Laboratoire d'optique et biosciences
Laboratoire d'optique et biosciences
Laboratoire d'optique et biosciences

Articles

  • Mueller polarimetric imaging for fast macroscopic mapping of microscopic collagen matrix remodeling by smooth muscle cells
    • Chashchina Olga
    • Mezouar Hachem
    • Vizet Jérémy
    • Raoux Clothilde
    • Park Junha
    • Ramón-Lozano Clara
    • Schanne-Klein Marie-Claire
    • Barakat Abdul I
    • Pierangelo Angelo
    Scientific Reports, Nature Publishing Group , 2021, 11 . Smooth muscle cells (SMCs) are critical players in cardiovascular disease development and undergo complex phenotype switching during disease progression. However, SMC phenotype is difficult to assess and track in co-culture studies. To determine the contractility of SMCs embedded within collagen hydrogels, we performed polarized light imaging and subsequent analysis based on Mueller matrices. Measurements were made both in the absence and presence of endothelial cells (ECs) in order to establish the impact of EC-SMC communication on SMC contractility. The results demonstrated that Mueller polarimetric imaging is indeed an appropriate tool for assessing SMC activity which significantly modifies the hydrogel retardance in the presence of ECs. These findings are consistent with the idea that EC-SMC communication promotes a more contractile SMC phenotype. More broadly, our findings suggest that Mueller polarimetry can be a useful tool for studies of spatial heterogeneities in hydrogel remodeling by SMCs. (10.1038/s41598-021-85164-y)
    DOI : 10.1038/s41598-021-85164-y
  • Ultrafast dynamics of heme distortion in the O2-sensor of a thermophilic anaerobe bacterium
    • Petrova Olga N
    • Yoo Byung-Kuk
    • Lamarre Isabelle
    • Selles Julien
    • Nioche Pierre
    • Negrerie Michel
    Communications Chemistry, Nature Research , 2021, 4 (1), pp.31 . Heme-Nitric oxide and Oxygen binding protein domains (H-NOX) are found in signaling pathways of both prokaryotes and eukaryotes and share sequence homology with soluble guanylate cyclase, the mammalian NO receptor. In bacteria, H-NOX is associated with kinase or methyl accepting chemotaxis domains. In the O2-sensor of the strict anaerobe Caldanaerobacter tengcongensis (Ct H-NOX) the heme appears highly distorted after O2 binding, but the role of heme distortion in allosteric transitions was not yet evidenced. Here, we measure the dynamics of the heme distortion triggered by the dissociation of diatomics from Ct H-NOX using transient electronic absorption spectroscopy in the picosecond to millisecond time range. We obtained a spectroscopic signature of the heme flattening upon O2 dissociation. The heme distortion is immediately (<1 ps) released after O2 dissociation to produce a relaxed state. This heme conformational change occurs with different proportions depending on diatomics as follows: CO < NO < O2. Our time-resolved data demonstrate that the primary structural event of allostery is the heme distortion in the Ct H-NOX sensor, contrastingly with hemoglobin and the human NO receptor, in which the primary structural events are respectively the motion of the proximal histidine and the rupture of the iron-histidine bond. (10.1038/s42004-021-00471-9)
    DOI : 10.1038/s42004-021-00471-9
  • Ligand Binding to Dynamically Populated G‐Quadruplex DNA
    • Aznauryan Mikayel
    • Noer Sofie Louise
    • Pedersen Camilla
    • Mergny Jean‐louis
    • Teulade-Fichou Marie‐paule
    • Birkedal Victoria
    ChemBioChem, Wiley-VCH Verlag , 2021, 22 (10), pp.1811-1817 . Several small‐molecule ligands specifically bind and stabilize G‐quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen‐DC3 and a combination of single‐molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre‐folded G4 structures and that Phen‐DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand‐induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligand‐induced G4 folding and trapping of dynamically populated short‐lived conformation states. Thus, ligand‐induced stabilization does not necessarily require the initial presence of stably folded G4s. (10.1002/cbic.202000792)
    DOI : 10.1002/cbic.202000792
  • Influence of core extension and side chain nature in targeting G-quadruplex structures with perylene monoimide derivatives
    • Busto Natalia
    • García-Calvo José
    • Cuevas José Vicente
    • Herrera Antonio
    • Mergny Jean-Louis
    • Pons Sebastian
    • Torroba Tomás
    • García Begoña
    Bioorganic Chemistry, Elsevier , 2021, 108 (18), pp.104660 . Abstract Recent studies indicate that i‐DNA, a four‐stranded cytosine‐rich DNA also known as the i‐motif, is actually formed in vivo; however, a systematic study on sequence effects on stability has been missing. Herein, an unprecedented number of different sequences (271) bearing four runs of 3–6 cytosines with different spacer lengths has been tested. While i‐DNA stability is nearly independent on total spacer length, the central spacer plays a special role on stability. Stability also depends on the length of the C‐tracts at both acidic and neutral pHs. This study provides a global picture on i‐DNA stability thanks to the large size of the introduced data set; it reveals unexpected features and allows to conclude that determinants of i‐DNA stability do not mirror those of G‐quadruplexes. Our results illustrate the structural roles of loops and C‐tracts on i‐DNA stability, confirm its formation in cells, and allow establishing rules to predict its stability. (10.1016/j.bioorg.2021.104660)
    DOI : 10.1016/j.bioorg.2021.104660
  • Characterization of Light-Induced, Short-Lived Interacting Radicals in the Active Site of Flavoprotein Ferredoxin-NADP + Oxidoreductase
    • Zhuang Bo
    • Seo Daisuke
    • Aleksandrov Alexey
    • Vos Marten H.
    Journal of the American Chemical Society, American Chemical Society , 2021, 143 (7), pp.2757-2768 . Radicals of flavin adenine dinucleotide (FAD), as well as tyrosine and tryptophan, are widely involved as key reactive intermediates during electron transfer (ET) reactions in flavoproteins. Due to the high reactivity of these species, and their corresponding short lifetime, characterization of these intermediates in functional processes of flavoproteins is usually challenging, but can be achieved by ultrafast spectroscopic studies of light-activatable flavoproteins. In ferredoxin-NADP + oxidoreductase from Bacillus subtilis (BsFNR), fluorescence of the FAD cofactor that very closely interacts with a neighboring tyrosine residue (Tyr50), is strongly quenched. Here we study short-lived photoproducts of this enzyme and its variants with Tyr50 replaced by tryptophan or glycine. Using time-resolved fluorescence and absorption spectroscopies, we show that upon the excitation of WT BsFNR, ultrafast ET from Tyr50 to the excited FAD cofactor occurs in ~260 fs, an order of magnitude faster than the decay by charge recombination, facilitating the characterization of the reaction intermediates in the charge-separated state with respect to other recently studied systems. These studies are corroborated by experiments on the Y50W mutant protein, which yield photoproducts qualitatively similar to those observed in other tryptophan bearing flavoproteins. By combining the experimental results with molecular dynamics simulations and quantum mechanics calculations, we investigate in detail the effect of protein environment and relaxations on the spectral properties of those radical intermediates, and demonstrate that the spectral features of radical anionic FAD are highly sensitive to its environment, and in particular to the dynamics and nature of the counter-ions formed in the photoproducts. Altogether, comprehensive characterizations are provided for important radical intermediates that are generally involved in functional processes of flavoproteins. (10.1021/jacs.0c09627)
    DOI : 10.1021/jacs.0c09627
  • Effects of sequence and base composition on the CD and TDS profiles of i-DNA
    • Iaccarino Nunzia
    • Cheng Mingpan
    • Qiu Dehui
    • Pagano Bruno
    • Amato Jussara
    • Porzio Anna Di
    • Zhou Jun
    • Randazzo Antonio
    • Mergny Jean‐louis
    Angewandte Chemie International Edition, Wiley-VCH Verlag , 2021, 60, pp.10295-10303 . (10.1002/anie.202016822)
    DOI : 10.1002/anie.202016822
  • Identification of the vibrational marker of tyrosine cation radical using ultrafast transient infrared spectroscopy of flavoprotein systems
    • Pirisi Katalin
    • Nag Lipsa
    • Fekete Zsuzsanna
    • Iuliano James N
    • Tolentino Collado Jinnette
    • Clark Ian P
    • Pécsi Ildikó
    • Sournia Pierre
    • Liebl Ursula
    • Greetham Gregory M
    • Tonge Peter J
    • Meech Stephen R
    • Vos Marten H.
    • Lukacs Andras
    Photochemical & Photobiological Sciences, Springer , 2021 . Tryptophan and tyrosine radical intermediates play crucial roles in many biological charge transfer processes. Particularly in flavoprotein photochemistry, short-lived reaction intermediates can be studied by the complementary techniques of ultrafast visible and infrared spectroscopy. The spectral properties of tryptophan radical are well established, and the formation of neutral tyrosine radicals has been observed in many biological processes. However, only recently, the formation of a cation tyrosine radical was observed by transient visible spectroscopy in a few systems. Here, we assigned the infrared vibrational markers of the cationic and neutral tyrosine radical at 1483 and 1502 cm −1 (in deuterated buffer), respectively, in a variant of the bacterial methyl transferase TrmFO, and in the native glucose oxidase. In addition, we studied a mutant of AppABLUF blue-light sensor domain from Rhodobacter sphaeroides in which only a direct formation of the neutral radical was observed. Our studies highlight the exquisite sensitivity of transient infrared spectroscopy to low concentrations of specific radicals. (10.1007/s43630-021-00024-y)
    DOI : 10.1007/s43630-021-00024-y
  • The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD
    • Gargaun Elena
    • Falcone Sestina
    • Sole Guilhem
    • Durigneux Julien
    • Urtizberea Andoni
    • Cuisset Jean Marie
    • Benkhelifa-Ziyyat Sofia
    • Julien Laura
    • Boland Anne
    • Sandron Florian
    • Meyer Vincent
    • Deleuze Jean François
    • Salgado David
    • Desvignes Jean-Pierre
    • Béroud Christophe
    • Chessel Anatole
    • Blesius Alexia
    • Krahn Martin
    • Levy Nicolas
    • Leturcq France
    • Pietri-Rouxel France
    Biomedicines, MDPI , 2021, 9 (2), pp.219 . In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion. (10.3390/biomedicines9020219)
    DOI : 10.3390/biomedicines9020219
  • Thermal and pH stabilities of i-DNA: confronting in vitro experiments with models and in-cell NMR data
    • Cheng Mingpan
    • Qiu Dehui
    • Tamon Liezel
    • Ištvánková Eva
    • Víšková Pavlína
    • Amrane Samir
    • Guédin Aurore
    • Chen Jielin
    • Lacroix Laurent
    • Ju Huangxian
    • Trantírek Lukáš
    • Sahakyan Aleksandr B
    • Zhou Jun
    • Mergny Jean‐louis
    Angewandte Chemie International Edition, Wiley-VCH Verlag , 2021, 60, pp.10286-10294 . (10.1002/anie.202016801)
    DOI : 10.1002/anie.202016801
  • Rapid Evaluation of Novel Therapeutic Strategies Using a 3D Collagen-Based Tissue-Like Model
    • Maury Pauline
    • Porcel Erika
    • Mau Adrien
    • Lux François
    • Tillement Olivier
    • Mahou Pierre
    • Schanne-Klein Marie-Claire
    • Lacombe Sandrine
    Frontiers in Bioengineering and Biotechnology, Frontiers , 2021 . 2D cell cultures are commonly used to rapidly evaluate the therapeutic potential of various treatments on living cells. However, the effects of the extracellular matrix (ECM) including the 3D arrangement of cells and the complex physiology of native environment are missing, which makes these models far from in vivo conditions. 3D cell models have emerged in preclinical studies to simulate the impact of the ECM and partially bridge the gap between monolayer cultures and in vivo tissues. To date, the difficulty to handle the existing 3D models, the cost of their production and their poor reproducibility have hindered their use. Here, we present a reproducible and commercially available “3D cell collagen-based model” (3D-CCM) that allows to study the influence of the matrix on nanoagent uptake and radiation effects. The cell density in these samples is homogeneous. The oxygen concentration in the 3D-CCM is tunable, which opens the opportunity to investigate hypoxic effects. In addition, thanks to the intrinsic properties of the collagen, the second harmonic imaging microscopy may be used to probe the whole volume and visualize living cells in real-time. Thus, the architecture and composition of 3D-CCMs as well as the impact of various therapeutic strategies on cells embedded in the ECM is observed directly. Moreover, the disaggregation of the collagen matrix allows recovering of cells without damaging them. It is a major advantage that makes possible single cell analysis and quantification of treatment effects using clonogenic assay. In this work, 3D-CCMs were used to evaluate the correlative efficacies of nanodrug exposure and medical radiation on cells contained in a tumor like sample. A comparison with monolayer cell cultures was performed showing the advantageous outcome and the higher potential of 3D-CCMs. This cheap and easy to handle approach is more ethical than in vivo experiments, thus, giving a fast evaluation of cellular responses to various treatments. (10.3389/fbioe.2021.574035)
    DOI : 10.3389/fbioe.2021.574035
  • Euryarchaeal genomes are folded into SMC-dependent loops and domains, but lack transcription-mediated compartmentalization
    • Cockram Charlotte
    • Thierry Agnès
    • Gorlas Aurore
    • Lestini Roxane
    • Koszul Romain
    Molecular Cell, Cell Press , 2021, 81 (3), pp.459-472.e10 . Hi-C has become a routine method for probing the 3D organization of genomes. However, when applied to prokaryotes and archaea, the current protocols are expensive and limited in their resolution. We develop a cost-effective Hi-C protocol to explore chromosome conformations of these two kingdoms at the gene or operon level. We first validate it on E. coli and V. cholera, generating sub-kilobase-resolution contact maps, and then apply it to the euryarchaeota H. volcanii, Hbt. salinarum, and T. kodakaraensis. With a resolution of up to 1 kb, we explore the diversity of chromosome folding in this phylum. In contrast to crenarchaeota, these euryarchaeota lack (active/inactive) compartment-like structures. Instead, their genomes are composed of self-interacting domains and chromatin loops. In H. volcanii, these structures are regulated by transcription and the archaeal structural maintenance of chromosomes (SMC) protein, further supporting the ubiquitous role of these processes in shaping the higher-order organization of genomes. (10.1016/j.molcel.2020.12.013)
    DOI : 10.1016/j.molcel.2020.12.013
  • Insights into G-Quadruplex–Hemin Dynamics Using Atomistic Simulations: Implications for Reactivity and Folding
    • Stadlbauer Petr
    • Islam Barira
    • Otyepka Michal
    • Chen Jielin
    • Monchaud David
    • Zhou Jun
    • Mergny Jean-Louis
    • Šponer Jiří
    Journal of Chemical Theory and Computation, American Chemical Society , 2021, 17 (3), pp.1883 - 1899 . Guanine quadruplex nucleic acids (G4s) are involved in key biological processes such as replication or transcription. Beyond their biological relevance, G4s find applications as biotechnological tools since they readily bind hemin and enhance its peroxidase activity, creating a G4-DNAzyme. The biocatalytic properties of G4-DNAzymes have been thoroughly studied and used for biosensing purposes. Despite hundreds of applications and massive experimental efforts, the atomistic details of the reaction mechanism remain unclear. To help select between the different hypotheses currently under investigation, we use extended explicit-solvent molecular dynamics (MD) simulations to scrutinize the G4/hemin interaction. We find that besides the dominant conformation in which hemin is stacked atop the external G-quartets, hemin can also transiently bind to the loops and be brought to the external G-quartets through diverse delivery mechanisms. The simulations do not support the catalytic mechanism relying on a wobbling guanine. Similarly, the catalytic role of the iron-bound water molecule is not in line with our results; however, given the simulation limitations, this observation should be considered with some caution. The simulations rather suggest tentative mechanisms in which the external G-quartet itself could be responsible for the unique H 2 O 2-promoted biocatalytic properties of the G4/hemin complexes. Once stacked atop a terminal G-quartet, hemin rotates about its vertical axis while readily sampling shifted geometries where the iron transiently contacts oxygen atoms of the adjacent G-quartet. This dynamics is not apparent from the ensemble-averaged structure. We also visualize transient interactions between the stacked hemin and the G4 loops. Finally, we investigated interactions between hemin and on-pathway folding intermediates of the parallel-stranded G4 fold. The simulations suggest that hemin drives the folding of parallel-stranded G4s from slip-stranded intermediates, acting as a G4 chaperone. Limitations of the MD technique are briefly discussed. (10.1021/acs.jctc.0c01176)
    DOI : 10.1021/acs.jctc.0c01176
  • Human Papillomavirus G-Rich Regions as Potential Antiviral Drug Targets
    • Carvalho Josué
    • Lopes-Nunes Jéssica
    • Campello Maria Paula Cabral
    • Paulo António
    • Milici Janice
    • Meyers Craig
    • Mergny Jean-Louis
    • Salgado Gilmar
    • Queiroz João
    • Cruz Carla
    Nucleic Acid Therapeutics, Mary Ann Liebert, Inc. publishers , 2021, 31 (1), pp.68-81 . Abstract G‐quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G‐quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4‐prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G‐quadruplex or not. In this article, we adapted the well‐known FRET‐melting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual‐labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G‐quadruplexes. In this so‐called FRET‐MC (melting competition) assay, G4‐forming competitors lead to a marked decrease of the ligand‐induced stabilization effect (∆ T m ), while non‐specific competitors (e.g., single‐ or double‐stranded sequences) have little effect. Sixty‐five known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized. (10.1089/nat.2020.0869)
    DOI : 10.1089/nat.2020.0869
  • Targeting nucleolin by RNA G-quadruplex-forming motif
    • Figueiredo Joana
    • Miranda André
    • Lopes-Nunes Jéssica
    • Carvalho Josué
    • Alexandre Daniela
    • Valente Salete
    • Mergny Jean‐louis
    • Cruz Carla
    Biochemical Pharmacology, Elsevier , 2021, 189, pp.114418 . (10.1016/j.bcp.2021.114418)
    DOI : 10.1016/j.bcp.2021.114418
  • Cyclodipeptide synthases of the NYH subfamily recognize tRNA using an α-helix enriched with positive residues
    • Croitoru Anastasia
    • Babin Morgan
    • Myllykallio Hannu
    • Gondry Muriel
    • Aleksandrov Alexey
    Biochemistry, American Chemical Society , 2021, 60 (1), pp.64-76 . Cyclodipeptide synthases (CDPSs) perform nonribosomal protein synthesis using two aminoacyl-tRNA substrates to produce cyclodipeptides. There is no available structural detail on the CDPS:tRNA interaction to date. Using AlbC, a CDPS that produces cyclo(L-Phe-L-Phe), the interaction between AlbC with its Phe-tRNA substrate was investigated. Simulations of models of the AlbC:tRNA complex, proposed by rigid body docking or homology modeling, demonstrated that interactions with residues of an AlbC alpha helix, α4, significantly contribute to the binding free energy of AlbC to tRNA. Individual residue contributions to the tRNA binding free energy of the discovered binding mode explain well available biochemical data, and the results of in vivo assay experiments performed in this work and guided by simulations. In molecular dynamics simulations the phenylalanyl group predominantly occupied the two positions observed in the experimental structure of AlbC in the dipeptide intermediate state, suggesting that tRNAs of the first and second substrates interact with AlbC in a similar manner. Overall, given the high sequence and structural similarity among the members of the CDPS NYH protein subfamily, the mechanism of the protein:tRNA interaction is expected to be pertinent to a wide range of tRNA interacting proteins. (10.1021/acs.biochem.0c00761)
    DOI : 10.1021/acs.biochem.0c00761
  • Core–Shell Pure Collagen Threads Extruded from Highly Concentrated Solutions Promote Colonization and Differentiation of C3H10T1/2 Cells
    • Picaut Lise
    • Trichet Lea
    • Hélary Christophe
    • Ducourthial Guillaume
    • Bonnin Marie-Ange
    • Haye Bernard
    • Ronsin Olivier
    • Schanne-Klein Marie-Claire
    • Duprez Delphine
    • Baumberger Tristan
    • Mosser Gervaise
    ACS Biomaterials Science and Engineering, ACS , 2021, 7 (2), pp.626–635 . The elaboration of scaffolds able to efficiently promote cell differentiation toward a given cell type remains challenging. Here, we engineered dense type I collagen threads with the aim of providing scaffolds with specific morphological and mechanical properties for C3H10T1/2 mesenchymal stem cells. Extrusion of pure collagen solutions at different concentrations (15, 30, and 60 mg/mL) in a PBS 5× buffer generated dense fibrillated collagen threads. For the two highest concentrations, threads displayed a core-shell structure with a marked fibril orientation of the outer layer along the longitudinal axis of the threads. Young's modulus and ultimate tensile stress as high as 1 and 0.3 MPa, respectively, were obtained for the most concentrated collagen threads without addition of any cross-linkers. C3H10T1/2 cells oriented themselves with a mean angle of 15-24° with respect to the longitudinal axis of the threads. Cells penetrated the 30 mg/mL scaffolds but remained on the surface of the 60 mg/mL ones. After three weeks of culture, cells displayed strong expression of the tendon differentiation marker Tnmd, especially for the 30 mg/mL threads. These results suggest that both the morphological and mechanical characteristics of collagen threads are key factors in promoting C3H10T1/2 differentiation into tenocytes, offering promising levers to optimize tissue engineering scaffolds for tendon regeneration. (10.1021/acsbiomaterials.0c01273)
    DOI : 10.1021/acsbiomaterials.0c01273
  • FRET-MC: a fluorescence melting competition assay for studying G4 structures in vitro
    • Luo Yu
    • Granzhan Anton
    • Verga Daniela
    • Mergny Jean-Louis
    Biopolymers, Wiley , 2021, 112 (4), pp.e23415 . G-quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G-quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4-prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G-quadruplex or not. In this article, we adapted the well-known FRETmelting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual-labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G-quadruplexes. In this so-called FRET-MC (melting competition) assay, G4-forming competitors lead to a marked decrease of the ligand-induced stabilization effect (∆Tm), while non-specific competitors (e.g., single-or double-stranded sequences) have little effect. Sixtyfive known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized. (10.1002/bip.23415)
    DOI : 10.1002/bip.23415
  • The catalytic properties of DNA G-quadruplexes rely on their structural integrity
    • Chen Jielin
    • Cheng Mingpan
    • Wang Jiawei
    • Qiu Dehui
    • Monchaud David
    • Mergny Jean‐louis
    • Ju Huangxian
    • Zhou Jun
    Chinese Journal of Catalysis, Elsevier , 2021, 42 (7), pp.1102-1107 . The influence of the G-quartet structural integrity on the catalytic activity of the G-quadruplex (G4) was investigated by comparing the G4-DNAzyme performances of a series of G4s with a G-vacancy site and a G-triplex (G-tri). The results presented herein not only confirm that the structural integrity of the 3’-end G-quartet is necessary for G4s to be catalytically competent but also show how to remediate G-vacancy-mediated catalytic activity losses via the addition of guanine surrogates in an approach referred to as G-vacancy complementation strategy that is applicable to parallel G4s only. Furthermore, this study demonstrates that the terminal G-quartet could act as a proximal coordinating group and cooperate with the flanking nucleotide to activate the hemin cofactor. (10.1016/S1872-2067(20)63744-5)
    DOI : 10.1016/S1872-2067(20)63744-5
  • Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus
    • Zhuang Bo
    • Nag Lipsa
    • Sournia Pierre
    • Croitoru Anastasia
    • Ramodiharilafy Rivo
    • Lambry Jean-Christophe
    • Myllykallio Hannu
    • Aleksandrov Alexey
    • Liebl Ursula
    • Vos Marten
    Photochemical & Photobiological Sciences, Springer , 2021, 20 (5), pp.663–670 . Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor–acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations (10.1007/s43630-021-00052-8)
    DOI : 10.1007/s43630-021-00052-8
  • L’imagerie optique de nanoparticules luminescentes : de la détection de biomolécules au diagnostic in vitro
    • Mousseau Fanny
    • Yu Chao
    • Alexandrou Antigoni
    • Bouzigues Cédric
    Photoniques, EDP Sciences , 2021 (106), pp.30-33 . Pour détecter des biomolécules et pathogènes (protéines, virus, bactéries, …) avec des sensibilités satisfaisantes, il est actuellement nécessaire d’utiliser des appareils de laboratoires coûteux. En combinant les remarquables propriétés optiques des ions lanthanides à un lecteur simple couplé à un smartphone, nous démontrons comment développer un système de détection portable, rapide et ultrasensible. (10.1051/photon/202110630)
    DOI : 10.1051/photon/202110630
  • Synthesis of acyclic nucleoside phosphonates targeting Flavin-Dependent Thymidylate Synthase in Mycobacterium tuberculosis
    • Biteau Nicolas G
    • Roy Vincent
    • Lambry J.-C.
    • Becker Hubert F
    • Myllykallio Hannu
    • Agrofoglio Luigi A
    Bioorganic and Medicinal Chemistry Letters, Elsevier , 2021, 46, pp.116351 . Flavin-Dependent Thymidylate Synthase (FDTS) encoded by ThyX gene was discovered as a new class of thymidylate synthase involved in the de novo synthesis of dTMP named only in 30 % of human pathogenic bacteria. This target was pursed for the development of new antibacterial agents against multiresistant pathogens. We have developed a new class of ANPs based on the mimic of two natural’s cofactors (dUMP and FAD) as inhibitors against Mycobacterium tuberculosis ThyX. Several synthetic efforts were performed to optimize regioselective N1-alkylation, cross-coupling metathesis and Sonogashira cross-coupling. Compound 19c showed a poor 31.8% inhibitory effect on ThyX at 200 μM. (10.1016/j.bmc.2021.116351)
    DOI : 10.1016/j.bmc.2021.116351
  • Methylselenol Produced In Vivo from Methylseleninic Acid or Dimethyl Diselenide Induces Toxic Protein Aggregation in Saccharomyces cerevisiae
    • Dauplais Marc
    • Bierla Katarzyna
    • Maizeray Coralie
    • Lestini Roxane
    • Lobinski Ryszard
    • Plateau Pierre
    • Szpunar Joanna
    • Lazard Myriam
    International Journal of Molecular Sciences, MDPI , 2021, 22 (5), pp.2241 . Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of MSeA and GSH) in S.cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation. (10.3390/ijms22052241)
    DOI : 10.3390/ijms22052241
  • SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction
    • Lavigne Marc
    • Helynck Olivier
    • Rigolet Pascal
    • Boudria-Souilah Rofia
    • Nowakowski Mireille
    • Baron Bruno
    • Brülé Sébastien
    • Hoos Sylviane
    • Raynal Bertrand
    • Guittat Lionel
    • Beauvineau Claire
    • Petres Stéphane
    • Granzhan Anton
    • Guillon Jean
    • Pratviel Geneviève
    • Teulade-Fichou Marie-Paule
    • England Patrick
    • Mergny Jean‐louis
    • Munier-Lehmann Hélène
    Nucleic Acids Research, Oxford University Press , 2021, 49 (13), pp.7695–7712 . The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds. (10.1093/nar/gkab571)
    DOI : 10.1093/nar/gkab571
  • Digital micromirror device for holographic and Fourier optics applications
    • Douet Brice
    • Tedoldi Téo
    • Kabacinski Adeline
    • Morana Ambra
    • Gallot Guilhem
    • Bouganne Raphaël
    Emergent Scientist, EDP Sciences , 2021, 5 (4), pp.1-7 . The electromagnetic wavefront diffracted by an object carries information about the shape of the object from which the wave was emitted. Being able to record the phase and intensity of such a wave thus allows to reconstruct the object from the information carried by the wave, even if the object is no longer present. Among the reconstruction techniques, holography plays a big part. However the waves may experience a great variety of distortions on their way from the object to the measurement apparatus. Thus being able to shape the wavefront at will is key in holography. Micromirror light modulators are powerful tools for that matter and are well known for holographic applications. This paper explores the fundamental principles for digitally reconstructing a precise image of an object, but also for digitally correcting an imperfectly shaped wavefront, by exploiting the diffraction properties of light on a reflective surface. The methods presented here have been implemented as part of practical work for 2nd year students at the Ecole Polytechnique (last year of undergraduate program). (10.1051/emsci/2021003)
    DOI : 10.1051/emsci/2021003
  • Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA–induced metabolic stress in reconstructed human skin
    • Ung Thi Phuong Lien
    • Lim Seongbin
    • Solinas Xavier
    • Mahou Pierre
    • Chessel Anatole
    • Marionnet Claire
    • Bornschlögl Thomas
    • Beaurepaire Emmanuel
    • Bernerd Françoise
    • Pena Ana-Maria
    • Stringari Chiara
    Scientific Reports, Nature Publishing Group , 2021, 11, pp.22171 . Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P) + /NAD(P)H and FAD/FADH 2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring. (10.1038/s41598-021-00126-8)
    DOI : 10.1038/s41598-021-00126-8