Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter.

Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.

Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4-binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step.

G-quadruplex structure of the C. elegans telomeric repeat: a two tetrads basket type conformation stabilized by a non-canonical C-T base-pair.

The Caenorhabditis elegans model has greatly contributed to the understanding of the role of G-quadruplexes in genomic instability. The GGCTTA repeats of the C. elegans telomeres resemble the GGGTTA repeats of the human telomeres. However, the comparison of telomeric sequences (Homo sapiens, Tetrahymena, Oxytricha, Bombyx mori and Giardia) revealed that small changes in these repeats can drastically change the topology of the folded G-quadruplex. In the present work we determined the structure adopted by the C. elegans telomeric sequence d[GG(CTTAGG)3]. The investigated C. elegans telomeric sequence is shown to fold into an intramolecular two G-tetrads basket type G-quadruplex structure that includes a C-T base pair in the diagonal loop. This work sheds light on the telomeric structure of the widely used C. elegans animal model.

G-Quadruplex binding optimization by gold(iii) insertion into the center of a porphyrin.

Porphyrins represent a valuable class of ligands for G-quadruplex nucleic acids. Herein, we evaluate the binding of cationic porphyrins metallated with gold(iii) to G-quadruplex DNA and we compare it with other porphyrin derivatives. The G-quadruplex stabilization capacity and the selectivity of the various porphyrins were evaluated by biophysical and biochemical assays. The porphyrins were also tested as inhibitors of telomerase. It clearly appeared that the insertion of gold(iii) ion in the center of the porphyrin increases the binding affinity of the porphyrin for the G-quadruplex target. Together with modelling studies, it is possible to propose that the insertion of the square planar gold(iii) ion adds an extra positive charge on the complex and decreases the electron density in the porphyrin aromatic macrocycle, both properties being in favour of stronger electrostatic and π-staking interactions.

Synthesis, Binding Properties, and Differences in Cell Uptake of†G-Quadruplex Ligands Based on Carbohydrate Naphthalene Diimide Conjugates.

The G-quadruplexes (G4s) are currently being explored as therapeutic targets in cancer and other pathologies. Six carbohydrate naphthalene diimide conjugates (carb-NDIs) have been synthesized as G4 ligands to investigate their potential selectivity in G4 binding and cell penetration. Carb-NDIs have shown certain selectivity for G4 structures against DNA duplexes, but different sugar moieties do not induce a preference for a specific G4 topology. Interestingly, when monosaccharides were attached through a short ethylene linker to the NDI scaffold, their cellular uptake was two- to threefold more efficient than that when the sugar was directly attached through its anomeric position. Moreover, a correlation between more efficient cell uptake of these carb-NDIs and their higher toxicity in cancerous cell lines has been observed. Carb-NDIs seem to be mainly translocated into cancer cells through glucose transporters (GLUT), of which GLUT4 plays a major role.

A Flexible Terpyridine Derivative Interacts Specifically with G-Quadruplexes.

G-quadruplexes formed by nucleic acids are implicated in pathologies ranging from cancers to neurodegenerative diseases. We evaluated interactions of 29 bi- and terpyridine derivatives with G-quadruplexes and duplexes. FRET-melting, circular dichroism, and (1) H NMR spectroscopy showed that one terpyridine derivative interacted very selectively with G-quadruplexes. This G-quadruplex ligand inhibited helicase activity and should influence G-quadruplex-related biological processes.

Efficient Inhibition of Telomerase by Nickel-Salophen Complexes.

Four nickel(II)-salophen complexes containing alkyl-imidazolium chains connected at the ortho or meta positions were prepared: N,N'-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)phenylenediamine (1), N,N'-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino)phenylenediamine (2), N,N'-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino)methyl-3H-imidazol-1-iumphenylenediamine (3), and N,N'-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)methyl-3H-imidazol-1-iumphenylenediamine (4). They protect G-quadruplex DNA (G4 -DNA) against thermal denaturation and show KA values in the range of 7.4×10(5) to 4×10(7) m(-1) for G4 -DNA models. Complex 4 exhibits an IC50 value of 70 nm for telomerase inhibition.

Harmonization of QSAR Best Practices and Molecular Docking Provides an Efficient Virtual Screening Tool for Discovering New G-Quadruplex Ligands.

Telomeres and telomerase are key players in tumorogenesis. Among the various strategies proposed for telomerase inhibition or telomere uncapping, the stabilization of telomeric G-quadruplex (G4) structures is a very promising one. Additionally, G4 stabilizing ligands also act over tumors mediated by the alternative elongation of telomeres. Accordingly, the discovery of novel compounds able to act on telomeres and/or inhibit the telomerase enzyme by stabilizing DNA telomeric G4 structures as well as the development of approaches efficiently prioritizing such compounds constitute active areas of research in computational medicinal chemistry and anticancer drug discovery. In this direction, we applied a virtual screening strategy based on the rigorous application of QSAR best practices and its harmonized integration with structure-based methods. More than 600,000 compounds from commercial databases were screened, the first 99 compounds were prioritized, and 21 commercially available and structurally diverse candidates were purchased and submitted to experimental assays. Such strategy proved to be highly efficient in the prioritization of G4 stabilizer hits, with a hit rate of 23.5%. The best G4 stabilizer hit found exhibited a shift in melting temperature from FRET assay of +7.3 °C at 5 µM, while three other candidates also exhibited a promising stabilizing profile. The two most promising candidates also exhibited a good telomerase inhibitory ability and a mild inhibition of HeLa cells growth. None of these candidates showed antiproliferative effects in normal fibroblasts. Finally, the proposed virtual screening strategy proved to be a practical and reliable tool for the discovery of novel G4 ligands which can be used as starting points of further optimization campaigns.

Assessment of selectivity of G-quadruplex ligands via an optimised FRET melting assay.

Four-stranded G-quadruplex (G4) DNA structures are promising drug targets as these non-canonical structures appear to regulate gene expression and telomere growth. Although all types of G4 are stabilised by quartets of four guanosines, differences in loops, grooves, and flanking bases, result in an impressive structural diversity among G4 structures that may allow selective recognition by small molecule ligands. We adapted the previously described Förster resonance energy transfer (FRET) melting assay to evaluate the selectivity of G4 ligands for different G-quadruplex topologies. We demonstrated that the incorporation of FAM and Tamra fluorescent dyes and the presence of PEG influenced the structures adopted by certain sequences with G-quadruplex-forming potential. Optimisation of the measurement conditions ensured the folding and thermal stability of a selected set of G4 DNA oligonucleotides in a measurable temperature range with and without ligand. The optimised method enabled comparison of well known G4 ligands such as TmPyP4, Braco19, pyridostatin, 360A, PhenDC3, and TrisQ.

The nickel(II) complex of guanidinium phenyl porphyrin, a specific G-quadruplex ligand, targets telomeres and leads to POT1 mislocalization in culture cells.

With the aim of finding selective and biologically active G-quadruplex ligands, modified porphyrin with bulky cationic substituents, meso-5,10,15,20-tetrakis(4-guanidinophenyl)porphyrin tetrahydrochloride, referred to as guanidinium phenyl porphyrin, was prepared. The corresponding nickel(II) and cobalt(III) metallated porphyrins were also synthesized. Interaction with quadruplexes was examined by means of fluorescence resonance energy transfer melting and surface plasmon resonance-based assays: the three compounds proved to bind to G-quadruplex DNA in a similar and highly selective way. Guanidinium phenyl porphyrin and its nickel(II) metallated derivative exhibit moderate cytotoxicity toward cells in culture. Strikingly, the nickel porphyrin derivative was able to displace hPOT1 shelterin protein from telomeres in human cells. Nickel(II) guanidinium phenyl porphyrin, a cationic bulky porphyrin is a powerful specific G-quadruplex DNA ligand. It enters the cells and induces shelterin modification.

Cobalt(III)porphyrin to target G-quadruplex DNA.

G-quadruplex DNA ligands attract much attention because of their potential use in biology. Indeed they may interfere with G-quadrulex nucleic acid function in cells. Most of the G-quadruplex ligands so far reported (including also metal complexes) are large planar aromatic compounds that interact by π-π stacking with an external G-quartet of quadruplex. Porphyrins are well-known G-quadruplex ligands. We report herein a new porphyrin scaffold (meso-tetrakis(4-(N-methyl-pyridinium-2-yl)phenyl)porphyrin) able to strongly and selectively bind to G-quadruplex DNA. We show that even when this porphyrin is metallated with cobalt(III), i.e. it carries two water molecules as axial ligands on the cobalt ion, on each face of the porphyrin, the interaction occurs by a π-stacking-like mode with an external G-quartet of quadruplex DNA.

Interaction of polycationic Ni(II)-salophen complexes with G-quadruplex DNA.

A series of nine Ni(II) salophen complexes involving one, two, or three alkyl-imidazolium side-chains was prepared. The lengths of the side-chains were varied from one to three carbons. The crystal structure of one complex revealed a square planar geometry of the nickel ion. Fluorescence resonance energy transfer melting of G-quadruplex structures in the presence of salophen complex were performed. The G-quadruplex DNA structures were stabilized in the presence of the complexes, but a duplex DNA was not. The binding constants of the complexes for parallel and antiparallel G-quadruplex DNA, as well as hairpin DNA, were measured by surface plasmon resonance. The compounds were selective for G-quadruplex DNA, as reflected by equilibrium dissociation constant KD values in the region 0.1-1 µM for G-quadruplexes and greater than 2 µM for duplex DNA. Complexes with more and shorter side-chains had the highest binding constants. The structural basis for the interaction of the complexes with the human telomeric G-quadruplex DNA was investigated by computational studies: the aromatic core of the complex stacked over the last tetrad of the G-quadruplex with peripherical cationic side chains inserted into opposite grooves. Biochemical studies (telomeric repeat amplification protocol assays) indicated that the complexes significantly inhibited telomerase activity with IC50 values as low as 700 nM; the complexes did not significantly inhibit polymerase activity.

Impact of G-quadruplex structures and intronic polymorphisms rs17878362 and rs1642785 on basal and ionizing radiation-induced expression of alternative p53 transcripts.

G-quadruplex (G4) structures in intron 3 of the p53 pre-mRNA modulate intron 2 splicing, altering the balance between the fully spliced p53 transcript (FSp53, encoding full-length p53) and an incompletely spliced transcript retaining intron 2 (p53I2 encoding the N-terminally truncated Δ40p53 isoform). The nucleotides forming G4s overlap the polymorphism rs17878362 (A1 wild-type allele, A2 16-base pair insertion) which is in linkage disequilibrium with rs1642785 in intron 2 (c.74+38 G>C). Biophysical and biochemical analyses show rs17878362 A2 alleles form similar G4 structures as A1 alleles although their position is shifted with respect to the intron 2 splice acceptor site. In addition basal FSp53 and p53I2 levels showed allele specific differences in both p53-null cells transfected with reporter constructs or lymphoblastoid cell lines. The highest FSp53 and p53I2 levels were associated with combined rs1642785-GG/rs17878362-A1A1 alleles, whereas the presence of rs1642785-C with either rs17878362 allele was associated with lower p53 pre-mRNA, total TP53, FSp53 and p53I2 levels, due to the lower stability of transcripts containing rs1642785-C. Treatment of lymphoblastoid cell with the G4 binding ligands 360A or PhenDC3 or with ionizing radiation increased FSp53 levels only in cells with rs17878362 A1 alleles, suggesting that under this G4 configuration full splicing is favoured. These results demonstrate the complex effects of intronic TP53 polymorphisms on G4 formation and identify a new role for rs1642785 on mRNA splicing and stability, and thus on the differential expression of isoform-specific transcripts of the TP53 gene.

Electrochemical discrimination between G-quadruplex and duplex DNA.

Analytical tools enabling the discrimination between duplex DNA and G-quadruplex DNA are necessary to unravel the biological function(s) of G-quadruplexes. A methodology relying on the electrochemical response of the electroactive hexaammineruthenium(III) cation at DNA-modified surfaces is presented. A characteristic voltammetric peak is evidenced for all the investigated G-quadruplex sequences, encompassing various types of folding and numbers of quartets. In contrast, no such peak is detected for dsDNA sequences. The occurrence of the voltammetric peak is the consequence of a strong association between the hexaammineruthenium ligand and the surface-immobilized G-quadruplexes. The peak potential points to a significant contribution of nonelectrostatic interactions between the electroactive ligand and G-quadruplexes. The very good efficiency of the discrimination methodology is demonstrated by comparing a G-quadruplex and its corresponding duplex.

On the interaction between [Ru(NH3)6]3+ and the G-quadruplex forming thrombin binding aptamer sequence.

The interaction between the thrombin binding aptamer (TBA), a G-quadruplex forming DNA sequence, and the electroactive hexaammineruthenium(III) cation has been studied by electrochemical methods and circular dichroism spectroscopy. When TBA is immobilised on a gold surface in a typical aptasensor configuration, the [Ru(NH3)6](3+) cation can be bound to the electrode surface through its interaction with the TBA sequence. This interaction is strong enough to enable the ruthenium complex to remain at the surface when the electrode is immersed in an electrolyte free of [Ru(NH3)6](3+), meaning that the complex does not diffuse back into the solution. A stoichiometry of 2 [Ru(NH3)6](3+) per TBA strand has been determined, indicating that the interaction differs from the conventional, non-specific electrostatic charge compensation, for which a 5 to 1 ratio would be expected between the triply charged cation and the 15 bases sequence. It is shown that this interaction takes place not only at the surface, but also when both TBA and hexaammineruthenium(III) are dissolved in solution. Under such conditions, a similar stoichiometry of 2 [Ru(NH3)6](3+) per TBA strand has been evidenced by two independent methods, namely circular dichroism spectroscopy and differential pulse voltammetry.

Elongated thrombin binding aptamer: a G-quadruplex cation-sensitive conformational switch.

Aptamer-based biosensors offer promising perspectives for high performance, specific detection of proteins. The thrombin binding aptamer (TBA) is a G-quadruplex-forming DNA sequence, which is frequently elongated at one end to increase its analytical performances in a biosensor configuration. Herein, we investigate how the elongation of TBA at its 5' end affects its structure and stability. Circular dichroism spectroscopy shows that TBA folds in an antiparallel G-quadruplex conformation with all studied cations (Ba(2+), Ca(2+), K(+), Mg(2+), Na(+), NH(4)(+), Sr(2+) and the [Ru(NH(3))(6)](2+/3+) redox marker) whereas other structures are adopted by the elongated aptamers in the presence of some of these cations. The stability of each structure is evaluated on the basis of UV spectroscopy melting curves. Thermal difference spectra confirm the quadruplex character of all conformations. The elongated sequences can adopt a parallel or an antiparallel structure, depending on the nature of the cation; this can potentially confer an ion-sensitive switch behavior. This switch property is demonstrated with the frequently employed redox complex [Ru(NH(3))(6)](3+), which induces the parallel conformation at very low concentrations (10 equiv per strand). The addition of large amounts of K(+) reverts the conformation to the antiparallel form, and opens interesting perspectives for electrochemical biosensing or redox-active responsive devices.