Thioflavin T As A Fluorescence Light-Up Probe For G4 Formation

Thioflavin T (ThT) becomes fluorescent in the presence of the G-quadruplex structure such as that formed by the human telomeric motif. In this report, we extend and generalize these observations and show that this dye may be used as a convenient and specific quadruplex probe. In the presence of most, but not all, G4-forming sequences, we observed a large increase in ThT fluorescence emission, whereas the presence of control duplexes and single strands had a more limited effect on emission. This differential behavior allowed us to design a high-throughput assay to detect G4 formation. Hundreds of different oligonucleotides may be tested in parallel for G4 formation with a simple fluorescence plate reader. We applied this technique to a family of aptamers not previously recognized as G4-forming sequences and demonstrated that ThT fluorescence signal may be used to predict G4 formation

Fishing for G-Quadruplexes in solution with a perylene diimide derivative labeled with biotins

A new fluorescent, non‐cytotoxic perylene diimide derivative with two biotins at the peri position, PDI2B, has been synthesized. This molecule is able to interact selectively with G‐quadruplexes with scarce or no affinity towards single‐ or double‐stranded DNA. These features have made it possible to design a simple, effective, safe, cheap, and selective method for fishing G‐quadruplex structures in solution by use of PDI2B and streptavidin coated magnetic beads. The new cyclic method reported leads to the recovery of more than 80 % of G‐quadruplex structures from solution, even in the presence of an excess of single‐stranded or duplex DNA as competitors. Moreover, PDI2B is a G4 ligand that can display higher thermal stabilization and greater affinity for 2‐ over 3‐tetrad quadruplexes, which constitutes a novel type of behavior.“la Caixa” Foundation (LCF/PR/PR12/ 11070003), MINECO (CTQ2014-58812-C2-2-R and CTQ2015- 71353-R, FEDER Funds), and Junta de Castilla y Lejn, Consejer &a de Educacijn y Cultura y Fondo Social Europeo (Project BU051U16), Spain, is gratefully acknowledged. N.B. is grateful to Dr. Oscar Mendoza of the ARNA Laboratory for useful discussion and also to the financial support of the Jos8 Castillejo Program by the Spanish Ministry of Education, Culture and Sports (JC2015-00403). J.L.M. acknowledges support from Conseil Regional d’Aquitaine, Agence Nationale de la Recherche (ANR Quarpdiem) and the Symbit program [CZ.02.1.01/0.0/0.0/ 15_003/0000477] financed by the ERDF

Design, Synthesis, and Evaluation of 2,9-Bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline Derivatives as G-Quadruplex Ligands.

International audienceGenomic sequences able to form guanine quadruplexes (G4) are found in oncogene promoters, in telomeres, and in 5'- and 3'-untranslated regions as well as introns of messenger RNAs. These regions are potential targets for drugs designed to treat cancer. Herein, we present the design and syntheses of ten new phenanthroline derivatives and characterization of their interactions with G4-forming oligonucleotides. We evaluated ligand-induced stabilization and specificity and selectivity of ligands for various G4 conformations using FRET-melting experiments. We investigated the interaction of compound 1 a (2,9-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-1,10-phenanthroline), which combined the greatest stabilizing effect and specificity for G4, with human telomeric sequences using FRET, circular dichroism, and ESI-MS. In addition, we showed that compound 1 a interferes with the G4 helicase activity of Saccharomyces cerevisiae Pif1. Interestingly, compound 1 a was significantly more cytotoxic toward two human leukemic cell lines than to normal human blood mononuclear cells. These novel phenanthroline derivatives will be a starting point for further development and optimization of potent G4 ligands that have potential as anticancer agents

Common G-Quadruplex Binding Agents Found to Interact With i-Motif-Forming DNA: Unexpected Multi-Target-Directed Compounds

G-quadruplex (G4) and i-motif (iM) are four-stranded non-canonical nucleic acid structural arrangements. Recent evidences suggest that these DNA structures exist in living cells and could be involved in several cancer-related processes, thus representing an attractive target for anticancer drug discovery. Efforts toward the development of G4 targeting compounds have led to a number of effective bioactive ligands. Herein, employing several biophysical methodologies, we studied the ability of some well-known G4 ligands to interact with iM-forming DNA. The data showed that the investigated compounds are actually able to interact with both DNA in vitro, thus acting de facto as multi-target-directed agents. Interestingly, while all the compounds stabilize the G4, some of them significantly reduce the stability of the iM. The present study highlights the importance, when studying G4-targeting compounds, of evaluating also their behavior toward the i-motif counterpart

DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential.

Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy.The Balasubramanian laboratory is core-funded by Cancer Research UK (C14303/A17197) and further supported by a Cancer Research UK programme grant (C9681/A18618). S.B. is a Wellcome Trust Senior Investigator (099232/Z/12/Z)

G4-Hunter : un nouvel algorithme pour la prédiction des G-quadruplexes

Biologically relevant G4 DNA structures are formed throughout the genome including immunoglobulin switch regions, promoter sequences and telomeric repeats. They can arise when single-stranded G-rich DNA or RNA sequences are exposed during replication, transcription or recombination. Computational analysis using predictive algorithms suggests that the human genome contains approximately 370 000 potential G4-forming sequences. These predictions are generally limited to the standard G3+N(1−7)G3+N(1−7)G3+N(1−7)G3+ description. However, many stable G4s defy this description and escape this consensus; this is the reason why broadening this description should allow the prediction of more G4 loci. We propose an objective score function, G4- hunter, which predicts G4 folding propensity from a linear nucleic acid sequence. The new method focus on guanines clusters and GC asymmetry, taking into account the whole genomic region rather than individual quadruplexes sequences. In parallel with this computational technique, a large scale in vitro experimental work has also been developed to validate the performance of our algorithm in silico on one hundred of different sequences. G4- hunter exhibits unprecedented accuracy and sensitivity and leads us to reevaluate significantly the number of G4-prone sequences in the human genome. G4-hunter also allowed us to predict potential G4 sequences in HIV and Dictyostelium discoideum, which could not be identified by previous computational methods.Des séquences compatibles avec la formation de G4 sont présentes au niveau de certaines régions clés du génome telles que les extrémités des chromosomes, mais également les régions de commutation de classe des immunoglobulines, les promoteurs de certains gènes dont des oncogènes et des séquences transcrites. Plus de 370 000 cibles potentielles ont été prédites lors des analyses bioinformatiques du génome humain. Cependant, ces prédictions ne sont pas exhaustives étant limitées par la formulation des algorithmes de prédiction utilisés. En effet, les séquences recherchées suivent la formule consensus suivante G3+N(1−7)G3+N(1−7)G3+N(1−7)G3+. Ainsi, en apportant plus de souplesse dans la description du quadruplex nous pourrons identifier et localiser plus de cibles potentielles. C’est pourquoi, nous proposons un nouvel algorithme G4-Hunter qui permettra l’identification la plus exhaustive possible de séquences cibles en prenant en compte la totalité de la région et non plus uniquement la cible potentielle. Par ailleurs, une étude expérimentale à grande échelle (sur une centaine de séquences cibles) a été menée afin de valider et tester la robustesse de G4-Hunter. A l’aide de ce nouvel outil, nous avons pu identifier de nouvelles séquences cibles non identifiées par les approches déjà existantes au sein des génomes humain, HIV et Dictyostelium discoideum

G4-Hunter : a new algorithm for G-quadruplexes prediction’s

Des séquences compatibles avec la formation de G4 sont présentes au niveau de certaines régions clés du génome telles que les extrémités des chromosomes, mais également les régions de commutation de classe des immunoglobulines, les promoteurs de certains gènes dont des oncogènes et des séquences transcrites. Plus de 370 000 cibles potentielles ont été prédites lors des analyses bioinformatiques du génome humain. Cependant, ces prédictions ne sont pas exhaustives étant limitées par la formulation des algorithmes de prédiction utilisés. En effet, les séquences recherchées suivent la formule consensus suivante G3+N(1−7)G3+N(1−7)G3+N(1−7)G3+. Ainsi, en apportant plus de souplesse dans la description du quadruplex nous pourrons identifier et localiser plus de cibles potentielles. C’est pourquoi, nous proposons un nouvel algorithme G4-Hunter qui permettra l’identification la plus exhaustive possible de séquences cibles en prenant en compte la totalité de la région et non plus uniquement la cible potentielle. Par ailleurs, une étude expérimentale à grande échelle (sur une centaine de séquences cibles) a été menée afin de valider et tester la robustesse de G4-Hunter. A l’aide de ce nouvel outil, nous avons pu identifier de nouvelles séquences cibles non identifiées par les approches déjà existantes au sein des génomes humain, HIV et Dictyostelium discoideum.Biologically relevant G4 DNA structures are formed throughout the genome including immunoglobulin switch regions, promoter sequences and telomeric repeats. They can arise when single-stranded G-rich DNA or RNA sequences are exposed during replication, transcription or recombination. Computational analysis using predictive algorithms suggests that the human genome contains approximately 370 000 potential G4-forming sequences. These predictions are generally limited to the standard G3+N(1−7)G3+N(1−7)G3+N(1−7)G3+ description. However, many stable G4s defy this description and escape this consensus; this is the reason why broadening this description should allow the prediction of more G4 loci. We propose an objective score function, G4- hunter, which predicts G4 folding propensity from a linear nucleic acid sequence. The new method focus on guanines clusters and GC asymmetry, taking into account the whole genomic region rather than individual quadruplexes sequences. In parallel with this computational technique, a large scale in vitro experimental work has also been developed to validate the performance of our algorithm in silico on one hundred of different sequences. G4- hunter exhibits unprecedented accuracy and sensitivity and leads us to reevaluate significantly the number of G4-prone sequences in the human genome. G4-hunter also allowed us to predict potential G4 sequences in HIV and Dictyostelium discoideum, which could not be identified by previous computational methods

Topology of a DNA G-quadruplex structure formed in the HIV-1 promoter: a potential target for anti-HIV drug development.

International audienceNucleic acid sequences containing guanine tracts are able to adopt noncanonical four-stranded nucleic acid structures called G-quadruplexes (G4s). These structures are based on the stacking of two or more G-tetrads; each tetrad is a planar association of four guanines held together by eight hydrogen bonds. In this study, we analyzed a conserved G-rich region from HIV-1 promoter that is known to regulate the transcription of the HIV-1 provirus. Strikingly, our analysis of an alignment of 1684 HIV-1 sequences from this region showed a high conservation of the ability to form G4 structures despite a lower conservation of the nucleotide primary sequence. Using NMR spectroscopy, we determined the G4 topology adopted by a DNA sequence from this region (HIV-PRO1: 5' TGGCCTGGGCGGGACTGGG 3'). This DNA fragment formed a stable two G-tetrad antiparallel G4 with an additional Watson-Crick CG base pair. This hybrid structure may be critical for HIV-1 gene expression and is potentially a novel target for anti-HIV-1 drug development

Mapping and characterization of G-quadruplexes in the genome of the social amoeba Dictyostelium discoideum

International audienceG-quadruplexes (G4) are non-canonical DNA and/or RNA secondary structures formed in guanine-rich regions. Given their over-representation in specific regions in the genome such as promoters and telomeres, they are likely to play important roles in key processes such as transcription, replication or RNA maturation. Putative G4-forming sequences (G4FS) have been reported in humans, yeast, bacteria, viruses and many organisms. Here we present the first mapping of G-quadruplex sequences in Dictyostelium discoideum, the social amoeba. ‘Dicty’ is an ameboid protozoan with a small (34 Mb) and extremely AT rich genome (78%). As a consequence, very few G4-prone motifs are expected. An in silico analysis of the Dictyostelium genome with the G4Hunter software detected 249–1055 G4-prone motifs, depending on G4Hunter chosen threshold. Interestingly, despite an even lower GC content (as compared to the whole Dicty genome), the density of G4 motifs in Dictyostelium promoters and introns is significantly higher than in the rest of the genome. Fourteen selected sequences located in important genes were characterized by a combination of biophysical and biochemical techniques. Our data show that these sequences form highly stable G4 structures under physiological conditions. Five Dictyostelium genes containing G4-prone motifs in their promoters were studied for the effect of a new G4-binding porphyrin derivative on their expression. Our results demonstrated that the new ligand significantly decreased their expression. Overall, our results constitute the first step to adopt Dictyostelium discoideum as a ‘G4-poor’ model for studies on G-quadruplexes