L’objectif du projet est de comprendre l’effet du peptide vecteur (R/W)9 sur les cellules tumorales EF, modèles du sarcome d’Ewing. En effet, ce peptide a la capacité de remodeler le cytosquelette d’actine dans ces cellules, ainsi que de réduire leur motilité et leur aptitude à croître en indépendance d’ancrage (Delaroche D. JBC 2010).La première étape de ce travail a été la caractérisation in vitro de l’interaction directe avec l’actine par cross-linking chimique et spectrométrie de masse (Clavier S. EuProt 2014). Ensuite, pour avancer dans la compréhension de l’effet du peptide (R/W)9, deux approches ont été développées.La première approche, basée sur du photocross-linking in cellulo ou in lysat, vise à identifier des partenaires intracellulaires du peptide vecteur. Pour cela, nous avons mis au point et validé biologiquement une version photoactivable du peptide (R/W)9. Puis, avant de passer sur cellules entières, nous nous sommes assurés de la faisabilité de la réaction de photocross-linking in vitro sur un système d’interaction modèle que nous avons également utilisé pour développer un logiciel capable d’interpréter les spectres MS/MS d’espèces photocross-linkées (Xlink-Identifier, Collaboration Dr Du X). Des expériences de purification d’affinité ont également été menées en immobilisant le peptide (R/W)9 sur des billes de streptavidine ensuite mises en présence de lysat cellulaire. Les protéines capturées ont été identifiées par spectrométrie de masse haut-débit. La seconde approche est de la protéomique différentielle avec un marquage SILAC et a pour objectif de mettre en évidence l’influence du peptide vecteur sur l’expression des protéines. / The aim of the project is to understand the effect of (R/W)9 cell penetrating peptide (CPP) on EF tumoral cells, an Ewing sarcoma model cell line. Actually, this peptide is able to remodel the actin cytoskeleton of these cells, to decrease their motility as well as their ability to grow without anchorage (Delaroche D. JBC 2010).The first step of this work was to characterize the in vitro interaction with actin using chemical cross-linking and mass spectrometry (Clavier S. Euprot 2014). Then, in order to get a deeper understanding of (R/W)9 peptide effect, two approaches were developed. The goal of the first approach based on in cellulo or in lysate photocross-linking is to identify (R/W)9 CPP’s partners. To do this, we designed and biologically validated a photoactivable version of (R/W)9 peptide. Then, before starting to work with living cells, we checked the feasibility of in vitro photocross-linking on a model interacting system that we also used to develop a software able to interpret MS/MS spectra of photocross-linked species. (Xlink-Identifier, Collaboration with Dr. X. Du.). Affinity purification experiments were also performed by incubating streptavidin magnetic beads bearing (R/W)9 peptide with cell lysates. Captured proteins were identified using high-throughput mass spectrometry. The second approach is differential proteomic with SILAC labelling and aims at assessing the influence of (R/W)9 CPP on proteins expression.

<p>Synthetic and physicochemical studies on appropriately functionalized ODN-conjugates have been performed to evaluate their abilities to act as antisense agents against RNA or as intramolecular DNA cross-linking agents. Intercalating aromatic systems [phenazine (Pnz), dipyridophenazine (DPPZ)] and metallointercalators such as Ru²⁺(phen)₂(DPPZ) and Ru²⁺(tpy)(DPPZ)<b>L</b> [where <b>L</b> = chemically or photochemically labile ligand, phen = phenanthroline, tpy = terpyridine], which are covalently tethered to the oligo-deoxynucleotides (ODNs), have been chosen for this purpose. The ODN-conjugates were typically prepared by automated solid phase synthesis using phosphoramidite building blocks, or on solid supports, both functionalized with the chromophore groups. The photosensitive metal complex, Ru²⁺(tpy)(DPPZ)(CH₃CN), has been incorporated by post-synthetic coupling to the amino-linker modified ODNs <i>via</i> an amide bond. The intercalating ability of the tethered chromophores gave enhanced stability of the duplexes and triplexes formed with ODN-conjugates and their complementary targets: DNA, RNA, or double-stranded DNA. The conjugation of DPPZ chromophore to ODN (at 3', 5' or at the middle) led us to incorporate Ru²⁺(phen)₂(DPPZ) through the DPPZ ligand, for the first time. The corresponding (Ru²⁺-ODN)•DNA duplexes showed dramatic stabilization (ΔT_m = 19.4 – 22.0ºC). The CD and DNase I footprinting experiments suggest that the stabilization is owing to metallointercalation by threading of the Ru²⁺(phen)₂ moiety through the ODN•DNA duplex core, thus "stapling" the two helical strands from the minor to major groove. On the other hand, Ru²⁺(tpy)(DPPZ)(CH₃CN)-ODN conjugates represent a new class of oligonucleotides containing the photoactivatible Ru²⁺ complexes, which can successfully crosslink to the complementary strand. The mechanism of cross-linking upon photoirradiation of [Ru²⁺(tpy)(DPPZ)(CH₃CN)-ODN]•DNA involves <i>in situ</i> conversion to the reactive [Ru²⁺(tpy)(DPPZ)(H₂O)-ODN]•DNA which are subsequently cross-linked through the G residue of the complementary DNA strand. All starting materials and products have been purified by HPLC and/or by PAGE and subsequently characterized by MALDI-TOF as well as ESI mass spectroscopy. Terminal conjugation of the planar Pnz and DPPZ groups through the flexible linkers were also shown to improve thermal stability of the ODN•RNA hybrid duplexes without alteration of the initial AB-type global helical structure as revealed from CD experiments. As a result, RNase H mediated cleavage of the RNA strand in the intercalator-tethered ODN•RNA duplexes was more efficient compared to the natural counterpart. The RNase H cleavage pattern was also found to be dependent on the chemical nature of the chromophore. It appeared that introduction of a tether at the 3'-end of the ODN can be most easily tolerated by the enzyme regardless of the nature of the appending chromophore. The tethered DPPZ group has also been shown to chelate Cu²⁺ and Fe³⁺, like phenanthroline group, followed by the formation of redox-active metal complex which cleaves the complementary DNA strand in a sequence-specific manner. This shows that the choice of appropriate ligand is useful to (i) attain improved intercalation giving Tm enhancement, and (ii) sequence-specifically inactivate target RNA or DNA molecules using multiple modes of chemistry (RNase H mediated cleavage, free-radical, oxidative pathways or photocross-linkage).</p>

Synthetic and physicochemical studies on appropriately functionalized ODN-conjugates have been performed to evaluate their abilities to act as antisense agents against RNA or as intramolecular DNA cross-linking agents. Intercalating aromatic systems [phenazine (Pnz), dipyridophenazine (DPPZ)] and metallointercalators such as Ru2+(phen)2(DPPZ) and Ru2+(tpy)(DPPZ)<b>L</b> [where <b>L</b> = chemically or photochemically labile ligand, phen = phenanthroline, tpy = terpyridine], which are covalently tethered to the oligo-deoxynucleotides (ODNs), have been chosen for this purpose. The ODN-conjugates were typically prepared by automated solid phase synthesis using phosphoramidite building blocks, or on solid supports, both functionalized with the chromophore groups. The photosensitive metal complex, Ru2+(tpy)(DPPZ)(CH3CN), has been incorporated by post-synthetic coupling to the amino-linker modified ODNs via an amide bond. The intercalating ability of the tethered chromophores gave enhanced stability of the duplexes and triplexes formed with ODN-conjugates and their complementary targets: DNA, RNA, or double-stranded DNA. The conjugation of DPPZ chromophore to ODN (at 3', 5' or at the middle) led us to incorporate Ru2+(phen)2(DPPZ) through the DPPZ ligand, for the first time. The corresponding (Ru2+-ODN)•DNA duplexes showed dramatic stabilization (ΔTm = 19.4 – 22.0ºC). The CD and DNase I footprinting experiments suggest that the stabilization is owing to metallointercalation by threading of the Ru2+(phen)2 moiety through the ODN•DNA duplex core, thus "stapling" the two helical strands from the minor to major groove. On the other hand, Ru2+(tpy)(DPPZ)(CH3CN)-ODN conjugates represent a new class of oligonucleotides containing the photoactivatible Ru2+ complexes, which can successfully crosslink to the complementary strand. The mechanism of cross-linking upon photoirradiation of [Ru2+(tpy)(DPPZ)(CH3CN)-ODN]•DNA involves in situ conversion to the reactive [Ru2+(tpy)(DPPZ)(H2O)-ODN]•DNA which are subsequently cross-linked through the G residue of the complementary DNA strand. All starting materials and products have been purified by HPLC and/or by PAGE and subsequently characterized by MALDI-TOF as well as ESI mass spectroscopy. Terminal conjugation of the planar Pnz and DPPZ groups through the flexible linkers were also shown to improve thermal stability of the ODN•RNA hybrid duplexes without alteration of the initial AB-type global helical structure as revealed from CD experiments. As a result, RNase H mediated cleavage of the RNA strand in the intercalator-tethered ODN•RNA duplexes was more efficient compared to the natural counterpart. The RNase H cleavage pattern was also found to be dependent on the chemical nature of the chromophore. It appeared that introduction of a tether at the 3'-end of the ODN can be most easily tolerated by the enzyme regardless of the nature of the appending chromophore. The tethered DPPZ group has also been shown to chelate Cu2+ and Fe3+, like phenanthroline group, followed by the formation of redox-active metal complex which cleaves the complementary DNA strand in a sequence-specific manner. This shows that the choice of appropriate ligand is useful to (i) attain improved intercalation giving Tm enhancement, and (ii) sequence-specifically inactivate target RNA or DNA molecules using multiple modes of chemistry (RNase H mediated cleavage, free-radical, oxidative pathways or photocross-linkage).