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7 results on '"Phanélie Perche"'

1. Cationic Oligospermine-Oligonucleotide Conjugates Provide Carrier-free Splice Switching in Monolayer Cells and Spheroids

Author

Marc Nothisen, Phanélie Perche-Létuvée, Jean-Paul Behr, Jean-Serge Remy, and Mitsuharu Kotera

Subjects
Therapeutics. Pharmacology, RM1-950
Abstract

We report the evaluation of 18-mer 2′-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5′ end to the oligospermine units (Sn-: n = 5, 15, 20, 25, and 30 [number of spermine units]) as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein-labeled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in three-dimensional (3D) cell culture (multicellular tumor spheroids [MCTSs]) that mimics living tissues. Whereas vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared with the assay in monolayer culture, an efficient exon skipping without significant toxicity was observed with oligospermine-grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labeled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS, whereas the vector-complexed SSO penetrated only the cells of the spheroid’s outer layer. Keywords: cationic oligonucleotide-oligospermine conjugate, carrier-free oligonucleotide delivery, multicellular tumor spheroids, MCTS, splice switching oligonucleotides, SSO, exon skipping

Published
2018
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2. Cationic Oligospermine-Oligonucleotide Conjugates Provide Carrier-free Splice Switching in Monolayer Cells and Spheroids

Author

Jean-Paul Behr, Mitsuharu Kotera, Jean-Serge Remy, Phanélie Perche-Létuvée, Marc Nothisen, Conception et application de molécules bioactives (CAMB), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Gilbert-Laustriat : Biomolécules, Biotechnologie, Innovation Thérapeutique, Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'études dynamiques et structurales de la sélectivité (LEDSS), and Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], carrier-free oligonucleotide delivery, Spermine, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, 010402 general chemistry, 01 natural sciences, Article, law.invention, Flow cytometry, splice switching oligonucleotides, 03 medical and health sciences, chemistry.chemical_compound, Confocal microscopy, law, Drug Discovery, cationic oligonucleotide-oligospermine conjugate, Fluorescence microscope, medicine, ComputingMilieux_MISCELLANEOUS, SSO, medicine.diagnostic_test, Oligonucleotide, lcsh:RM1-950, MCTS, Transfection, multicellular tumor spheroids, 0104 chemical sciences, 030104 developmental biology, lcsh:Therapeutics. Pharmacology, chemistry, Cytoplasm, Cell culture, Biophysics, Molecular Medicine, exon skipping
Abstract

We report the evaluation of 18-mer 2′-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5′ end to the oligospermine units (Sn-: n = 5, 15, 20, 25, and 30 [number of spermine units]) as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein-labeled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in three-dimensional (3D) cell culture (multicellular tumor spheroids [MCTSs]) that mimics living tissues. Whereas vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared with the assay in monolayer culture, an efficient exon skipping without significant toxicity was observed with oligospermine-grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labeled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS, whereas the vector-complexed SSO penetrated only the cells of the spheroid’s outer layer. Keywords: cationic oligonucleotide-oligospermine conjugate, carrier-free oligonucleotide delivery, multicellular tumor spheroids, MCTS, splice switching oligonucleotides, SSO, exon skipping

Published
2018
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3. Spectroscopic evidence for cofactor-substrate interaction in the radical-SAM enzyme TYW1

Author

Farhad Forouhar, Serge Gambarelli, Ricardo Garcia-Serres, Jean-Marc Latour, Mohamed G. Atta, Phanélie Perche-Letuvée, Velavan Kathirvelu, Conception d’Architectures Moléculaires et Processus Electroniques (CAMPE), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Columbia University [New York], ANR-10-BLAN-0737,INSERAD,Modifications post-transcriptionnelles des ARNs de transfert par insertion radicalaire.(2010), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
0301 basic medicine, Iron-Sulfur Proteins, Pyrococcus abyssi, S-Adenosylmethionine, Stereochemistry, Carboxy-Lyases, Radical, Archaeal Proteins, Coenzymes, Guanosine, Cofactor, Substrate Specificity, Inorganic Chemistry, 03 medical and health sciences, chemistry.chemical_compound, Spectroscopy, Mossbauer, Pyruvic Acid, Moiety, [CHIM]Chemical Sciences, Substrate Interaction, 030102 biochemistry & molecular biology, biology, Electron Spin Resonance Spectroscopy, Nucleosides, [CHIM.CATA]Chemical Sciences/Catalysis, Protein Structure, Tertiary, chemistry, Docking (molecular), biology.protein, Biocatalysis, Mutagenesis, Site-Directed, [PHYS.PHYS.PHYS-CHEM-PH]Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], Radical SAM, Wybutosine
Abstract

International audience; TYW1 is a metalloenzyme involved in the modifications of guanosine 37 of Phe-tRNA of Eukaryota and Archaea. It catalyzes the second step of Wybutosine biosynthesis, which consists of the formation of the tricyclic compound imG-14 from m1G using pyruvate and SAM (S-adenosyl-methionine) as co-substrates. Two [4Fe-4S] clusters are needed in the catalytic process. One effects the reductive binding of SAM, which initiates the radical reaction that inserts a C-C moiety into m1G. The other [4Fe-4S] cluster binds the pyruvate molecule that provides the C-C motif. Using a combination of EPR and Mössbauer spectroscopy, we have been able to probe the binding of both cofactors to the FeS clusters. The results highlight an interaction between pyruvate and SAM, indicating that they bind in close vicinity inside the catalytic pocket. They also indicate a chelating binding mode of pyruvate to the accessible Fe site of the corresponding FeS cluster. This binding mode has been used to construct a docking model of holoTYW1 with pyruvate and SAM, which is consistent with the spectroscopic findings.

Published
2017
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4. Cell-penetrating cationic siRNA and lipophilic derivatives efficient at nanomolar concentrations in the presence of serum and albumin

Author

Jean-Paul Behr, Marc Nothisen, Jean-Serge Remy, Mitsuharu Kotera, Jérémy Bagilet, and Phanélie Perche

Subjects
Serum, Gene knockdown, Chemistry, Cell, Oligonucleotides, Albumin, Cationic polymerization, Pharmaceutical Science, Lipids, In vitro, medicine.anatomical_structure, Biochemistry, Luciferases, Firefly, In vivo, Albumins, Cell Line, Tumor, medicine, Humans, Gene silencing, Spermine, Luciferase, Gene Silencing, RNA, Small Interfering
Abstract

Despite its considerable interest in human therapy, in vivo siRNA delivery is still suffering from hurdles of vectorization. We have shown recently efficient gene silencing by non-vectorized cationic siRNA. Here, we describe the synthesis and in vitro evaluation of new amphiphilic cationic siRNA. C 12 -, (C 12 ) 2 - and cholesteryl–spermine x –siRNA were capable of luciferase knockdown at nanomolar concentrations without vectorization ( i.e. one to two orders of magnitude more potent than commercially available cholesteryl siRNA). Moreover, incubation in the presence of serum did not impair their efficiency. Finally, amphiphilic cationic siRNA was pre-loaded on albumin. In A549Luc cells in the presence of serum, these siRNA conjugates were highly effective and had low toxicity.

Published
2013
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5. Wybutosine biosynthesis: structural and mechanistic overview

Author

Farhad Forouhar, Phanélie Perche-Letuvée, Thibaut Molle, Mohamed G. Atta, Etienne Mulliez, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Columbia University [New York], Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
Models, Molecular, S-Adenosylmethionine, Saccharomyces cerevisiae Proteins, Archaeal Proteins, Molecular Sequence Data, Saccharomyces cerevisiae, Review, Fe-S clusters, Biology, Protein Structure, Secondary, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Humans, Radical-SAM enzymes, Molecular Biology, Wybutosine, 030304 developmental biology, Genetics, 0303 health sciences, YW biosynthesis, tRNA Methyltransferases, Base Sequence, 030302 biochemistry & molecular biology, Nucleosides, Cell Biology, Archaea, Protein Structure, Tertiary, chemistry, Nucleic Acid Conformation, tRNA modification
Abstract

International audience; Over the last 10 years, significant progress has been made in understanding the genetics, enzymology and structural components of the wybutosine (yW) biosynthetic pathway. These studies have played a key role in expanding our understanding of yW biosynthesis and have revealed unexpected evolutionary ties, which are presently being unraveled. The enzymes catalyzing the 5 steps of this pathway, from genetically encoded guanosine to wybutosine base, provide an ensemble of amazing reaction mechanisms that are to be discussed in this review article.

Published
2015
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6. 4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate*

Author

Martin Clémancey, Vincent Maurel, Ricardo Garcia-Serres, Jean-Marc Latour, Mohamed G. Atta, Marc Fontecave, Gustav Berggren, Thierry Douki, Serge Gambarelli, Velavan Kathirvelu, Etienne Mulliez, Jean Armengaud, Phanélie Perche-Letuvée, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), Laboratoire Lésions des Acides Nucléiques (LAN), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Laboratoire de Biochimie des Systèmes Perturbés (LBSP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Collège de France - Chaire Chimie des processus biologiques, Laboratoire de Chimie des Processus Biologiques (LCPB), Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Chaire Chimie des processus biologiques

Subjects
Iron-Sulfur Proteins, Pyrococcus abyssi, S-Adenosylmethionine, Carboxy-Lyases, 01 natural sciences, Biochemistry, Ribosome, Mass Spectrometry, Substrate Specificity, chemistry.chemical_compound, RNA, Transfer, Pyruvic Acid, Cluster Analysis, Cloning, Molecular, Chromatography, High Pressure Liquid, chemistry.chemical_classification, 0303 health sciences, ATP synthase, biology, Guanosine, Transfer RNA, Oxidoreductases, endocrine system, TRNA modification, Saccharomyces cerevisiae Proteins, Stereochemistry, Ultraviolet Rays, Archaeal Proteins, Molecular Sequence Data, Saccharomyces cerevisiae, 010402 general chemistry, Catalysis, 03 medical and health sciences, [CHIM.COOR]Chemical Sciences/Coordination chemistry, Amino Acid Sequence, Cysteine, Molecular Biology, 030304 developmental biology, Sequence Homology, Amino Acid, Electron Spin Resonance Spectroscopy, Cell Biology, biology.organism_classification, 0104 chemical sciences, Enzyme, chemistry, Models, Chemical, biology.protein, Enzymology, Wybutosine
Abstract

International audience; Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called "Radical-SAM." These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX(3)CX(2)C motif, and S-adenosyl-L-methionine (SAM) to generate a 5'-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.

Published
2012
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7. MMT, Npeoc-protected spermine, a valuable synthon for the solid phase synthesis of oligonucleotide oligospermine conjugates via guanidine linkers

Author

Phanélie Perche, Jean-Serge Remy, and Mitsuharu Kotera

Subjects
Stereochemistry, Oligonucleotide, Organic Chemistry, Clinical Biochemistry, Synthon, Oligonucleotides, Thiourea, Pharmaceutical Science, Spermine, Trityl Compounds, Biochemistry, Chemical synthesis, Combinatorial chemistry, chemistry.chemical_compound, Solid-phase synthesis, chemistry, Drug Discovery, Molecular Medicine, Transition Temperature, Guanidine, Polyamine, Molecular Biology
Abstract

Solid phase spermine oligomerization via guanidine linkers was achieved using activated thiourea coupling reaction with primary amino group. Disymmetric spermine synthon was efficiently synthesised in eight steps from spermine. MMT group was used as coupling monitor and resulting oligomeric spermines were conjugated to oligonucleotides.

Published
2010

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