNA origami is a powerful method to fold DNA into rationally designed nanostructures that holds great promise for bionanotechnology. However, the folding mechanism has yet to be fully resolved, principally due to a lack of data with single molecule resolution. To address this issue, we have investigated in detail, using atomic force microscopy, the morphological evolution of hundreds of individual rectangular origamis in solution as a function of temperature.

Synthesis of programmable reaction-diffusion fronts using DNA catalyzers.

We 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.

Automated design of programmable enzyme-driven DNA circuits.

Molecular programming allows for the bottom-up engineering of biochemical reaction networks in a controlled in vitro setting. These engineered biochemical reaction networks yield important insight in the design principles of biological systems and can potentially enrich molecular diagnostic systems. The DNA polymerase-nickase-exonuclease (PEN) toolbox has recently been used to program oscillatory and bistable biochemical networks using a minimal number of components.

High-throughput and long-term observation of compartmentalized biochemical oscillators.

We report the splitting of an oscillating DNA circuit into ∼700 droplets with picoliter volumes. Upon incubation at constant temperature, the droplets display sustained oscillations that can be observed for more than a day. Superimposed to the bulk behaviour, we find two intriguing new phenomena - slow desynchronization between the compartments and kinematic spatial waves - and investigate their possible origin.

Pressure-assisted selective preconcentration in a straight nanochannel.

We investigate the preconcentration profiles of a fluorescein and bovine serum albumin derivatized with this fluorescent tag in a microfluidic chip bearing a nanoslit. A new preconcentration method in which a hydrodynamic pressure is added to both electroosmotic and electrophoretic contributions is proposed to monitor the location of the preconcentration frontline. A simple predictive model of this pressure-assisted electropreconcentration is proposed for the evolution of the flow profile along this micro/nano/microfluidic structure.

Spatial waves in synthetic biochemical networks.

We report the experimental observation of traveling concentration waves and spirals in a chemical reaction network built from the bottom up. The mechanism of the network is an oscillator of the predator-prey type, and this is the first time that predator-prey waves have been observed in the laboratory. The molecular encoding of the nonequilibrium behavior relies on small DNA oligonucleotides that enforce the network connectivity and three purified enzymes that control the reactivity.

A nanoliter-scale open chemical reactor.

An open chemical reactor is a container that exchanges matter with the exterior. Well-mixed open chemical reactors, called continuous stirred tank reactors (CSTR), have been instrumental for investigating the dynamics of out-of-equilibrium chemical processes, such as oscillations, bistability, and chaos. Here, we introduce a microfluidic CSTR, called μCSTR, that reduces reagent consumption by six orders of magnitude.

A microfluidic device for continuous cancer cell culture and passage with hydrodynamic forces.

We demonstrate a novel and robust microfluidic chip with combined functions of continuous culture and output of PC-3 prostate cancer cells. With digital controls, polydimethylsiloxane (PDMS) flexible diaphragms are able to apply hydrodynamic shear forces on cultures, detaching a fraction of attached cancer cells from the surface for output while leaving others for reuse in subsequent cultures. The fractions of detached cells and remaining cells can be precisely controlled.

Sequence-independent and reversible photocontrol of transcription/expression systems using a photosensitive nucleic acid binder.

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.

Fourier transform to analyse reaction-diffusion dynamics in a microsystem.

An integrated approach relying on a microsystem is introduced to easily extract, from a single experiment and with a global robust bi-exponential fit, an extensive set of thermodynamic, kinetic, and diffusion parameters governing associations in solution.