Insights into the G-rich VEGF-binding aptamer V7t1: when two G-quadruplexes are better than one!

The G-quadruplex-forming VEGF-binding aptamer V7t1 was previously found to be highly polymorphic in a K+-containing solution and, to restrict its conformational preferences to a unique, well-defined form, modified nucleotides (LNA and/or UNA) were inserted in its sequence. We here report an in-depth biophysical characterization of V7t1 in a Na+-rich medium, mimicking the extracellular environment in which VEGF targeting should occur, carried out combining several techniques to analyse the conformational behaviour of the aptamer and its binding to the protein. Our results demonstrate that, in the presence of high Na+ concentrations, V7t1 behaves in a very different way if subjected or not to annealing procedures, as evidenced by native gel electrophoresis, size exclusion chromatography and dynamic light scattering analysis. Indeed, not-annealed V7t1 forms both monomeric and dimeric G-quadruplexes, while the annealed oligonucleotide is a monomeric species. Remarkably, only the dimeric aptamer efficiently binds VEGF, showing higher affinity for the protein compared to the monomeric species. These findings provide new precious information for the development of improved V7t1 analogues, allowing more efficient binding to the cancer-related protein and the design of effective biosensors or theranostic devices based on VEGF targeting

Ligand selectivity in stabilising tandem parallel folded G-quadruplex motifs in human telomeric DNA sequences

Biophysical studies of ligand interactions with three human telomeric repeat sequences (d(AGGG(TTAGGG)n, n = 3, 7 and 11)) show that an oxazole-based ‘click’ ligand, which induces parallel folded quadruplexes, preferentially stabilises longer telomeric repeats providing evidence for selectivity in binding at the interface between tandem quadruplex motifs

Vertically-resolved particle size distribution within and above the mixing layer over the Milan metropolitan area

Vertical aerosol profiles were directly measured over the city of Milan during three years (2005–2008) of field campaigns. An optical particle counter, a portable meteorological station and a miniaturized cascade impactor were deployed on a tethered balloon. More than 300 vertical profiles were measured, both in winter and summer, mainly in conditions of clear, dry skies. <br><br> The mixing height was determined from the observed vertical aerosol concentration gradient, and from potential temperature and relative humidity profiles. Results show that inter-consistent mixing heights can be retrieved highlighting good correlations between particle dispersion in the atmosphere and meteorological parameters. Mixing height growth speed was calculated for both winter and summer showing the low potential atmospheric dispersion in winter. <br><br> Aerosol number size distribution and chemical composition profiles allowed us to investigate particle behaviour along height. Aerosol measurements showed changes in size distribution according to mixing height. Coarse particle profiles (<i>d</i><sub><i>p</i></sub>>1.6 μm) were distributed differently than the fine ones (<i>d</i><sub><i>p</i></sub><1.6 μm) were, at different heights of the mixing layer. The sedimentation process influenced the coarse particle profiles, and led to a reduction in mean particle diameter for those particles observed by comparing data above the mixing height with ground data (−14.9±0.6% in winter and −10.7±1.0% in summer). Conversely, the mean particle diameter of fine particles increased above the mixing height under stable atmospheric conditions; the average increase, observed by comparing data above the mixing height with ground data, was +2.1±0.1% in winter and +3.9±0.3% in summer. A hierarchical statistical model was created to describe the changes in the size distribution of fine particles along height. The proposed model can be used to estimate the typical vertical profile characterising launches within pre-specified groups starting from: aerosol size and meteorological conditions measured at ground-level, and a mixing height estimation. The average increase of fine particle diameter, estimated on the basis of the model, was +1.9±0.5% in winter and +6.1±1.2% in summer, in keeping with experimental findings

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3′-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or n-mers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3)8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences

Pattern recognition and anomaly detection by self-organizing maps in a multi month e-nose survey at an industrial site

Currently people are aware of the risk related to pollution exposure. Thus odor annoyances are considered a warning about the possible presence of toxic volatile compounds. Malodor often generates immediate alarm among citizens, and electronic noses are convenient instruments to detect mixture of odorant compounds with high monitoring frequency. In this paper we present a study on pattern recognition on ambient air composition in proximity of a gas and oil pretreatment plant by elaboration of data from an electronic nose implementing 10 metal-oxide-semiconductor (MOS) sensors and positioned outdoor continuously during three months. A total of 80,017 e-nose vectors have been elaborated applying the self-organizing map (SOM) algorithm and then k-means clustering on SOM outputs on the whole data set evidencing an anomalous data cluster. Retaining data characterized by dynamic responses of the multisensory system, a SOM with 264 recurrent sensor responses to air mixture sampled at the site and four main air type profiles (clusters) have been identified. One of this sensor profiles has been related to the odor fugitive emissions of the plant, by using ancillary data from a total volatile organic compound (VOC) detector and wind speed and direction data. The overall and daily cluster frequencies have been evaluated, allowing us to identify the daily duration of presence at the monitoring site of air related to industrial emissions. The refined model allowed us to confirm the anomaly detection of the sensor responses