2020 (7)
Droplet-based microfluidics has permeated many areas of life sciences including biochemistry, biology and medicine. Water-in-oil droplets act as independent femto- to nano-liter reservoirs, enabling the parallelization of (bio)chemical reactions with a minimum sample input. Among the range of applications spanned by droplet microfluidics, digital detection of biomolecules, using Poissonian isolation of single molecules in compartments, has gained considerable attention due to the high accuracy, sensitivity and robustness of these methods. However, while the droplet throughput can be very high, the sample throughput of these methods is poor in comparison to well plate-based assays. This limitation comes from the necessity to convert independently each sample into a monodisperse emulsion. In this paper, we report a versatile device that performs the quick sequential partitioning of up to 15 samples using a …
MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water …
High-throughput, in vitro approaches for the evolution of enzymes rely on a random micro-encapsulation to link phenotypes to genotypes, followed by screening or selection steps. In order to optimise these approaches, or compare one to another, one needs a measure of their performance at extracting the best variants of a library. Here, we introduce a new metric, the Selection Quality Index (SQI), which can be computed from a simple mock experiment, performed with a known initial fraction of active variants. In contrast to previous approaches, our index integrates the effect of random co-encapsulation, and comes with a straightforward experimental interpretation. We further show how this new metric can be used to extract general protocol efficiency trends or reveal hidden selection mechanisms such as a counterintuitive form of beneficial poisoning in the compartmentalized self-replication protocol.
High-throughput, in vitro approaches for the evolution of enzymes rely on a random micro-encapsulation to link phenotypes to genotypes, followed by screening or selection steps. In order to optimise these approaches, or compare one to another, one needs a measure of their performance at extracting the best variants of a library. Here, we introduce a new metric, the Selection Quality Index (SQI), which can be computed from a simple mock experiment, performed with a known initial fraction of active variants. In contrast to previous approaches, our index integrates the effect of random co-encapsulation, and comes with a straightforward experimental interpretation. We further show how this new metric can be used to extract general protocol efficiency trends or reveal hidden selection mechanisms such as a counterintuitive form of beneficial poisoning in the compartmentalized self-replication protocol.
Nanopore sequencing is a powerful single molecule DNA sequencing technology which provides a high throughput and long sequence reads. Nevertheless, its relatively high native error rate limits the direct detection of point mutations in individual reads of amplicon libraries, as these mutations are difficult to distinguish from the sequencing noise. We propose a computational method to reduce noise in nanopore detection of point variations. Our approach uses the fact that all reads are expected to be very similar to a wild type sequence, for which we experimentally characterize the position-specific systematic sequencing error pattern. We then use this information to reweight, in individual reads from the variant library, the confidence given to nucleotides read that do not match the wild type. We tested this method on two sets of known variants of Klen Taq, where the true mutation rate was 3.3 mutations per kb, well below the sequencing noise. We observed that the actual mutations became more distinguishable from sequencing noise after correction. This approach can be used, for example to help the clustering of variants, or to decrease the number of reads necessary to call a consensus. The computational method is simple to implement and requires only a few thousands reads of the wild type sequence of interest, which can be easily obtained by multiplexing in a single minION run. The approach does not require any modification in the experimental protocol for sequencing and can be simply implemented downstream standard base calling.
Nanopore sequencing is a powerful single molecule DNA sequencing technology which offers high throughput and long sequence reads. Nevertheless, its high native error rate limits the direct detection of point mutations in individual reads of amplicon libraries, as these mutations are difficult to distinguish from the sequencing noise. In this work, we developed SINGLe (SNPs In Nanopore reads of Gene Libraries), a computational method to reduce the noise in nanopore reads of amplicons containing point variations. Our approach uses the fact that all reads are very similar to a wild type sequence, for which we experimentally characterize the position-specific systematic sequencing error pattern. We then use this information to reweight the confidence given to nucleotides that do not match the wild type in individual variant reads. We tested this method in a set of variants of KlenTaq, where the true mutation rate was well below the sequencing noise. SINGLe improves between 4 and 9 fold the signal to noise ratio, in comparison to the data returned by the basecaller guppy. Downstream, this approach improves variants clustering and consensus calling. SINGLe is simple to implement and requires only a few thousands reads of the wild type sequence of interest, which can be easily obtained by multiplexing in a single minION run. It does not require any modification in the experimental protocol, it does not imply a large loss of sequencing throughput, and it can be incorporated downstream of standard basecalling.
Ubiquitous post-transcriptional regulators in eukaryotes, microRNAs currently emerge as promising biomarkers of physiological and patholog-ical processes. Multiplex and digital detection of microRNAs represents a major challenge toward the use of microRNA signatures in clinical settings. The classical RT-PCR quantification approach retains important limitations due to the need for thermocycling and a reverse transcription step. Simpler, isothermal alternatives have been proposed yet, none could be adapted to both a digital and multiplex format. This is either due to a lack of sensitivity that forbids single molecule detection, or molecular crosstalk reactions that are responsible for nonspecific amplification. Building on an ultrasensitive isothermal amplification mechanism, we present a strategy to suppress crosstalk reactions, allow-ing for robust isothermal and multiplex detection of microRNA targets. Our approach …
2019 (4)
The amplification cycle of many replicators (natural or artificial) involves the usage of a host compartment, inside of which the replicator expresses phenotypic compounds necessary to carry out its genetic replication. For example, viruses infect cells, where they express their own proteins and replicate. In this process, the host cell boundary limits the diffusion of the viral protein products, thereby ensuring that phenotypic compounds, such as proteins, promote the replication of the genes that encoded them. This role of maintaining spatial colocalization, also called genotype-phenotype linkage, is a critical function of compartments in natural selection. In most cases, however, individual replicating elements do not distribute systematically among the hosts, but are randomly partitioned. Depending on the replicator-to-host ratio, more than one variant may thus occupy some compartments, blurring the genotype-phenotype …
MicroRNA, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design DNA circuit that converts, threshold, amplifies and report the presence of a specific microRNA, down to the femtomolar concentration. Using a leak-absorption mechanism, we were able to suppress non-specific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNA: by partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.
The emerging field of high-throughput compartmentalized in vitro evolution is a promising new approach to protein engineering. In these experiments, libraries of mutant genotypes are randomly distributed and expressed in microscopic compartments—droplets of an emulsion. The selection of desirable variants is performed according to the phenotype of each compartment. The random partitioning leads to a fraction of compartments receiving more than one genotype making the whole process a lab implementation of the group selection. From a practical point of view (where efficient selection is typically sought), it is important to know the impact of the increase in the mean occupancy of compartments on the selection efficiency. We carried out a theoretical investigation of this problem in the context of selection dynamics for an infinite non-mutating subdivided population that randomly colonizes an infinite number of …
The potential of microRNAs (miRNAs) as biomarker candidates in clinical practice for diagnosis, prognosis and treatment response prediction, especially in liquid biopsies, has led to a tremendous demand for techniques that can detect these molecules rapidly and accurately. Hence, numerous achievements have been reported recently in miRNA research. In this review, we discuss the challenges associated with the emerging field of miRNA detection, which are linked to the intrinsic properties of miRNAs, advantages and drawbacks of the currently available technologies and their potential applications in clinical research. We summarize the most promising nucleic acid amplification techniques applied to the in vitro detection of miRNAs, with a particular emphasis on the state of the art for isothermal alternatives to RT-qPCR. We detail the sensitivity, specificity and quantitativity of these approaches, as well as their …
2018 (1)
In recent years, DNA computing frameworks have been developed to create dynamical systems which can be used for information processing. These emerging synthetic biochemistry tools can be leveraged to gain a better understanding of fundamental biology but can also be implemented in biosensors and unconventional computing. Most of the efforts so far have focused on changing the topologies of DNA molecular networks or scaling them up. Several issues have thus received little attention and remain to be solved to turn them into real life technologies. In particular, the ability to easily interact in real-time with them is a key requirement. The previous attempts to achieve this aim have used microfluidic approaches, such as valves, which are cumbersome. We show that electrochemical triggering using DNA-grafted micro-fabricated gold electrodes can be used to give …
2017 (9)
Information stored in synthetic nucleic acids sequences can be used in vitro to create complex reaction networks with precisely programmed chemical dynamics. Here, we scale up this approach to program networks of microscopic particles (agents) dispersed in an enzymatic solution. Agents may possess multiple stable states, thus maintaining a memory and communicate by emitting various orthogonal chemical signals, while also sensing the behaviour of neighbouring agents. Using this approach, we can produce collective behaviours involving thousands of agents, for example retrieving information over long distances or creating spatial patterns. Our systems recapitulate some fundamental mechanisms of distributed decision making and morphogenesis among living organisms and could find applications in cases where many individual clues need to be combined to …
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 …
Information stored in synthetic nucleic acids sequences can be used in vitro to create complex reaction networks with precisely programmed chemical dynamics. Here, we scale up this approach to program networks of microscopic particles (agents) dispersed in an enzymatic solution. Agents may possess multiple stable states, thus maintaining a memory and communicate by emitting various orthogonal chemical signals, while also sensing the behaviour of neighbouring agents. Using this approach, we can produce collective behaviours involving thousands of agents, for example retrieving information over long distances or creating spatial patterns. Our systems recapitulate some fundamental mechanisms of distributed decision making and morphogenesis among living organisms and could find applications in cases where many individual clues need to be combined to reach a decision, for example in molecular …
Figure 1| Principle of DNA domino circuits. Left: billions of DNA ‘fuel’strands and DNA ‘breadboards’ with attached components are fioating in solution. Right: an AND gate where five types of DNA strands with different sequences (in colours) protrude above the breadboard (top). When the first, purple, input strand is added to the solution it binds to its input site and generates a domino reaction along the line of green strands that consumes yellow fuel strands from the solution, ending up in the brown sink strand (centre). When the second, blue, input is added, a similar domino reaction occurs in another part of the breadboard that ends up inducing a fiuorescence signal at the red output strand.
The amplification cycle of many replicators (natural or artificial) involves the usage of a host compartment, inside which the replicator express phenotypic compounds necessary to carry out its genetic replication. The compartment limits the diffusion of the intermediate compounds, thereby also maintaining the genotypephenotype linkage enabling natural selection. In most cases however, genotypic variants are distributed randomly among the hosts. More than one variant may thus occupy each compartment, blurring the genotypephenotype linkage: how does this affect the effectiveness of natural selection? We derive selection equations for a variety of such random multiple occupancy situations, in particular considering the effect of replicator population polymorphism and internal replication dynamics. We conclude that the deleterious effect of random multiple occupancy is relatively benign, and may even completely vanish is some specific cases. In addition, given that higher mean occupancy allows larger populations to be channeled through the selection process, and thus provide a better exploration of phenotypic diversity, we show that it may represent a valid strategy in both natural and technological cases.
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 …
Biochemical systems in which multiple components take part in a given reaction are of increasing interest. Because the interactions between these different components are complex and difficult to predict from basic reaction kinetics, it is important to test for the effect of variations in the concentration for each reagent in a combinatorial manner. For example, in PCR, an increase in the concentration of primers initially increases template amplification, but large amounts of primers result in primer–dimer by-products that inhibit the amplification of the template. Manual titration of biochemical mixtures rapidly becomes costly and laborious, forcing scientists to settle for suboptimal concentrations. Here we present a droplet-based microfluidics platform for mapping of the concentration space of up to three reaction components followed by detection with a fluorescent readout. The concentration of each reaction component is …
The amplification cycle of many replicators (natural or artificial) involves the usage of a host compartment, inside which the replicator express phenotypic compounds necessary to carry out its genetic replication. The compartment limits the diffusion of the intermediate compounds, thereby also maintaining the genotypephenotype linkage enabling natural selection. In most cases however, genotypic variants are distributed randomly among the hosts. More than one variant may thus occupy each compartment, blurring the genotypephenotype linkage: how does this affect the effectiveness of natural selection? We derive selection equations for a variety of such random multiple occupancy situations, in particular considering the effect of replicator population polymorphism and internal replication dynamics. We conclude that the deleterious effect of random multiple occupancy is relatively benign, and may even completely vanish is some specific cases. In addition, given that higher mean occupancy allows larger populations to be channeled through the selection process, and thus provide a better exploration of phenotypic diversity, we show that it may represent a valid strategy in both natural and technological cases.
In electronic chips, multiple simple components are carefully positioned to collectively perform an integrated computation. Since the introduction of two-dimensional DNA origami nanostructures in 2006 1, researchers have considered using this matrix-like, uniquely addressable molecular array of nanometric pixels to implement chemical analogues of this concept. However, to ensure proper information flow in this molecular version of an electronic breadboard remains challenging. Now, writing in Nature Nanotechnology, Chatterjee et al. 2 propose the use of an external fuel source to drive this flow by chain-polymerizing the tethered DNA components. This approach brings modularity, robustness and speed to the molecular chips, taking them one step closer to practical computational applications.Molecular programming, the art of creating computational devices using networks of biomolecular reactions 3, has a …
2016 (3)
Micro and nano systems (MNS) and Nano scaled devices, that are capable of handling fluids and to interact with DNA and proteins enable bio analysis at the “ultimate” molecular level and are prone to be coupled to IC Technology. This paper includes recent developments in this area, aimed at illustrating the diversity and potential of the MNS approach:(1) micromachined tweezers with sharp tips successfully captured a bundle of DNA molecules, allowing real-time observation of DNA degradation dynamics under therapeutic irradiations;(2) a simple method to fabricate FET silicon nanowires using only standard micro-electromechanical system (MEMS) processes is able to perform molecular level medical diagnosis;(3) a bio motor system, composed by microtubules and kinesin, reconstructed on a chip, can distinguish normal and abnormal tau-proteins related to …
Molecular programming takes advantage of synthetic nucleic acid biochemistry to assemble networks of reactions, in vitro, with the double goal of better understanding cellular regulation and providing information-processing capabilities to man-made chemical systems. The function of molecular circuits is deeply related to their topological structure, but dynamical features (rate laws) also play a critical role. Here we introduce a mechanism to tune the nonlinearities associated with individual nodes of a synthetic network. This mechanism is based on programming deactivation laws using dedicated saturable pathways. We demonstrate this approach through the conversion of a single-node homoeostatic network into a bistable and reversible switch. Furthermore, we prove its generality by adding new functions to the library of reported man-made molecular devices …
Analog molecular circuits can exploit the nonlinear nature of biochemical reaction networks to compute low-precision outputs with fewer resources than digital circuits. This analog computation is similar to that employed by gene-regulation networks. Although digital systems have a tractable link between structure and function, the nonlinear and continuous nature of analog circuits yields an intricate functional landscape, which makes their design counter-intuitive, their characterization laborious and their analysis delicate. Here, using droplet-based microfluidics, we map with high resolution and dimensionality the bifurcation diagrams of two synthetic, out-of-equilibrium and nonlinear programs: a bistable DNA switch and a predator–prey DNA oscillator. The diagrams delineate where function is optimal, dynamics bifurcates and models fail. Inverse problem solving on these large-scale data sets indicates interference …
2015 (4)
In the past three decades, DNA has emerged as a versatile polymer to build and program at the nanoscale, allowing the construction of a rich variety of nanostructures. The programmability of DNA has also paved the way for the interdisciplinary field of molecular programming, which seeks to understand how to best program molecules‒inspired by the vast information processing capabilities of cells. Here we focus on recent efforts in LIMMS aimed at combining microsystems and molecular programs, demonstrating how the dimensions and throughput offered by the former complement aptly the molecular control of the latter.
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.
In this paper, we introduce our approach for evolving reaction networks. It is an efficient derivative of the neuroevolution of augmenting topologies algorithm directed at the evolution of biochemical systems or molecular programs. Our method addresses the problem of meaningful crossovers between two chemical reaction networks of different topologies. It also builds on features such as speciation to speed up the search, to the point where it can deal with complete, realistic mathematical models of the biochemical processes. We demonstrate this framework by evolving credible biochemical answers to challenging autonomous molecular problems: in vitro batch oscillatory networks that match specific oscillation shapes. Our experimental results suggest that the search space is efficiently covered and that, by using crossover and preserving topological innovations, significant …
2014 (7)
In living organisms, the integration of signals from the environment and the molecular computing leading to a cellular response are orchestrated by Gene Regulatory Networks (GRN). However, the molecular complexity of in vivo genetic regulation makes it next to impossible to describe in a quantitative manner. Reproducing, in vitro, reaction networks that could mimic the architecture and behavior of in vivo networks, yet lend themselves to mathematical modeling, represents a useful strategy to understand, and even predict, the function of GRN. In this paper, we define a set of in vitro, DNA-based molecular transformations that can be linked to each other in such a way that the product of one transformation can activate or inhibit the production of one or several other DNA compounds. Therefore, these reactions can be wired in arbitrary networks. This approach …
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 …
In the past few years, there have been many exciting advances in the field of molecular programming, reaching a point where implementation of non-trivial systems, such as neural networks or switchable bistable networks, is a reality. Such systems require nonlinearity, be it through signal amplification, digitalization or the generation of autonomous dynamics such as oscillations. The biochemistry of DNA systems provides such mechanisms, but assembling them in a constructive manner is still a difficult and sometimes counterintuitive process. Moreover, realistic prediction of the actual evolution of concentrations over time requires a number of side reactions, such as leaks, cross-talks or competitive interactions, to be taken into account. In this case, the design of a system targeting a given function takes much trial and error before the correct architecture can be found. To speed up this …
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. Previous work has reported the automatic construction of in silico descriptions of biochemical networks derived from the PEN toolbox, paving the way for generating networks of arbitrary size and complexity in vitro. Here, we report an automated approach that further bridges the gap between an in silico description and in vitro realization. A biochemical network of arbitrary complexity can be globally …
DNA computing has the potential to create powerful devices, but, in the context of well-mixed systems, sequentiality of operations is hard to achieve. To enforce such sequentiality, we propose a generic delay gate that can be interfaced with virtually any DNA system. Since it is system-independent, our delay gate can be used as an off-the-shelf library to accelerate the design of increasingly complex systems. Additionally, we checked the feasibility of our design by testing various in vitro implementations. We also present a theoretical proof of concept of its applicability by using it to complement an existing DNA module library, the DNA toolbox, to design new systems.
In the past few years, there have been many exciting advances in the field of molecular programming, reaching a point where implementation of non-trivial systems, such as neural networks or switchable bistable networks, is a reality. Such systems require nonlinearity, be it through signal amplification, digitalization or the generation of autonomous dynamics such as oscillations. The biochemistry of DNA systems provides such mechanisms, but assembling them in a constructive manner is still a difficult and sometimes counterintuitive process. Moreover, realistic prediction of the actual evolution of concentrations over time requires a number of side reactions, such as leaks, cross-talks or competitive interactions, to be taken into account. In this case, the design of a system targeting a given function takes much trial and error before the correct architecture can be found. To speed up this process, we have created DNA …
In the past few years, there have been many exciting advances in the field of molecular programming, reaching a point where implementation of non-trivial systems, such as neural networks or switchable bistable networks, is a reality. Such systems require nonlinearity, be it through signal amplification, digitalization or the generation of autonomous dynamics such as oscillations. The biochemistry of DNA systems provides such mechanisms, but assembling them in a constructive manner is still a difficult and sometimes counterintuitive process. Moreover, realistic prediction of the actual evolution of concentrations over time requires a number of side reactions, such as leaks, cross-talks or competitive interactions, to be taken into account. In this case, the design of a system targeting a given function takes much trial and error before the correct architecture can be found. To speed up this process, we have created DNA …
DNA computing has the potential to create powerful devices, but, in the context of well-mixed systems, sequentiality of operations is hard to achieve. To enforce such sequentiality, we propose a generic delay gate that can be interfaced with virtually any DNA system. Since it is system-independent, our delay gate can be used as an off-the-shelf library to accelerate the design of increasingly complex systems. Additionally, we checked the feasibility of our design by testing various in vitro implementations. We also present a theoretical proof of concept of its applicability by using it to complement an existing DNA module library, the DNA toolbox, to design new systems.
2013 (12)
DNA has proved to be an exquisite substrate to compute at the molecular scale. However, nonlinear computations (such as amplification, comparison or restoration of signals) remain costly in term of strands and are prone to leak. Kim et al. showed how competition for an enzymatic resource could be exploited in hybrid DNA/enzyme circuits to compute a powerful nonlinear primitive: the winner-take-all (WTA) effect. Here, we first show theoretically how the nonlinearity of the WTA effect allows the robust and compact classification of four patterns with only 16 strands and three enzymes. We then generalize this WTA effect to DNA-only circuits and demonstrate similar classification capabilities with only 23 strands.
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. This approach provides a route to study the influence of small volume effects in biology, and paves the way to technological applications of compartmentalized molecular programs controlling complex dynamics.
The reductionist approach has revolutionized biology in the past 50 years. Yet its limits are being felt as the complexity of cellular interactions is gradually revealed by high-throughput technology. In order to make sense of the deluge of “omic data”, a hypothesis-driven view is needed to understand how biomolecular interactions shape cellular networks. We review recent efforts aimed at building in vitro biochemical networks that reproduce the flow of genetic regulation. We highlight how those efforts have culminated in the rational construction of biochemical oscillators and bistable memories in test tubes. We also recapitulate the lessons learned about in vivo biochemical circuits such as the importance of delays and competition, the links between topology and kinetics, as well as the intriguing resemblance between cellular reaction networks and ecosystems.
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. Wave velocities in the range 80–400 μm min–1 were measured. A reaction–diffusion model in quantitative agreement with the experiments is proposed. Three fundamental parameters are easy to tune in nucleic acid reaction networks: the topology of the network, the rate constants of the individual reactions, and the diffusion coefficients of the individual species. For this reason, we expect such networks to bring …
Nucleic acid-based circuits are rationally designed in vitro assemblies that can perform complex preencoded programs. They can be used to mimic in silico computations. Recent works emphasized the modularity and robustness of these circuits, which allow their scaling-up. Another new development has led to dynamic, time-responsive systems that can display emergent behaviors like oscillations. These are closely related to biological architectures and provide an in vitro model of in vivo information processing. Nucleic acid circuits have already been used to handle various processes for technological or biotechnological purposes. Future applications of these chemical smart systems will benefit from the rapidly growing ability to design, construct, and model nucleic acid circuits of increasing size.
This chapter highlights the historical evolution from small‐molecule systems to biology and generalized experimental chemistries. It reviews the discoveries and theoretical advances that eventually allowed the dynamic description of molecular assemblies. The chapter provides examples of natural implementations of biological reaction networks. It also highlights some of the most recent schemes for the rational molecular programming of complex out‐of‐equilibrium behaviors. The chapter introduces some examples of implementations based on these experimental schemes. It focuses on the building of out‐of‐equilibrium chemical systems, instead of the more general area of molecular programming. Several fundamental properties must be carefully taken into account when designing synthetic biochemical dynamic circuits: non‐equilibrium, nonlinearity, modularity, and …
In models of games, the indirect interactions between players, such as body language or knowledge about the other’s playstyle, are often omitted. They are, however, a rich source of information in real life, and increase the complexity of possible strategies. In the game of rock-paper-scissors, the simple monitoring of the opponent’s move before it was played is a sufficient condition to trigger an arms race of detection and misinformation among evolved individuals. The most interesting aspect of those results is that they were obtained by evolving purely chemical reaction networks thanks to an adapted version of the famous NEAT algorithm. More specifically, those individuals were represented as biochemical systems built on the DNA toolbox, a paradigm that allows both easy in-vitro implementation and predictive in-silico simulation. This guarantees that the specific motives that emerged …
In models of games, the indirect interactions between players, such as body language or knowledge about the other’s playstyle, are often omitted. They are, however, a rich source of information in real life, and increase the complexity of possible strategies. In the game of rock-paper-scissors, the simple monitoring of the opponent’s move before it was played is a sufficient condition to trigger an arms race of detection and misinformation among evolved individuals. The most interesting aspect of those results is that they were obtained by evolving purely chemical reaction networks thanks to an adapted version of the famous NEAT algorithm. More specifically, those individuals were represented as biochemical systems built on the DNA toolbox, a paradigm that allows both easy in-vitro implementation and predictive in-silico simulation. This guarantees that the specific motives that emerged in this competition w…
DNA has proved to be an exquisite substrate to compute at the molecular scale. However, nonlinear computations (such as amplification, comparison or restoration of signals) remain costly in term of strands and are prone to leak. Kim et al. showed how competition for an enzymatic resource could be exploited in hybrid DNA/enzyme circuits to compute a powerful nonlinear primitive: the winner-take-all (WTA) effect. Here, we first show theoretically how the nonlinearity of the WTA effect allows the robust and compact classification of four patterns with only 16 strands and three enzymes. We then generalize this WTA effect to DNA-only circuits and demonstrate similar classification capabilities with only 23 strands.
DNA has proved to be an exquisite substrate to compute at the molecular scale. However, nonlinear computations (such as amplification, comparison or restoration of signals) remain costly in term of strands and are prone to leak. Kim et al. showed how competition for an enzymatic resource could be exploited in hybrid DNA/enzyme circuits to compute a powerful nonlinear primitive: the winner-take-all (WTA) effect. Here, we first show theoretically how the nonlinearity of the WTA effect allows the robust and compact classification of four patterns with only 16 strands and three enzymes. We then generalize this WTA effect to DNA-only circuits and demonstrate similar classification capabilities with only 23 strands.
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. This approach provides a route to study the influence of small volume effects in biology, and paves the way to technological applications of compartmentalized molecular programs controlling complex dynamics.
This paper describes the use of molecular programming techniques to build synthetic in vitro and spatially distributed reactions networks with tailored topologies. The basic workflow is to use synthetic DNA strands to encode the topologies of molecular interactions of the reaction network. The actual dynamic of the system is provided by enzymatic reactions controlled and templated by these DNA strands. Here we focus on the implementation of a molecular predator-prey ecosystem. We thus create two autocatalytic DNA amplifications reactions and connect them through predation–the second DNA-species consumes the first one to fuel its growth. We also ensure that these species have a limited lifetime in the test tube. We are therefore able to detect sustained oscillations of the two molecular species, as predicted and observed for real ecosystems. This is the first time that predator prey oscillations are observed in a …
2012 (7)
Cells rely on limited resources such as enzymes or transcription factors to process signals and make decisions. However, independent cellular pathways often compete for a common molecular resource. Competition is difficult to analyze because of its nonlinear global nature, and its role remains unclear. Here we show how decision pathways such as transcription networks may exploit competition to process information. Competition for one resource leads to the recognition of convex sets of patterns, whereas competition for several resources (overlapping or cascaded regulons) allows even more general pattern recognition. Competition also generates surprising couplings, such as correlating species that share no resource but a common competitor. The mechanism we propose relies on three primitives that are ubiquitous in cells: multiinput motifs, competition for a resource, and positive …
Water-in-oil microdroplets offer microreactors for compartmentalized biochemical reactions with high throughput. Recently, the combination with a sol-gel switch ability, using agarose-in-oil microdroplets, has increased the range of possible applications, allowing for example the capture of amplicons in the gel phase for the preservation of monoclonality during a PCR reaction. Here, we report a new method for generating such agarose-in-oil microdroplets on a microfluidic device, with minimized inlet dead volume, on-chip cooling, and in situ monitoring of biochemical reactions within the gelified microbeads. We used a flow-focusing microchannel network and successfully generated agarose microdroplets at room temperature using the “push-pull” method. This method consists in pushing the oil continuous phase only, while suction is applied to the device outlet. The agarose phase …
Reaction networks displaying bistability provide a chemical mechanism for long-term memory storage in cells, as exemplified by many epigenetic switches. These biological systems are not only bistable but switchable, in the sense that they can be flipped from one state to the other by application of specific molecular stimuli. We have reproduced such functions through the rational assembly of dynamic reaction networks based on basic DNA biochemistry. Rather than rewiring genetic systems as synthetic biology does in vivo, our strategy consists of building simplified dynamic analogs in vitro, in an artificial, well-controlled milieu. We report successively a bistable system, a two-input switchable memory element, and a single-input push-push memory circuit. These results suggest that it is possible to build complex time-responsive molecular circuits by following a modular …
Biological organisms use intricate networks of chemical reactions to control molecular processes and spatiotemporal organization. In turn, these living systems are embedded in self-organized structures of larger scales, for example, ecosystems. Synthetic in vitro efforts have reproduced the architectures and behaviors of simple cellular circuits. However, because all these systems share the same dynamic foundations, a generalized molecular programming strategy should also support complex collective behaviors, as seen, for example, in animal populations. We report here the bottom-up assembly of chemical systems that reproduce in vitro the specific dynamics of ecological communities. We experimentally observed unprecedented molecular behaviors, including predator–prey oscillations, competition-induced chaos, and symbiotic synchronization. These synthetic …
Genetic regulation networks orchestrate many complex cellular behaviors. Dynamic operations that take place within cells are thus dependent on the gene expression machinery, enabled by powerful enzymes such as polymerases, ribosomes, or nucleases. These generalist enzymes typically process many different substrates, potentially leading to competitive situations: by saturating the common enzyme, one substrate may down-regulate its competitors. However, most theoretical or experimental models simply omit these effects, focusing on the pattern of genetic regulatory interactions as the main determinant of network function. We show here that competition effects have important outcomes, which can be spotted within the global dynamics of experimental systems. Further we demonstrate that enzyme saturation creates a layer of cross couplings that may foster, but also hamper, the …
We present a simple yet efficient technique to monitor the dynamics of DNA-based reaction circuits. This technique relies on the labeling of DNA oligonucleotides with a single fluorescent modification. In this quencher-free setup, the signal is modulated by the interaction of the 3′-terminus fluorophore with the nucleobases themselves. Depending on the nature of the fluorophore’s nearest base pair, fluorescence intensity is decreased or increased upon hybridization. By tuning the 3′-terminal nucleotides, it is possible to obtain opposite changes in fluorescence intensity for oligonucleotides whose hybridization site is shifted by a single base. Quenching by nucleobases provides a highly sequence-specific monitoring technique, which presents a high sensitivity even for small oligonucleotides. Compared with other sequence-specific detection methods, it is relatively non-invasive …
Reaction networks displaying bistability provide a chemical mechanism for long-term memory storage in cells, as exemplified by many epigenetic switches. These biological systems are not only bistable but switchable, in the sense that they can be flipped from one state to the other by application of specific molecular stimuli. We have reproduced such functions through the rational assembly of dynamic reaction networks based on basic DNA biochemistry. Rather than rewiring genetic systems as synthetic biology does in vivo, our strategy consists of building simplified dynamic analogs in vitro, in an artificial, well-controlled milieu. We report successively a bistable system, a two-input switchable memory element, and a single-input push-push memory circuit. These results suggest that it is possible to build complex time-responsive molecular circuits by following a modular approach to the design of dynamic in vitro …
2011 (6)
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Living organisms perform and control complex behaviours by using webs of chemical reactions organized in precise networks. This powerful system concept, which is at the very core of biology, has recently become a new foundation for bioengineering. Remarkably, however, it is still extremely difficult to rationally create such network architectures in artificial, non‐living and well‐controlled settings. We introduce here a method for such a purpose, on the basis of standard DNA biochemistry. This approach is demonstrated by assembling de novo an efficient chemical oscillator: we encode the wiring of the corresponding network in the sequence of small DNA templates and obtain the predicted dynamics. Our results show that the rational cascading of standard elements opens the possibility to implement complex behaviours in vitro. Because of the simple and well …
Figure 1: Scientists try to capture the complexity of the genetic expression of a protein (left) in artificial in vitro settings (right).(Left) Enzymes (red) in the nucleus transcribe the genetic code onto RNA, stopping at the repressor protein (pink). Messenger RNA (mRNA) carries this information outside the nucleus, where it is translated by ribosomes (enzymes) into proteins (blue). Enzymes also break down proteins (and RNA, not shown) into smaller amino acids.(Right) Karzbrun et al. designed a simple genetic network, made up from the machinery of bacteria, as a starting point to model the same process:“R” enzymes participate in transcription and translation;“X” enzymes break down the mRNA and proteins. Scientists try to capture the complexity of the genetic expression of a protein (left) in artificial in vitro settings (right).(Left) Enzymes (red) in the nucleus transcribe the genetic code onto RNA …
Hydrogen atoms of proteinmolecule have setdom been assigned by X-ray … Hydrogen atoms havebeenassigned by X-ray sLructural analyses perfonncd at … Kazuki Takeda U}ziversity) informationofthe outer shell e]ectrons iscritical forunderstariding theactivity of clectron canier proteins.In addition, orientations of bound waters significantly affect redox potentialsof the proteins.Therefore,visualization of … SPring-8. HoweveT, many jrnprovcments oll the data colrections were … , Yu Hirano,KunioMiki (GraduateSchool ofSbience,1<],oto … Teso]ution X-raycrystal structures, this signaT should refiect hydrogenbond alteration at the watcr-channel gateinHelix-X. thussuggesting the dynamic … 2SD-05 Itsfi”+opeeAEXh=XAeneoptrUktoopS- ptvatta\co MN Theoreticaland computational quantum structura] bioLogyt’or understanding functionalmechanisms of bio]ogical macromo]ecu]arsvsterns Masaru Tatenod,Jiyoung Kangi, Ryo Nakaki? (i …
Living organisms perform and control complex behaviours by using webs of chemical reactions organized in precise networks. This powerful system concept, which is at the very core of biology, has recently become a new foundation for bioengineering. Remarkably, however, it is still extremely difficult to rationally create such network architectures in artificial, non‐living and well‐controlled settings. We introduce here a method for such a purpose, on the basis of standard DNA biochemistry. This approach is demonstrated by assembling de novo an efficient chemical oscillator: we encode the wiring of the corresponding network in the sequence of small DNA templates and obtain the predicted dynamics. Our results show that the rational cascading of standard elements opens the possibility to implement complex behaviours in vitro. Because of the simple and well‐controlled environment, the corresponding chemical …
Figure 1: Scientists try to capture the complexity of the genetic expression of a protein (left) in artificial in vitro settings (right).(Left) Enzymes (red) in the nucleus transcribe the genetic code onto RNA, stopping at the repressor protein (pink). Messenger RNA (mRNA) carries this information outside the nucleus, where it is translated by ribosomes (enzymes) into proteins (blue). Enzymes also break down proteins (and RNA, not shown) into smaller amino acids.(Right) Karzbrun et al. designed a simple genetic network, made up from the machinery of bacteria, as a starting point to model the same process:“R” enzymes participate in transcription and translation;“X” enzymes break down the mRNA and proteins. Scientists try to capture the complexity of the genetic expression of a protein (left) in artificial in vitro settings (right).(Left) Enzymes (red) in the nucleus transcribe the genetic code onto RNA, stopping at the repressor protein (pink). Messen… Show more
2009 (3)
We previously developed micron-sized, femtoliter (fL) reaction chamber array made of polydimethylsiloxane and polyacrylamide. Downsizing of the reaction volume to fL level made it possible to carry out single-molecule assay (SMA) of biological reactions in a straightforward manner. In this review, preparation of the fL chamber array and its application to SMA of enzyme, DNA, and rotary motor protein under an optical microscope are described.
We previously developed micron-sized, femtoliter (fL) reaction chamber array made of polydimethylsiloxane and polyacrylamide. Downsizing of the reaction volume to fL level made it possible to carry out single-molecule assay (SMA) of biological reactions in a straightforward manner. In this review, preparation of the fL chamber array and its application to SMA of enzyme, DNA, and rotary motor protein under an optical microscope are described.
We propose a device to electrically trap and lyse single bacterial cells in an array format for high-throughput analysis. The applied electric field is highly deformed and concentrated toward the inside of the microwell structure patterned on the plane electrode, which effectively generates dielectrophoresis (DEP) to attract a single cell to a nearby microchamber. We sucsessfully trapped single cells (Escherichia coli) into the microwell array by DEP and lysed the trapped cells by applying high electric pulse.