Biomedical subjects

J L Mergny

Publications and source records attributed to J L Mergny.

At least 19 recordsLinked to original sources

PubMed2004

DNA structural changes as the basis for a nanomolecular device.

There is currently great interest in the design of nanodevices that are capable of performing movements. Protein molecular machines are abundant in biology but it has recently been proposed that nucleic acids could also act as nanomolecular machines in model systems. Several types of movements have been described with DNA machines: rotation, extension-contraction and "scissor-like" opening and closing. Here we analyze the properties of a simple and robust device composed of a single 21-base-long oligonucleotide which relies on a duplex/quadruplex equilibrium fueled by the sequential addition of DNA single-strands, generating a DNA duplex as a by-product. The interconversion between two well-defined topological states induces a five nanometer two-stroke, linear motor type movement, which is detected by FRET spectroscopy.

Computers, Molecular↗

PubMed2002

Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands.

Telomeres of human chromosomes contain a G-rich 3'-overhang that adopts an intramolecular G-quadruplex structure in vitro which blocks the catalytic reaction of telomerase. Agents that stabilize G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalyzed by telomerase and can therefore act as antitumor agents. We have identified by Fluorescence Resonance Energy Transfer a new series of quinoline-based G-quadruplex ligands that also exhibit potent and specific anti-telomerase activity with IC50 in the nanomolar concentration range. Long term treatment of tumor cells at subapoptotic dosage induces a delayed growth arrest that depends on the initial telomere length. This growth arrest is associated with telomere erosion and the appearance of the senescent cell phenotype (large size and expression of beta-galactosidase activity). Our data show that a G-quadruplex interacting agent is able to impair telomerase function in a tumor cell thus providing a basis for the development of new anticancer agents.

Apoptosis↗

PubMed2001

Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2-20 degrees C at 1 microM dye concentration. This increase in T(m) value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC(50) value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.

DNA↗

PubMed2001

Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes.

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC(50) values of 18-100 nM in a standard TRAP assay.

DNA↗

PubMed2001

Fluorescence resonance energy transfer as a probe for G-quartet formation by a telomeric repeat.

The secondary structure of guanine-rich oligodeoxynucleotides has been investigated with fluorescent probes. Intramolecular folding of a telomeric oligonucleotide into a quadruplex led to fluorescence resonance energy transfer (FRET) between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the DNA, respectively. Depending on oligonucleotide length, quenching efficiency varied between 0.45 and 0.72 at 20 degrees C. The conjugation of the dyes to the oligonucleotide had a limited, but significant, influence on the thermodynamics of G-quartet formation. Intramolecular folding was demonstrated from the concentration independence of fluorescence resonance energy transfer over a wide concentration range. Folding of the oligonucleotide was confirmed by UV absorption, UV melting, and circular dichroism experiments. The folding of the G-quartet could be followed at concentrations as low as 100 pM. Fluorescence resonance energy transfer can thus be used to reveal the formation of multistranded DNA structures.

Chromosomes↗

PubMed2001

Interaction of an acridine dimer with DNA quadruplex structures.

Acridines↗

PubMed2000

Interaction of human DNA topoisomerase I with G-quartet structures.

Because of their role in the control of the topological state of DNA, topoisomerases are ubiquitous and vital enzymes, which participate in nearly all events related to DNA metabolism including replication and transcription. We show here that human topoisomerase I (Topo I) plays an unexpected role of 'molecular matchmaker' for G-quartet formation. G-quadruplexes are multi-stranded structures held together by square planes of four guanines ('G-quartets') interacting by forming Hoogsteen hydrogen bonds. Topo I is able to promote the formation of four-stranded intermolecular DNA structures when added to single-stranded DNA containing a stretch of at least five guanines. We provide evidence that these complexes are parallel G-quartet structures, mediated by tetrads of hydrogen-bonded guanine. In addition, Topo I binds specifically to pre-formed parallel and anti-parallel G4-DNA.

Base Sequence↗

PubMed2000

Chemical modification of pyrimidine TFOs: effect on i-motif and triple helix formation.

In order to form more stable triple helical structures or to prevent their degradation in cells, oligonucleotide analogs are routinely used, either in the backbone or among the bases. The target sequence chosen for this study is a 16-base-long oligopurine-oligopyrimidine region present in the human neurotrophin 4/5 gene. Seven different chemical modifications were tested for their effect on (i) triple helix formation and (ii) i-DNA stability. i-DNA is a tetrameric structure involving hemiprotonated C x C+ base pairs, which may act as a competing structure for triplex formation, especially in the case of a cytosine-rich third strand. At acid pH, oligophosphoramidates formed the most stable triple helix, whereas oligonucleotides including 5-propynyl-dU formed a stable i-motif which precluded triplex formation. Only two candidates stabilized triple helices at neutral pH: oligonucleotides with phosphoramidate linkage and phosphodiester oligonucleotides containing 5-methyl-dC and 5-propynyl-dU.

Base Sequence↗

PubMed2000

Pyrimidine morpholino oligonucleotides form a stable triple helix in the absence of magnesium ions.

Oligonucleotides can be used as sequence-specific DNA ligands by forming a local triple helix. In order to form more stable triple-helical structures or prevent their degradation in cells, oligonucleotide analogues that are modified at either the backbone or base level are routinely used. Morpholino oligonucleotides appeared recently as a promising modification for antisense applications. We report here a study that indicates the possibility of a triple helix formation with a morpholino pyrimidine TFO and its comparison with a phosphodiester and a phosphoramidate oligonucleotide. At a neutral pH and in the presence of a high magnesium ion concentration (10 mM), the phosphoramidate oligomer forms the most stable triple helix, whereas in the absence of magnesium ion but at a physiological monovalent cation concentration (0.14 M) only morpholino oligonucleotides form a stable triplex. To our knowledge, this is the first report of a stable triple helix in the pyrimidine motif formed by a noncharged oligonucleotide third strand (the morpholino oligonucleotide) and a DNA duplex. We show here that the structure formed with the morpholino oligomer is a bona fide triple helix and it is destabilized by high concentrations of potassium ions or divalent cations (Mg(2+)).

Base Sequence↗

PubMed2000

Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro.

Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)(n)repeat] with high affinity and bind double-stranded telomeric DNA with a 100xreduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66-64, 45 and 35 kDa, respectively. To identify these polypeptides we screened alambdagt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.

Base Composition↗

PubMed2000

Design of antisense and triplex-forming oligonucleotides.

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Animals↗

PubMed1999

Energetics of strand-displacement reactions in triple helices: a spectroscopic study.

DNA triple helices offer exciting new perspectives toward oligonucleotide-directed inhibition of gene expression. Purine and GT triplexes appear to be the most promising motifs for stable binding under physiological conditions compared to the pyrimidine motif, which forms at relatively low pH. There are, however, very little data available for comparison of the relative stabilities of the different classes of triplexes under identical conditions. We, therefore, designed a model system which allowed us to set up a competition between the oligonucleotides of the purine and pyrimidine motifs targeting the same Watson-Crick duplex. Several conclusions may be drawn: (i) a weak hypochromism at 260 nm is associated with purine triplex formation; (ii) delta H degree of GA, GT and TC triplex formation (at pH 7.0) was calculated as -0.1, -2.5 and -6.1 kcal/mol per base triplet, respectively. This unexpectedly low delta H degree for the purine triple helix formation implies that its delta G degree is nearly temperature-independent and it explains why these triplexes may still be observed at high temperatures. In contrast, the pyrimidine triplex is strongly favoured at lower temperatures; (iii) as a consequence, in a system where two third-strands compete for triplex formation, displacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This original purine-to-pyrimidine triplex conversion shows a significant hypochromism at 260 nm and a hyperchromism at 295 nm which is similar to the duplex-to-triplex conversion in the pyrimidine motif. Further evidence for this triplex-to-triplex conversion is provided by mung bean-nuclease foot-printing assay.

Base Pairing↗

PubMed1999

UV-A-induced decrease in nuclear factor-kappaB activity in human keratinocytes.

Previous reports have demonstrated an increase in nuclear factor-kappaB (NF-kappaB) activity in response to UV radiation. These studies have essentially focused on the DNA-damaging fraction of solar UV radiation (UV-B and UV-C). In contrast, the effects of UV-A radiation (320-400 nm) on NF-kappaB are not well known. In this study, we present evidence that UV-A radiation induces a marked decrease in NF-kappaB DNA-binding activity in NCTC 2544 human keratinocytes. In addition, NCTC 2544 keratinocytes pretreated with UV-A fail to respond to NF-kappaB inducers. Moreover, UV-A radiation induces a decrease in NF-kappaB-driven luciferase reporter gene expression in NCTC 2544 keratinocytes. The expression of the gene encoding IkappaBalpha (IkappaB is the NF-kappaB inhibitor), which is closely associated with NF-kappaB activity, is also reduced (3-fold) upon UV-A treatment. Our results indicate that the UV-A-induced decrease in NF-kappaB DNA-binding activity is associated with a decrease in the levels of the p50 and p65 protein subunits. This is the first evidence that an oxidative stress, such as UV-A radiation, may induce a specific decrease in NF-kappaB activity in mammalian cells, probably through degradation of NF-kappaB protein subunits. These findings suggest that UV-A could modulate the NF-kappaB-dependent gene expression.

Antioxidants↗

PubMed1999

Triplex formation by oligonucleotides containing 5-(1-propynyl)-2'-deoxyuridine: decreased magnesium dependence and improved intracellular gene targeting.

Oligonucleotides capable of sequence-specific triple helix formation have been proposed as DNA binding ligands useful for modulation of gene expression and for directed genome modification. However, the effectiveness of such triplex-forming oligonucleotides (TFOs) depends on their ability to bind to their target sites within cells, and this can be limited under physiologic conditions. In particular, triplex formation in the pyrimidine motif is favored by unphysiologically low pH and high magnesium concentrations. To address these limitations, a series of pyrimidine TFOs were tested for third-strand binding under a variety of conditions. Those containing 5-(1-propynyl)-2'-deoxyuridine (pdU) and 5-methyl-2'-deoxycytidine (5meC) showed superior binding characteristics at neutral pH and at low magnesium concentrations, as determined by gel mobility shift assays and thermal dissociation profiles. Over a range of Mg2+ concentrations, pdU-modified TFOs formed more stable triplexes than did TFOs containing 2'-deoxythymidine. At 1 mM Mg2+, a DeltaTm of 30 degreesC was observed for pdU- versus T-containing 15-mers (of generic sequence 5' TTTTCTTTTTTCTTTTCT 3') binding to the cognate A:T bp rich site, indicating that pdU-containing TFOs are capable of substantial binding even at physiologically low Mg2+ concentrations. In addition, the pdU-containing TFOs were superior in gene targeting experiments in mammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third-strand-directed psoralen adducts. These results suggest the utility of the pdU substitution in the pyrimidine motif for triplex-based gene targeting experiments.

Animals↗

PubMed1999

Fluorescence energy transfer as a probe for tetraplex formation: the i-motif.

The secondary structure of cytosine-rich oligodeoxynucleotides has been investigated with fluorescent probes. Intramolecular folding of an oligonucleotide into an i-DNA motif led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the DNA, respectively, provided that a suitable linker was chosen. The conjugation of the dyes to the oligonucleotide had an influence on the thermodynamics of i-motif formation as well as on the kinetics of folding. Intramolecular folding was demonstrated from the concentration independence of FRET over a wide concentration range. Folding of the oligonucleotide was confirmed by UV absorption melting experiments. The folding of the i-motif could be followed at concentrations as low as 50 pM. Fluorescence energy transfer can thus be used to reveal the formation of multistranded DNA structures.

Base Composition↗

PubMed1999

The development of telomerase inhibitors: the G-quartet approach.

Human telomeres, which consist of repeated TTAGGG sequences, have recently become the focus of intense and highly competitive biological research. This scientific interest lies in their unique biological functions: telomeres are essential for genome integrity and appear to play an important role in cellular aging and cancer. As telomerase appears to be selectively expressed in tumors versus normal cells, this enzyme represents a good target for inhibition. Different types of telomerase inhibitors have recently been described. We will present briefly the different strategies that have been proposed to achieve efficient telomerase inhibition, with a special emphasis on G-quartet ligands.

Enzyme Inhibitors↗

PubMed1998

Kinetics and thermodynamics of i-DNA formation: phosphodiester versus modified oligodeoxynucleotides.

At slightly acidic or even neutral pH, oligodeoxynucleotides that include a stretch of cytidines have been shown to form a tetrameric structure in which two parallel-stranded duplexes have their hemiprotonated C.C+base pairs face to face and fully intercalated, in a so-called i-motif. Cytosine-rich pyrimidine oligodeoxynucleotides can form an intramolecular i-motif. We have studied the ability of several DNA analogs to fold into this structure. Evidence for folding was provided by thermal denaturation. We have shown that phosphorothioate and phosphodiester oligodeoxynucleotides, but not methylphosphonate or PNA oligomers, may form the i-motif. Four different PS oligodeoxynucleotides were compared with their PO counterparts. In all cases, the melting temperature (Tm) of the phosphorothioate oligomer was equal or slightly inferior (by 2-3 degreesC) to the Tmof the natural oligodeoxynucleotide. For long oligodeoxynucleotides, a small change of pH leads to a completely different melting profile: the curves are reversible at pH 6.4 or lower, and a hysteresis is obtained at pH 6.8 or higher; cooling and heating curves were not superimposed, allowing us to determine the rate constants of association (kon) and dissociation (koff) as a function of the temperature: these rate constants give linear Arrhenius plots, in agreement with the prediction of the two-state model of association-dissociation. The activation energy Eonis strongly negative and, at neutral pH, the phosphorothioate associates and dissociates nine times faster than the phosphodiester oligodeoxynucleotide of identical sequence.

Base Sequence↗

PubMed1998

Following G-quartet formation by UV-spectroscopy.

Oligodeoxynucleotides which include stretches of guanines form a well-known tetrameric structure. We show that the recording of reversible absorbance changes at 295 nm allows to precisely monitor intramolecular guanine (G)-quartet formation and dissociation. Accurate Tm and thermodynamic values could be easily extracted from the data, whereas classical recordings at 260 nm led to a much larger uncertainty and in extreme cases, to completely inaccurate measurements. This inverted denaturation profile was observed for all G-quartet-forming oligonucleotides studied so far. This technique is very useful in all cases where intramolecular or intermolecular quadruplex formation is suspected.

DNA↗