Publications by authors named "Yannick Rondelez"

37 Publications

A small-molecule chemical interface for molecular programs.

Nucleic Acids Res 2021 07;49(13):7765-7774

Laboratoire Gulliver, CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin 75005 Paris, France.

In vitro molecular circuits, based on DNA-programmable chemistries, can perform an increasing range of high-level functions, such as molecular level computation, image or chemical pattern recognition and pattern generation. Most reported demonstrations, however, can only accept nucleic acids as input signals. Real-world applications of these programmable chemistries critically depend on strategies to interface them with a variety of non-DNA inputs, in particular small biologically relevant chemicals. We introduce here a general strategy to interface DNA-based circuits with non-DNA signals, based on input-translating modules. These translating modules contain a DNA response part and an allosteric protein sensing part, and use a simple design that renders them fully tunable and modular. They can be repurposed to either transmit or invert the response associated with the presence of a given input. By combining these translating-modules with robust and leak-free amplification motifs, we build sensing circuits that provide a fluorescent quantitative time-response to the concentration of their small-molecule input, with good specificity and sensitivity. The programmability of the DNA layer can be leveraged to perform DNA based signal processing operations, which we demonstrate here with logical inversion, signal modulation and a classification task on two inputs. The DNA circuits are also compatible with standard biochemical conditions, and we show the one-pot detection of an enzyme through its native metabolic activity. We anticipate that this sensitive small-molecule-to-DNA conversion strategy will play a critical role in the future applications of molecular-level circuitry.
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http://dx.doi.org/10.1093/nar/gkab470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8287923PMC
July 2021

Advances in multiplexed techniques for the detection and quantification of microRNAs.

Chem Soc Rev 2021 Mar 4;50(6):4141-4161. Epub 2021 Feb 4.

Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, Université de Paris, CNRS SNC5096, Equipe Labellisée Ligue Nationale Contre le Cancer, F-75006 Paris, France.

MicroRNA detection is currently a crucial analytical chemistry challenge: almost 2000 papers were referenced in PubMed in 2018 and 2019 for the keywords "miRNA detection method". MicroRNAs are potential biomarkers for multiple diseases including cancers, neurodegenerative and cardiovascular diseases. Since miRNAs are stably released in bodily fluids, they are of prime interest for the development of non-invasive diagnosis methods, such as liquid biopsies. Their detection is however challenging, as high levels of sensitivity, specificity and robustness are required. The analysis also needs to be quantitative, since the aim is to detect miRNA concentration changes. Moreover, a high multiplexing capability is also of crucial importance, since the clinical potential of miRNAs probably lays in our ability to perform parallel mapping of multiple miRNA concentrations and recognize typical disease signature from this profile. A plethora of biochemical innovative detection methods have been reported recently and some of them provide new solutions to the problem of sensitive multiplex detection. In this review, we propose to analyze in particular the new developments in multiplexed approaches to miRNA detection. The main aspects of these methods (including sensitivity and specificity) will be analyzed, with a particular focus on the demonstrated multiplexing capability and potential of each of these methods.
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http://dx.doi.org/10.1039/d0cs00609bDOI Listing
March 2021

Multiplex Digital MicroRNA Detection Using Cross-Inhibitory DNA Circuits.

ACS Sens 2020 08 25;5(8):2430-2437. Epub 2020 Jul 25.

Gulliver Laboratory, ESPCI Paris-Université PSL, 10 rue Vauquelin, 75005 Paris, France.

Ubiquitous post-transcriptional regulators in eukaryotes, microRNAs are currently emerging as promising biomarkers of physiological and pathological processes. Multiplex and digital detection of microRNAs represents a major challenge toward the use of microRNA signatures in clinical settings. The classical reverse transcription polymerase chain reaction quantification approach has important limitations because of the need for thermocycling and a reverse transcription step. Simpler, isothermal alternatives have been proposed, yet none could be adapted in both a digital and multiplex format. This is either because of a lack of sensitivity that forbids single molecule detection or molecular cross-talk reactions that are responsible for nonspecific amplification. Building on an ultrasensitive isothermal amplification mechanism, we present a strategy to suppress cross-talk reactions, allowing for robust isothermal and multiplex detection of microRNA targets. Our approach relies on target-specific DNA circuits interconnected with DNA-encoded inhibitors that repress nonspecific signal amplification. We demonstrate the one-step, isothermal, digital, and simultaneous quantification of various pairs of important microRNA targets.
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http://dx.doi.org/10.1021/acssensors.0c00593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460561PMC
August 2020

Quantifying the Performance of Micro-Compartmentalized Directed Evolution Protocols.

Life (Basel) 2020 Feb 13;10(2). Epub 2020 Feb 13.

Laboratoire Gulliver, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005 Paris, France.

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.
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http://dx.doi.org/10.3390/life10020017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175308PMC
February 2020

Isothermal digital detection of microRNAs using background-free molecular circuit.

Sci Adv 2020 01 22;6(4):eaay5952. Epub 2020 Jan 22.

Laboratoire Gulliver, CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005 Paris, France.

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-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.
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http://dx.doi.org/10.1126/sciadv.aay5952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976291PMC
January 2020

Streamlined digital bioassays with a 3D printed sample changer.

Analyst 2020 Jan 26;145(2):572-581. Epub 2019 Nov 26.

Laboratoire Gulliver, UMR7083 CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005 Paris, France.

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 single microfluidic chip. A 3D printed sample rotor is loaded with all samples and connected to a pressure source. Simple magnetic actuation is then used to inject the samples in the microfluidic chip without pressure disruption. This procedure generates monodisperse droplets with high sample-to-sample consistency. We also describe a fluorescent barcoding strategy that allows all samples to be collected, incubated, imaged and analyzed simultaneously, thus decreasing significantly the time of the assay. As an example of application, we perform a droplet digital PCR assay for the quantification of a DNA amplicon from 8 samples in less than 2 hours. We further validate our approach demonstrating the parallel quantification of 11 microRNAs from a human sample using an isothermal nucleic acid amplification chemistry. As an off-chip device, the sample changer can be connected to a variety of microfluidic geometries and therefore, used for a wide range of applications.
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http://dx.doi.org/10.1039/c9an01744eDOI Listing
January 2020

Emerging isothermal amplification technologies for microRNA biosensing: Applications to liquid biopsies.

Mol Aspects Med 2020 04 23;72:100832. Epub 2019 Nov 23.

Centre de Recherche des Cordeliers, INSERM, CNRS, Sorbonne. Université, USPC, Université Paris Descartes, Université Paris Diderot, F-75006, Paris, France; INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, 45 rue des Saints-Pères, Paris, 75006, France; Equipe labellisée Ligue Nationale contre le cancer, France. Electronic address:

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 potential for multiplexing. We also review the different detection formats to which these chemistries have been adapted, including analog readouts such as real-time monitoring, digital counting based on single-molecule amplification in compartments, and surface-based strategies.
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http://dx.doi.org/10.1016/j.mam.2019.11.002DOI Listing
April 2020

Selection strategies for randomly partitioned genetic replicators.

Phys Rev E 2019 Jun;99(6-1):062416

Laboratoire Gulliver, CNRS, ESPCI Paris, PSL Research University, 75005 Paris, France.

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 linkage and affecting 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 on selection 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.
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http://dx.doi.org/10.1103/PhysRevE.99.062416DOI Listing
June 2019

Spatiotemporal control of DNA-based chemical reaction network via electrochemical activation in microfluidics.

Sci Rep 2018 04 23;8(1):6396. Epub 2018 Apr 23.

BioMEMS, Univ. Lille, CNRS, ISEN, UMR 8520 - IEMN, F-59000, Lille, France.

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 instructions to these molecular systems. We demonstrate how this approach can be used to release at specific times and locations DNA- based instructions. In particular, we trigger reaction-diffusion autocatalytic fronts in microfluidic channels. While limited by the stability of the Au-S bond, this easy to implement, versatile and scalable technique can be used in any biology laboratory to provide new ways to interact with any DNA-based computing framework.
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http://dx.doi.org/10.1038/s41598-018-24659-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913268PMC
April 2018

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

Nat Chem 2017 10 1;9(10):990-996. Epub 2017 May 1.

Laboratoire Jean Perrin, Université Pierre et Marie Curie, 4 place Jussieu, 75005 Paris, France.

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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http://dx.doi.org/10.1038/nchem.2770DOI Listing
October 2017

Massively parallel and multiparameter titration of biochemical assays with droplet microfluidics.

Nat Protoc 2017 Sep 24;12(9):1912-1932. Epub 2017 Aug 24.

LIMMS, CNRS-Institute of Industrial Science, UMI 2820, The University of Tokyo, Tokyo, Japan.

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 read through its internal standard (barcode), which is fluorescent but chemically orthogonal. We describe in detail the workflow, which comprises the following: (i) production of the microfluidics chips, (ii) preparation of the biochemical mixes, (iii) their mixing and compartmentalization into water-in-oil emulsion droplets via microfluidics, (iv) incubation and imaging of the fluorescent barcode and reporter signals by fluorescence microscopy and (v) image processing and data analysis. We also provide recommendations for choosing the appropriate fluorescent markers, programming the pressure profiles and analyzing the generated data. Overall, this platform allows a researcher with a few weeks of training to acquire ∼10,000 data points (in a 1D, 2D or 3D concentration space) over the course of a day from as little as 100-1,000 μl of reaction mix.
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http://dx.doi.org/10.1038/nprot.2017.092DOI Listing
September 2017

DNA computing: Spatially localized DNA domino.

Nat Nanotechnol 2017 09 24;12(9):842-843. Epub 2017 Jul 24.

Laboratoire Gulliver, Ecole Supérieure de Physique et de Chimie Industrielles, PSL Research University, and CNRS, Paris, France.

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http://dx.doi.org/10.1038/nnano.2017.157DOI Listing
September 2017

Boosting functionality of synthetic DNA circuits with tailored deactivation.

Nat Commun 2016 11 15;7:13474. Epub 2016 Nov 15.

LIMMS/CNRS-IIS (UMI 2820), Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

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: a system with three addressable bits of memory, and the first DNA-encoded excitable circuit. Specific saturable deactivation pathways thus greatly enrich the functional capability of a given circuit topology.
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http://dx.doi.org/10.1038/ncomms13474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116077PMC
November 2016

Synthesis of programmable reaction-diffusion fronts using DNA catalyzers.

Phys Rev Lett 2015 Feb 9;114(6):068301. Epub 2015 Feb 9.

Laboratoire de photonique et de nanostructures, CNRS, route de Nozay, 91460 Marcoussis, France.

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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http://dx.doi.org/10.1103/PhysRevLett.114.068301DOI Listing
February 2015

Automated design of programmable enzyme-driven DNA circuits.

ACS Synth Biol 2015 Jun 10;4(6):735-45. Epub 2014 Nov 10.

▽Department of Applied Mathematics, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.

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 screened for parameter values that display the desired function and combining this approach with robustness analysis further increases the chance of successful in vitro implementation. Moreover, we present an automated design procedure for generating optimal DNA sequences, exhibiting key characteristics deduced from the in silico analysis. Our in silico method has been tested on a previously reported network, the Oligator, and has also been applied to the design of a reaction network capable of displaying adaptation in one of its components. Finally, we experimentally characterize unproductive sequestration of the exonuclease to phosphorothioate protected ssDNA strands. The strong nonlinearities in the degradation of active components caused by this unintended cross-coupling are shown computationally to have a positive effect on adaptation quality.
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http://dx.doi.org/10.1021/sb500300dDOI Listing
June 2015

Dynamic DNA-toolbox reaction circuits: a walkthrough.

Methods 2014 May 2;67(2):234-49. Epub 2014 Feb 2.

LIMMS/CNRS UMI2820 Institute of Industrial Science, The University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 155-0085, Japan. Electronic address:

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 provides an experimental way to reproduce the dynamic features of genetic regulation in a test tube. We introduce the rules to design the necessary DNA species, a guide to implement the chemical reactions and ways to optimize the experimental conditions. We finally show how this framework, or "DNA toolbox", can be used to generate an inversion module, though many other behaviors, including oscillators and bistable switches, can be implemented.
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http://dx.doi.org/10.1016/j.ymeth.2014.01.015DOI Listing
May 2014

Computer-assisted design for scaling up systems based on DNA reaction networks.

J R Soc Interface 2014 Apr 22;11(93):20131167. Epub 2014 Jan 22.

Graduate School of Information Science and Technology, University of Tokyo, , Tokyo, Japan.

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 Artificial Circuits Computer-Assisted Design (DACCAD), a computer-assisted design software that supports the construction of systems for the DNA toolbox. DACCAD is ultimately aimed to design actual in vitro implementations, which is made possible by building on the experimental knowledge available on the DNA toolbox. We illustrate its effectiveness by designing various systems, from Montagne et al.'s Oligator or Padirac et al.'s bistable system to new and complex networks, including a two-bit counter or a frequency divider as well as an example of very large system encoding the game Mastermind. In the process, we highlight a variety of behaviours, such as enzymatic saturation and load effect, which would be hard to handle or even predict with a simpler model. We also show that those mechanisms, while generally seen as detrimental, can be used in a positive way, as functional part of a design. Additionally, the number of parameters included in these simulations can be large, especially in the case of complex systems. For this reason, we included the possibility to use CMA-ES, a state-of-the-art optimization algorithm that will automatically evolve parameters chosen by the user to try to match a specified behaviour. Finally, because all possible functionality cannot be captured by a single software, DACCAD includes the possibility to export a system in the synthetic biology markup language, a widely used language for describing biological reaction systems. DACCAD can be downloaded online at http://www.yannick-rondelez.com/downloads/.
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http://dx.doi.org/10.1098/rsif.2013.1167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928947PMC
April 2014

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

Chem Commun (Camb) 2013 Sep 5;49(73):8090-2. Epub 2013 Aug 5.

LIMMS/CNRS-IIS, University of Tokyo, Komaba 4-6-2 Meguro-ku, Tokyo, Japan.

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.
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http://dx.doi.org/10.1039/c3cc44323jDOI Listing
September 2013

Scaling down DNA circuits with competitive neural networks.

J R Soc Interface 2013 Aug 12;10(85):20130212. Epub 2013 Jun 12.

LIMMS/CNRS-IIS, University of Tokyo, Tokyo, Japan.

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.
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http://dx.doi.org/10.1098/rsif.2013.0212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4043154PMC
August 2013

Spatial waves in synthetic biochemical networks.

J Am Chem Soc 2013 Oct 20;135(39):14586-92. Epub 2013 Jun 20.

LIMMS/CNRS-IIS, The University of Tokyo , Komaba 4-6-2, Meguro-ku, Tokyo 153-8505, Japan.

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 unprecedented opportunities for assaying the principles of spatiotemporal order formation in chemistry.
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http://dx.doi.org/10.1021/ja403584pDOI Listing
October 2013

In vitro regulatory models for systems biology.

Biotechnol Adv 2013 Nov 4;31(6):789-96. Epub 2013 May 4.

LIMMS/CNRS-IIS, Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

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.
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http://dx.doi.org/10.1016/j.biotechadv.2013.04.008DOI Listing
November 2013

Nucleic acids for the rational design of reaction circuits.

Curr Opin Biotechnol 2013 Aug 22;24(4):575-80. Epub 2012 Dec 22.

LIMMS/CNRS-IIS, Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

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.
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http://dx.doi.org/10.1016/j.copbio.2012.11.011DOI Listing
August 2013

Computing with competition in biochemical networks.

Phys Rev Lett 2012 Nov 13;109(20):208102. Epub 2012 Nov 13.

LIMMS/CNRS-IIS, Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Tokyo 153-8505, Japan.

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 feedback loops.
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http://dx.doi.org/10.1103/PhysRevLett.109.208102DOI Listing
November 2012

Predator-prey molecular ecosystems.

ACS Nano 2013 Jan 17;7(1):27-34. Epub 2012 Dec 17.

LIMMS/CNRS-IIS, Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Tokyo 153-8505, Japan.

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 systems are tailored through a novel, compact, and versatile design strategy, leveraging the programmability of DNA interactions under the precise control of enzymatic catalysis. Such self-organizing assemblies will foster a better appreciation of the molecular origins of biological complexity and may also serve to orchestrate complex collective operations of molecular agents in technological applications.
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http://dx.doi.org/10.1021/nn3043572DOI Listing
January 2013

Bottom-up construction of in vitro switchable memories.

Proc Natl Acad Sci U S A 2012 Nov 29;109(47):E3212-20. Epub 2012 Oct 29.

Laboratory for Integrated Micro-Mechatronic Systems, Centre National de la Recherche Scientifique/Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

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 behaviors. Our approach thus provides an unmatched opportunity to study topology/function relationships within dynamic reaction networks.
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http://dx.doi.org/10.1073/pnas.1212069109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511151PMC
November 2012

Quencher-free multiplexed monitoring of DNA reaction circuits.

Nucleic Acids Res 2012 Aug 29;40(15):e118. Epub 2012 Jun 29.

LIMMS/CNRS-IIS, Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

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 and compatible with the complex dynamics of DNA reaction circuits. As an application, we show the implementation of nucleobase quenching to monitor a DNA-based chemical oscillator, allowing us to follow in real time and quantitatively the dephased oscillations of the components of the network. This cost-effective monitoring technique should be widely implementable to other DNA-based reaction systems.
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http://dx.doi.org/10.1093/nar/gks621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424586PMC
August 2012

Competition for catalytic resources alters biological network dynamics.

Authors:
Yannick Rondelez

Phys Rev Lett 2012 Jan 5;108(1):018102. Epub 2012 Jan 5.

LIMMS/CNRS-IIS, Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

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 expected behavior of synthetic biology constructs.
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http://dx.doi.org/10.1103/PhysRevLett.108.018102DOI Listing
January 2012

A microfluidic device for on-chip agarose microbead generation with ultralow reagent consumption.

Biomicrofluidics 2012 9;6(4):44101. Epub 2012 Oct 9.

LIMMS/CNRS-IIS, Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

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 present at the inlet is thus aspirated in the device, and segmented in microdroplets. The cooling system consists of two copper wires embedded in the microfluidic device. The transition from agarose microdroplets to microbeads provides additional stability and facilitated manipulation. We demonstrate the potential of this method by performing on-chip a temperature-triggered DNA isothermal amplification in agarose microbeads. Our device thus provides a new way to generate microbeads with high throughput and no dead volume for biochemical applications.
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http://dx.doi.org/10.1063/1.4758460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482248PMC
October 2013

Programming an in vitro DNA oscillator using a molecular networking strategy.

Mol Syst Biol 2011 Feb;7:466

LIMMS/CNRS-IIS, Institute of Industrial Science, University of Tokyo, Meguro-ku, Tokyo, Japan.

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 network is easily amenable to quantitative mathematical analysis. These synthetic systems may thus accelerate our understanding of the underlying principles of biological dynamic modules.
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http://dx.doi.org/10.1038/msb.2010.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063689PMC
February 2011

Monocopper center embedded in a biomimetic cavity: from supramolecular control of copper coordination to redox regulation.

J Am Chem Soc 2007 Jul 20;129(28):8801-10. Epub 2007 Jun 20.

Laboratoire de Chimie, Electrochimie Moléculaires et Chimie Analytique, UMR CNRS 6521, Université de Bretagne Occidentale, CS 93837, 6 avenue Le Gorgeu, 29238 Brest cedex 3, France.

The electrochemical behavior of diversely substituted Cu-N3-calix[6]arene, enzyme-like, "funnel" complexes is analyzed. The Cu(II)/Cu(I) redox process is regulated by the supramolecular organization of the Cu coordination. The presence of a "shoetree" alkyl nitrile guest molecule inside the host cavity is a prerequisite for a dynamic redox behavior. Combination of supramolecular CH-pi weak interactions with the calixarene cavity and electronic/steric effects from the N3 substituting groups (pyridine, imidazole, pyrrolidine) enforces the preferential geometrical pattern adopted by Cu. This dictates the pathway of the electron-transfer process and, thus, the thermodynamics and kinetics of the redox reaction in the framework of a square-scheme mechanism. The present observations recall strongly the redox control exerted by the protein matrix on copper proteins through biological concepts such as induced fit mechanism, protein foldings, and entatic and allosteric effects.
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http://dx.doi.org/10.1021/ja071219hDOI Listing
July 2007
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