Publications by authors named "Guillaume Gines"

11 Publications

  • Page 1 of 1

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

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

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

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

A multiplex assay based on encoded microbeads conjugated to DNA NanoBeacons to monitor base excision repair activities by flow cytometry.

Biosens Bioelectron 2014 Aug 24;58:81-4. Epub 2014 Feb 24.

Laboratoire des Lésions des Acides Nucléiques, INAC/SCIB UMR_E3 CEA/UJF-Grenoble 1/CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France. Electronic address:

We reported here a new assay to detect base excision repair activities from purified enzymes, as well as in cell-free extracts. The multiplex format rests upon the encoding of magnetic beads with the fluorophore Alexa 488, thanks to a fast and original procedure. Fluorescently encoded microbeads are subsequently functionalized by lesion-containing DNA NanoBeacons labeled with the fluorophore/quencher pair Cyanine 5/BHQ2. Probes cleavage, induced by targeted enzymes leads to Cyanine 5 signal enhancement, which is finally quantified by flow cytometry. The multiplex assay was applied to the detection of restriction enzymes activities as well as base excision repair processes. Each test requires only 500fmol of DNA substrate, which constitutes great sensitivity compared to other BER functional assays. The present biosensor is able to detect both uracil DNA N-glycosylase (UNG) and AP-endonuclease 1 (APE1) within few nanograms of nuclear extract. Additionally, we demonstrated that the corresponding assay has potential application in DNA repair inhibitor search. Finally, the current multiplexed tool shows several advantages in comparison to other functional BER assays with no need of electrophoretic separation, straightforward, easy and reproducible functionalization of encoded microbeads and a high stability of DNA probes in cell-free extracts.
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http://dx.doi.org/10.1016/j.bios.2014.02.040DOI Listing
August 2014

On-bead fluorescent DNA nanoprobes to analyze base excision repair activities.

Anal Chim Acta 2014 Feb 9;812:168-75. Epub 2014 Jan 9.

Laboratoire des Lésions des Acides Nucléiques, SCIB-UMR E3 CEA-UJF/INAC/CEA Grenoble, Grenoble Cedex 09 38054, France. Electronic address:

DNA integrity is constantly threatened by endogenous and exogenous agents that can modify its physical and chemical structure. Changes in DNA sequence can cause mutations sparked by some genetic diseases or cancers. Organisms have developed efficient defense mechanisms able to specifically repair each kind of lesion (alkylation, oxidation, single or double strand break, mismatch, etc). Here we report the adjustment of an original assay to detect enzymes' activity of base excision repair (BER), that supports a set of lesions including abasic sites, alkylation, oxidation or deamination products of bases. The biosensor is characterized by a set of fluorescent hairpin-shaped nucleic acid probes supported on magnetic beads, each containing a selective lesion targeting a specific BER enzyme. We have studied the DNA glycosylase alkyl-adenine glycosylase (AAG) and the human AP-endonuclease (APE1) by incorporating within the DNA probe a hypoxanthine lesion or an abasic site analog (tetrahydrofuran), respectively. Enzymatic repair activity induces the formation of a nick in the damaged strand, leading to probe's break, that is detected in the supernatant by fluorescence. The functional assay allows the measurement of DNA repair activities from purified enzymes or in cell-free extracts in a fast, specific, quantitative and sensitive way, using only 1 pmol of probe for a test. We recorded a detection limit of 1 μg mL(-1) and 50 μg mL(-1) of HeLa nuclear extracts for APE1 and AAG enzymes, respectively. Finally, the on-bead assay should be useful to screen inhibitors of DNA repair activities.
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http://dx.doi.org/10.1016/j.aca.2013.12.038DOI Listing
February 2014
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