Biology subjects

Galas, J.-C.

Publications and source records attributed to Galas, J.-C..

6 recordsLinked to original sources

bioRxiv2021

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 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 24h but are latter disrupted by nucleases that give rise to parasitic amplification. To overcome this, we implement 3-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 cellular division rate and are sustainable for at least 3 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.

biophysics↗

bioRxiv2021

DNA-controlled Spatio-temporal Patterning of Active Matter

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 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 life-like materials with self-shaping properties.

biochemistry↗

bioRxiv2021

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. This property was used to engineer a synthetic material where contractions trigger 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 will promote the investigation of mechano-chemical transduction and the design of new materials with life-like properties.

biophysics↗

bioRxiv2020

Engineering an active extracellular medium with 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 spatio-temporal control. Here, we demonstrate that enzyme-DNA molecular programs combining these qualities are functional in an extracellular medium where human cells can grow. With this approach, we construct an internalization program that delivers fluorescent DNA inside living cells and remains functional for at least 48 h. Its non-equilibrium dynamics allows us to control both the time and position of cell internalization. In particular, a spatially inhomogeneous version of this program generates a tunable reaction-diffusion two-band pattern of cell internalization. This demonstrates that a synthetic extracellular program can provide temporal and positional information to living cells, emulating archetypal mechanisms observed during embryo development. We foresee that non-equilibrium reactive extracellular media could be advantageously applied to in vitro biomolecular tracking, tissue engineering or smart bandages.

biophysics↗

bioRxiv2019

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. Implementing this patterning stage in an autonomous synthetic material is a challenge that at least requires a programmable and long-lasting out-of-equilibrium chemistry compatible with a host material. Here we show that DNA/enzyme reactions can create reaction-diffusion patterns that are extraordinary long-lasting both in solution and inside an autonomous hydrogel. The life-time and stability of these patterns - here traveling fronts and two-band patterns - are significantly increased by blocking parasitic side reactions and by dramatically reducing the diffusion coefficient of specific DNA sequences. Immersed in oil, hydrogels pattern autonomously with limited evaporation, but can also exchange chemical information from other gels when brought in contact. Our primitive metabolic material thus recapitulates two important properties of living matter: a certain degree of autonomy that makes each piece of material an individual with its own metabolism and, at the same time, the capacity to interact with other individuals.

biochemistry↗

bioRxiv2019

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 languages, such as the PEN DNA toolbox. Here we show that the nicking enzyme Nb.BssSI allows to change the sequence design of EXPAR templates in a way that prevents the formation of parasites when dATP is removed from the solution. This method allows to make the EXPAR reaction robust to parasite contamination, a common feature in the laboratory, while keeping it compatible with PEN programs, which we demonstrate by engineering a parasite-proof bistable reaction network.

biochemistry↗