Stability of telomeric G-quadruplexes

In most eukaryotes, telomeric DNA consists of repeats of a short motif that includes consecutive guanines and may hence fold into G-quadruplexes. Budding yeasts have telomeres composed of longer repeats and show variation in the degree of repeat homogeneity. Although telomeric sequences from several organisms have been shown to fold into G-quadruplexes in vitro, surprisingly, no study has been dedicated to the comparison of G-quadruplex folding and stability of known telomeric sequences. Furthermore, to our knowledge, folding of yeast telomeric sequences into intramolecular G-quadruplexes has never been investigated. Using biophysical and biochemical methods, we studied sequences mimicking about four repetitions of telomeric motifs from a variety of organisms, including yeasts, with the aim of comparing the G-quadruplex folding potential of telomeric sequences among eukaryotes. G-quadruplex folding did not appear to be a conserved feature among yeast telomeric sequences. By contrast, all known telomeric sequences from eukaryotes other than yeasts folded into G-quadruplexes. Nevertheless, while G3T1-4A repeats (found in a variety of organisms) and G4T2,4 repeats (found in ciliates) folded into stable G-quadruplexes, G-quadruplexes formed by repetitions of G2T2A and G2CT2A motifs (found in many insects and in nematodes, respectively) appeared to be in equilibrium with non-G-quadruplex structures (likely hairpin-duplexes)

Guanine quadruplexes : formation, stability and interaction

Les quadruplexes de guanines (G4) sont des structures non canonique d’acides nucléiques à quatre brins formées à partir de séquences ADN ou ARN riches en guanines. Ces structures reposant sur la formation et l’empilement de quartets de guanines sont très polymorphes, leur formation pourrait être envisagé dans de nombreux domaines d’application, aussi bien pour les biotechnologies que les nanotechnologies. L’étude de G4 tétramoléculaires modifiés présentée dans ce manuscrit a participé à la compréhension du mécanisme d’association de ces complexes. En particulier, nous avons montré que l’insertion de 8-méthyle-2’-déoxyguanosine à l’extrémité 5’ de la séquence favorise l’association et la stabilité du G4. Par ailleurs, l’étude de l’ADN en série L (image de l’ADN naturel dans un miroir) a montré la formation d’un G4 tétramoléculaire avec les mêmes propriétés que son énantiomère, à l’exception de sa chiralité, qui est inversée. L’étude a révélé également une auto-exclusion de deux énantiomères (forme D et forme L) démontrant un assemblage contrôlé des brins parallèles. Ce travail de thèse a aussi permis d’introduire un système simple et stable de visualisation de G4 tétramoléculaire antiparallèle, appelé “ADN synaptique”, sur une nanostructure d’ADN origami. In vivo, ces structures pourraient être impliquées de façon transitoire dans de nombreux processus biologiques, en particulier au niveau des télomères. Nous avons réalisé, au cours de cette thèse, une étude comparative de la structure et de la stabilité des séquences télomériques connues de différents organismes. Cette étude a permis d’enrichir les données nécessaires au développement d’un algorithme prédisant la stabilité de G4. Enfin, nous avons développé une méthode facile et peu coûteuse de criblage (G4-FID) sur plaques 96 puits permettant d’identifier l’interaction de ligands avec différentes séquences biologiques pertinentes. La stabilisation du G4 dans certaines régions du génome via des ligands spécifiques pourrait limiter la prolifération de cellules tumorales et est donc intéressante pour les thérapies anticancéreuses.Guanine quadruplexes (G4) are non-canonical four-stranded nucleic acid structures formed by guanine-rich DNA and RNA sequences. Theses polymorphic structures are built from the stacking of several G-quartets and could be involved in many fields, in biotechnology as well as in nanotechnology. The study of modified tetramolecular G4 presented in this manuscript participated to the understanding of tetramolecular G4 formation. Especially, we showed that the insertion of 8-methyl-2’-deoxyguanosine at the 5’-end of the sequence accelerate G4 formation and increase its stability. Besides, we demonstrate here that short guanine rich L-DNA strands (mirror image of natural DNA) form a tetramolecular G4 with the same properties than their enantiomer, but with opposite chirality. The study revealed also self-exclusion between two enantiomers (D- and L- form), showing the controlled parallel self-assembly of different G-rich strands. This work introduced also a simple and stable system to observe tetramolecular antiparallel G4 formation, called “synaptic DNA”, into a DNA origami nanostructure. In vivo, such structures appear to be implicated in genome dynamics, and especially at telomeres. During this thesis, we dedicated a study to the comparison of G4 folding and stability of known telomeric sequences from different organisms. The present study allowed enriching the dataset necessary to build and refine algorithms predicting G4 stability. Last but not least, we developed a G4 ligand screening method onto 96-well plates allowing the comparison of different biological relevant sequences. The G4 stabilisation by specific ligands in some genome regions may prevent cancer cell proliferation, making it an attractive target for anticancer therapy

Guanine quadruplexes : formation, stability and interaction

Les quadruplexes de guanines (G4) sont des structures non canonique d’acides nucléiques à quatre brins formées à partir de séquences ADN ou ARN riches en guanines. Ces structures reposant sur la formation et l’empilement de quartets de guanines sont très polymorphes, leur formation pourrait être envisagé dans de nombreux domaines d’application, aussi bien pour les biotechnologies que les nanotechnologies. L’étude de G4 tétramoléculaires modifiés présentée dans ce manuscrit a participé à la compréhension du mécanisme d’association de ces complexes. En particulier, nous avons montré que l’insertion de 8-méthyle-2’-déoxyguanosine à l’extrémité 5’ de la séquence favorise l’association et la stabilité du G4. Par ailleurs, l’étude de l’ADN en série L (image de l’ADN naturel dans un miroir) a montré la formation d’un G4 tétramoléculaire avec les mêmes propriétés que son énantiomère, à l’exception de sa chiralité, qui est inversée. L’étude a révélé également une auto-exclusion de deux énantiomères (forme D et forme L) démontrant un assemblage contrôlé des brins parallèles. Ce travail de thèse a aussi permis d’introduire un système simple et stable de visualisation de G4 tétramoléculaire antiparallèle, appelé “ADN synaptique”, sur une nanostructure d’ADN origami. In vivo, ces structures pourraient être impliquées de façon transitoire dans de nombreux processus biologiques, en particulier au niveau des télomères. Nous avons réalisé, au cours de cette thèse, une étude comparative de la structure et de la stabilité des séquences télomériques connues de différents organismes. Cette étude a permis d’enrichir les données nécessaires au développement d’un algorithme prédisant la stabilité de G4. Enfin, nous avons développé une méthode facile et peu coûteuse de criblage (G4-FID) sur plaques 96 puits permettant d’identifier l’interaction de ligands avec différentes séquences biologiques pertinentes. La stabilisation du G4 dans certaines régions du génome via des ligands spécifiques pourrait limiter la prolifération de cellules tumorales et est donc intéressante pour les thérapies anticancéreuses.Guanine quadruplexes (G4) are non-canonical four-stranded nucleic acid structures formed by guanine-rich DNA and RNA sequences. Theses polymorphic structures are built from the stacking of several G-quartets and could be involved in many fields, in biotechnology as well as in nanotechnology. The study of modified tetramolecular G4 presented in this manuscript participated to the understanding of tetramolecular G4 formation. Especially, we showed that the insertion of 8-methyl-2’-deoxyguanosine at the 5’-end of the sequence accelerate G4 formation and increase its stability. Besides, we demonstrate here that short guanine rich L-DNA strands (mirror image of natural DNA) form a tetramolecular G4 with the same properties than their enantiomer, but with opposite chirality. The study revealed also self-exclusion between two enantiomers (D- and L- form), showing the controlled parallel self-assembly of different G-rich strands. This work introduced also a simple and stable system to observe tetramolecular antiparallel G4 formation, called “synaptic DNA”, into a DNA origami nanostructure. In vivo, such structures appear to be implicated in genome dynamics, and especially at telomeres. During this thesis, we dedicated a study to the comparison of G4 folding and stability of known telomeric sequences from different organisms. The present study allowed enriching the dataset necessary to build and refine algorithms predicting G4 stability. Last but not least, we developed a G4 ligand screening method onto 96-well plates allowing the comparison of different biological relevant sequences. The G4 stabilisation by specific ligands in some genome regions may prevent cancer cell proliferation, making it an attractive target for anticancer therapy

Quadruplexes de guanines (formation, stabilité et interaction)

Les quadruplexes de guanines (G4) sont des structures non canonique d acides nucléiques à quatre brins formées à partir de séquences ADN ou ARN riches en guanines. Ces structures reposant sur la formation et l empilement de quartets de guanines sont très polymorphes, leur formation pourrait être envisagé dans de nombreux domaines d application, aussi bien pour les biotechnologies que les nanotechnologies. L étude de G4 tétramoléculaires modifiés présentée dans ce manuscrit a participé à la compréhension du mécanisme d association de ces complexes. En particulier, nous avons montré que l insertion de 8-méthyle-2 -déoxyguanosine à l extrémité 5 de la séquence favorise l association et la stabilité du G4. Par ailleurs, l étude de l ADN en série L (image de l ADN naturel dans un miroir) a montré la formation d un G4 tétramoléculaire avec les mêmes propriétés que son énantiomère, à l exception de sa chiralité, qui est inversée. L étude a révélé également une auto-exclusion de deux énantiomères (forme D et forme L) démontrant un assemblage contrôlé des brins parallèles. Ce travail de thèse a aussi permis d introduire un système simple et stable de visualisation de G4 tétramoléculaire antiparallèle, appelé ADN synaptique , sur une nanostructure d ADN origami. In vivo, ces structures pourraient être impliquées de façon transitoire dans de nombreux processus biologiques, en particulier au niveau des télomères. Nous avons réalisé, au cours de cette thèse, une étude comparative de la structure et de la stabilité des séquences télomériques connues de différents organismes. Cette étude a permis d enrichir les données nécessaires au développement d un algorithme prédisant la stabilité de G4. Enfin, nous avons développé une méthode facile et peu coûteuse de criblage (G4-FID) sur plaques 96 puits permettant d identifier l interaction de ligands avec différentes séquences biologiques pertinentes. La stabilisation du G4 dans certaines régions du génome via des ligands spécifiques pourrait limiter la prolifération de cellules tumorales et est donc intéressante pour les thérapies anticancéreuses.Guanine quadruplexes (G4) are non-canonical four-stranded nucleic acid structures formed by guanine-rich DNA and RNA sequences. Theses polymorphic structures are built from the stacking of several G-quartets and could be involved in many fields, in biotechnology as well as in nanotechnology. The study of modified tetramolecular G4 presented in this manuscript participated to the understanding of tetramolecular G4 formation. Especially, we showed that the insertion of 8-methyl-2 -deoxyguanosine at the 5 -end of the sequence accelerate G4 formation and increase its stability. Besides, we demonstrate here that short guanine rich L-DNA strands (mirror image of natural DNA) form a tetramolecular G4 with the same properties than their enantiomer, but with opposite chirality. The study revealed also self-exclusion between two enantiomers (D- and L- form), showing the controlled parallel self-assembly of different G-rich strands. This work introduced also a simple and stable system to observe tetramolecular antiparallel G4 formation, called synaptic DNA , into a DNA origami nanostructure. In vivo, such structures appear to be implicated in genome dynamics, and especially at telomeres. During this thesis, we dedicated a study to the comparison of G4 folding and stability of known telomeric sequences from different organisms. The present study allowed enriching the dataset necessary to build and refine algorithms predicting G4 stability. Last but not least, we developed a G4 ligand screening method onto 96-well plates allowing the comparison of different biological relevant sequences. The G4 stabilisation by specific ligands in some genome regions may prevent cancer cell proliferation, making it an attractive target for anticancer therapy.BORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF

Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame.

Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G-G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex

Tetramolecular Quadruplex Stability and Assembly

International audienc

Electrical and Structural Properties of All-Sputtered Al/SiO2/p-GaN MOS Schottky Diode

The all-sputtered Al/SiO2/p-GaN metal-oxide-semiconductor (MOS) Schottky diode was fabricated by the cost-effective radio-frequency sputtering technique with a cermet target at 400 °C. Using scanning electron microscope (SEM), the thicknesses of the electrodes, insulator SiO2 layer, and p-GaN were found to be ~250 nm, 70 nm, and 1 µm, respectively. By Hall measurement of a p-Mg-GaN film on an SiO2/Si (100) substrate at room temperature, the hole’s concentration (Np) and carrier mobility (μ) were found to be Np = 4.32 × 1016 cm−3 and μ = 7.52 cm2·V−1·s−1, respectively. The atomic force microscope (AFM) results showed that the surface topography of the p-GaN film had smoother, smaller grains with a root-mean-square (rms) roughness of 3.27 nm. By I–V measurements at room temperature (RT), the electrical properties of the diode had a leakage current of ~4.49 × 10−8 A at −1 V, a breakdown voltage of −6 V, a turn-on voltage of ~2.1 V, and a Schottky barrier height (SBH) of 0.67 eV. By C–V measurement at RT, with a frequency range of 100–1000 KHz, the concentration of the diode’s hole increased from 3.92 × 1016 cm−3 at 100 kHz to 5.36 × 1016 cm−3 at 1 MHz, while the Fermi level decreased slightly from 0.109 to 0.099 eV. The SBH of the diode at RT in the C–V test was higher than in the I–V test because of the induced charges by dielectric layer. In addition, the ideality factor (n) and series resistance (Rs) determined by Cheung’s and Norde’s methods, other parameters for MOS diodes were also calculated by C–V measurement at different frequencies

PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes

Saccharomyces cerevisiae encodes two Pif1 family DNA helicases, Pif1 and Rrm3. Rrm3 promotes DNA replication past stable protein complexes at tRNA genes (tDNAs). We identify a new role for the Pif1 helicase: promotion of replication and suppression of DNA damage at tDNAs. Pif1 binds multiple tDNAs, and this binding is higher in rrm3Δ cells. Accumulation of replication intermediates and DNA damage at tDNAs is higher in pif1Δ rrm3Δ than in rrm3Δ cells. DNA damage at tDNAs in the absence of these helicases is suppressed by destabilizing R-loops while Pif1 and Rrm3 binding to tDNAs is increased upon R-loop stabilization. We propose that Rrm3 and Pif1 promote genome stability at tDNAs by displacing the stable multi-protein transcription complex and by removing R-loops. Thus, we identify tDNAs as a new source of R-loop-mediated DNA damage. Given their large number and high transcription rate, tDNAs may be a potent source of genome instability

HIV-1 Nucleocapsid Proteins as Molecular Chaperones for Tetramolecular Antiparallel G-Quadruplex Formation

International audienc

Electrical Characterization of RF Reactive Sputtered p–Mg-InxGa1−xN/n–Si Hetero-Junction Diodes without Using Buffer Layer

The modeling of p–InxGa1−xN/n–Si hetero junction diodes without using the buffer layer were investigated with the “top-top” electrode. The p–Mg-GaN and p–Mg-In0.05Ga0.95N were deposited directly on the n–Si (100) wafer by the RF reactive sputtering at 400 °C with single cermet targets. Al and Pt with the square size of 1 mm2 were used for electrodes of p–InxGa1−xN/n–Si diodes. Both devices had been designed to prove the p-type performance of 10% Mg-doped in GaN and InGaN films. By Hall measurement at the room temperature (RT), the holes concentration and mobility were determined to be Np = 3.45 × 1016 cm−3 and µ = 145 cm2/V·s for p–GaN film, Np = 2.53 × 1017 cm−3, and µ = 45 cm2/V·s for p–InGaN film. By the I–V measurement at RT, the leakage currents at −5 V and turn-on voltages were found to be 9.31 × 10−7 A and 2.4 V for p–GaN/n–Si and 3.38 × 10−6 A and 1.5 V for p–InGaN/n–Si diode. The current densities at the forward bias of 20 V were 0.421 and 0.814 A·cm−2 for p–GaN/n–Si and p–InGaN/n–Si devices. The electrical properties were measured at the temperature range of 25 to 150 °C. By calculating based on the TE mode, Cheungs’ and Norde methods, and other parameters of diodes were also determined and compared