P53 binds preferentially to non-B DNA structures formed by the pyrimidine-rich strands of GaA·TTC trinucleotide repeats associated with Friedreich’s ataxia

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich’s ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich’s ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs’ non-B DNA structures

Soluble collagen dissolution and assembling in pressurized carbon dioxide water solutions

Dissolution and gelation procedures have a great influence on gelation time, microstructure and mechanical properties of reconstituted collagen products. We have investigated the dissolution of atelocollagen in CO2/water solutions at low temperature (4 degrees C) at different CO2 pressures (0.3-0.9 MPa), as well as gelation kinetics and physico-chemical properties of the hydrogel obtained after CO2 removal. Compared to conventional methods, the CO2-assisted technique resulted in faster soluble collagen dissolution and faster gelation into transparent gels characterized by thin 10 nm fibrils. Electrophoresis and CD spectroscopy demonstrated that the process did not denature the soluble collagen. The possibility to obtain collagen dissolution and gelation without the use of chemical agent other than water and CO2 makes this process particularly appealing for biomedical applications

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

Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G3(TTAG3)3 forms an antiparallel quadruplex of the same basket type in solution containing either K+ or Na+ ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K+-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G3(TTAG3)3 motif. Both G3(TTAG3)3 and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K+-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K+-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG3(TTAG3)3 by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K+ ions

Diversity of Parallel Guanine Quadruplexes Induced by Guanine Substitutions

Recently, we reported an inhibitory effect of guanine substitutions on the conformational switch from antiparallel to parallel quadruplexes (G4) induced by dehydrating agents. As a possible cause, we proposed a difference in the sensitivity of parallel and antiparallel quadruplexes to the guanine substitutions in the resulting thermodynamic stability. Reports on the influence of guanine substitutions on the biophysical properties of intramolecular parallel quadruplexes are rare. Moreover, such reports are often complicated by the multimerisation tendencies of parallel quadruplexes. To address this incomplete knowledge, we employed circular dichroism spectroscopy (CD), both as stopped-flow-assisted fast kinetics measurements and end-point measurements, accompanied by thermodynamic analyses, based on UV absorption melting profiles, and electrophoretic methods. We showed that parallel quadruplexes are significantly more sensitive towards guanine substitutions than antiparallel ones. Furthermore, guanine-substituted variants, which in principle might correspond to native genomic sequences, distinctly differ in their biophysical properties, indicating that the four guanines in each tetrad of parallel quadruplexes are not equal. In addition, we were able to distinguish by CD an intramolecular G4 from intermolecular ones resulting from multimerisation mediated by terminal tetrad association, but not from intermolecular G4s formed due to inter-strand Hoogsteen hydrogen bond formation. In conclusion, our study indicates significant variability in parallel quadruplex structures, otherwise disregarded without detailed experimental analysis

DNA i-motif formation at neutral pH is driven by kinetic partitioning

International audienceAbstract Cytosine-rich DNA regions can form four-stranded structures based on hemi-protonated C.C+ pairs, called i-motifs (iMs). Using CD, UV absorption, NMR spectroscopy, and DSC calorimetry, we show that model (CnT3)3Cn (Cn) sequences adopt iM under neutral or slightly alkaline conditions for n > 3. However, the iMs are formed with long-lasting kinetics under these conditions and melt with significant hysteresis. Sequences with n > 6 melt in two or more separate steps, indicating the presence of different iM species, the proportion of which is dependent on temperature and incubation time. At ambient temperature, kinetically favored iMs of low stability are formed, most likely consisting of short C.C+ blocks. These species act as kinetic traps and prevent the assembly of thermodynamically favored, fully C.C+ paired iMs. A higher temperature is necessary to unfold the kinetic forms and enable their substitution by a slowly developing thermodynamic structure. This complicated kinetic partitioning process considerably slows down iM folding, making it much slower than the timeframes of biological reactions and, therefore, unlikely to have any biological relevance. Our data suggest kinetically driven iM species as more likely to be biologically relevant than thermodynamically most stable iM forms

Porphyrin-Based Design of Bioinspired Multitarget Quadruplex Ligands

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