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. First, we use DNA-based reaction networks to synthesize a French flag, an archetypal pattern composed of three chemically distinct zones with sharp borders whose synthetic analogue has remained elusive. A bistable network within a shallow concentration gradient creates an immobile, sharp and long-lasting concentration front through a reaction-diffusion mechanism. The combination of two bistable circuits generates a French flag pattern whose 'phenotype' can be reprogrammed by network mutation. Second, these concentration patterns control the macroscopic organization of DNA-decorated particles, inducing a French flag pattern of colloidal aggregation. This experimental framework could be used to test reaction-diffusion models and fabricate soft materials following an autonomous developmental programme.

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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. The intrinsic programmability of this RD system allows us to engineer, for the first time, orthogonal autocatalysts that counterpropagate 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.

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Out-of-equilibrium chemical systems may self-organize into structures displaying spatiotemporal order, such as traveling waves and Turing patterns. Because of its predictable chemistry, DNA has recently appeared as an interesting candidate to engineer these spatiotemporal structures. However, in addition to the intrinsic chemical parameters, initial and boundary conditions have a major impact on the final structure. Here we take advantage of microfluidics to design controlled reactors and investigate pursuit-and-evasion chemical waves generated by a DNA-based reaction network with Predator-Prey dynamics. We first propose two complementary microfabrication strategies to either control the initial condition or the two-dimensional geometry of the reactor where the waves develop. We subsequently use them to investigate the effect of curvature in wave propagation. We finally show that DNA-based waves can compute the optimal path within a maze. We thus suggest that coupling configurable microfluidics to programmable DNA-based dissipative reaction networks is a powerful route to investigate spatiotemporal order formation in chemistry.

Specific mutations in the XPD subunit of transcription factor IIH result in combined xeroderma pigmentosum (XP)/Cockayne syndrome (CS), a severe DNA repair disorder characterized at the cellular level by a transcriptional arrest following UV irradiation. This transcriptional arrest has always been thought to be the result of faulty transcription-coupled repair. In the present study, we showed that, following UV irradiation, XP-D/CS cells displayed a gross transcriptional dysregulation compared with "pure" XP-D cells or WT cells. Furthermore, global RNA-sequencing analysis showed that XP-D/CS cells repressed the majority of genes after UV, whereas pure XP-D cells did not. By using housekeeping genes as a model, we demonstrated that XP-D/CS cells were unable to reassemble these gene promoters and thus to restart transcription after UV irradiation. Furthermore, we found that the repression of these promoters in XP-D/CS cells was not a simple consequence of deficient repair but rather an active heterochromatinization process mediated by the histone deacetylase Sirt1. Indeed, RNA-sequencing analysis showed that inhibition of and/or silencing of Sirt1 changed the chromatin environment at these promoters and restored the transcription of a large portion of the repressed genes in XP-D/CS cells after UV irradiation. Our work demonstrates that a significant part of the transcriptional arrest displayed by XP-D/CS cells arises as a result of an active repression process and not simply as a result of a DNA repair deficiency. This dysregulation of Sirt1 function that results in transcriptional repression may be the cause of various severe clinical features in patients with XP-D/ CS that cannot be explained by a DNA repair defect.nucleotide excision repair | sirtuins | aging | progeria

The emerging field of high-throughput compartmentalized in vitro evolution, a promising new approach to protein engineering, can be described as a lab-scale mimicking of natural selection processes. Libraries of mutants genotypes are randomly distributed in emulsified droplets, and the selection of desirable variants is achieved by differential amplification according to the phenotype in each microcompartment. The random partitionning leads to a fraction of compartments receiving more than one molecule, which increase with the mean occupancy-i.e. the average number of genotype-bearing agents per compartment.In these compartments, genotypes with different phenotypes will pool their activity but also share the total number of produced copies, in effect rendering the selection frequency-dependent. From a practical point of view (where efficient selections are typically sought), it is then important to know the impact of an increase in mean occupancy. We carried out a theoretical investigation of this problem in the context of selection dynamics for a simple model with an infinite, non-mutating population that is periodically redistributed among an infinite number of identical compartments.We derive here an update equation for any distribution of phenotypical activities and any value of the mean occupancy. It can be interpreted as a generalization of Price's formula to the case of random clustering of the individuals. Using this result, we demonstrate that, for the linear additive fitness function, the best genotype is still selected regardless of the mean occupancy. Furthermore, the "natural" selection process is remarkably resilient to the presence of multiple genotypes per compartments, and slows down approximately inversely proportional to the mean occupancy at high values. We extend out results to more general expressions that cover nonadditive and non-linear fitnesses, as well non-Poissonian distribution among compartments. While the discussion uses the terminology of high-throughput directed evolution methods, our conclusions may also apply to natural genetic compartmentalized replicators, such as viruses or early transacting RNA replicators. *

Most protocols for the high-throughput directed evolution of enzymes rely on random encapsulation to link phenotype and genotype. In order to optimize these approaches, or compare one to another, one needs a measure of their performance at extracting the best variants. We introduce here a new metric named the Selection Quality Index (SQI), which can be computed from a simple mock experiment with a known initial fraction of active variants. As opposed to previous approaches, our index integrates the random co-encapsulation of entities in compartments and comes with a straightforward experimental interpretation. We further show how this new metric can be used to extract general trends of protocol efficiency, or reveal hidden mechanisms such as a counterintuitive form of beneficial poisoning in the Compartmentalized Self-Replication protocol.