Quadruplex (G4) nucleic acids, a family of secondary structures formed by guanine-rich sequences, exhibit an important structural polymorphism. We demonstrate here that G-rich DNA sequences may function as a double switch based on different triggers, provided that their quadruplex structures and stability display a high dependence on cation nature and concentration. A first switch is based on a remarkable antiparallel-to-parallel conversion, taking place in a few seconds at room temperature by addition of low KCl amounts to a sodium-rich sample. The second switch involves the conversion of alternative antiparallel quadruplex structures binding only one cation, formed in the presence of sub-millimolar potassium or strontium concentrations, to parallel structures by increasing the cation concentration. Incidentally, extremely low K(+) or Sr(2+) concentrations (≤5 equiv) are sufficient to induce G4 formation in a buffer devoid of other G4-promoting cations, and we suggest that the alternative structures observed contain only two tetrads. Such DNA systems are biological relevant targets, can be used in nanotechnology applications, and are valuable methodological tools for understanding DNA quadruplex folding, notably at low cation concentrations. We demonstrate that this behavior is not restricted to a narrow set of sequences but can also be found for other G-quadruplex-forming motifs, arguing for widespread applications.
G-quadruplexes are nucleic acid secondary structures for which many biological roles have been proposed but whose existence in vivo has remained elusive. To assess their formation, highly specific G-quadruplex ligands are needed. Here, we tested Phen-DC3 and Phen-DC6, two recently released ligands of the bisquinolinium class. In vitro, both compounds exhibit high affinity for the G4 formed by the human minisatellite CEB1 and inhibit efficiently their unwinding by the yeast Pif1 helicase. In vivo, both compounds rapidly induced recombination-dependent rearrangements of CEB1 inserted in the Saccharomyces cerevisiae genome, but did not affect the stability of other tandem repeats lacking G-quadruplex forming sequences. The rearrangements yielded simple-deletion, double-deletion or complex reshuffling of the polymorphic motif units, mimicking the phenotype of the Pif1 inactivation. Treatment of Pif1-deficient cells with the Phen-DC compounds further increased CEB1 instability, revealing additional G4 formation per cell. In sharp contrast, the commonly used N-methyl-mesoporphyrin IX G-quadruplex ligand did not affect CEB1 stability. Altogether, these results demonstrate that the Phen-DC bisquinolinium compounds are potent molecular tools for probing the formation of G-quadruplexes in vivo, interfere with their processing and elucidate their biological roles.
G-quadruplexes are guanine-rich nucleic acids that fold by forming successive quartets of guanines (the G-tetrads), stabilized by intra-quartet hydrogen bonds, inter-quartet stacking, and cation coordination. This specific although highly polymorphic type of secondary structure deviates significantly from the classical B-DNA duplex. G-quadruplexes are detectable in human cells and are strongly suspected to be involved in a number of biological processes at the DNA and RNA levels. The vast structural polymorphism exhibited by G-quadruplexes, together with their putative biological relevance, makes them attractive therapeutic targets compared to canonical duplex DNA. This chapter focuses on the essential and specific coordination of alkali metal cations by G-quadruplex nucleic acids, and most notably on studies highlighting cation-dependent dissimilarities in their stability, structure, formation, and interconversion. Section 1 surveys G-quadruplex structures and their interactions with alkali metal ions while Section 2 presents analytical methods used to study G-quadruplexes. The influence of alkali cations on the stability, structure, and kinetics of formation of G-quadruplex structures of quadruplexes will be discussed in Sections 3 and 4. Section 5 focuses on the cation-induced interconversion of G-quadruplex structures. In Sections 3 to 5, we will particularly emphasize the comparisons between cations, most often K(+) and Na(+) because of their prevalence in the literature and in cells.
G4-FID (G-quadruplex fluorescent intercalator displacement) is a simple and fast method that allows to evaluate the affinity of a compound for G-quadruplex DNA and its selectivity towards duplex DNA. This assay is based on the loss of fluorescence of thiazole orange (TO) upon competitive displacement from DNA by a putative ligand. We describe here the development of a high-throughput version of this assay performed in 96-well microplates, and fully transposable to 384-well microplates. The test was calibrated with a set of G-quadruplex ligands characterized for their ability to bind quadruplex within a large range of affinity. The comparison of the results obtained in microplates and in cuvettes was conducted indicating a full agreement. Additionally, the spectral range of the test was enlarged using two other fluorescent on/off probes whose absorption are red-shifted (TO-PRO-3) and blue-shifted (Hoechst 33258) as compared to that of TO. These labels enable to screen a large diversity of compounds with various optical properties, which was exemplified by evaluation of affinity and selectivity of the porphyrin TMPyP4 that could not be evaluated previously. Altogether, our study demonstrates that the HT-G4-FID assay offers the possibility to label a large variety of G-quadruplexes of biological interest and should enable screening of collections of putative G4-ligands of high structural diversity. It thus represents a powerful tool to bring into light new ligands able to discriminate between quadruplexes of different structures.
In recent years, an increasing number of reports have been focused on the structure and biological role of non-canonical nucleic acid secondary structures. Many of these studies involve the use of oligonucleotides that can often adopt a variety of structures depending on the experimental conditions, and hence change the outcome of an assay. The knowledge of the structure(s) formed by oligonucleotides is thus critical to correctly interpret the results, and gain insight into the biological role of these particular sequences. Herein we demonstrate that size-exclusion HPLC (SE-HPLC) is a simple yet surprisingly powerful tool to quickly and effortlessly assess the secondary structure(s) formed by oligonucleotides. For the first time, an extensive calibration and validation of the use of SE-HPLC to confidently detect the presence of different species displaying various structure and/or molecularity, involving >110 oligonucleotides forming a variety of secondary structures (antiparallel, parallel, A-tract bent and mismatched duplexes, triplexes, G-quadruplexes and i-motifs, RNA stem loops), is performed. Moreover, we introduce simple metrics that allow the use of SE-HPLC without the need for a tedious calibration work. We show that the remarkable versatility of the method allows to quickly establish the influence of a number of experimental parameters on nucleic acid structuration and to operate on a wide range of oligonucleotide concentrations. Case studies are provided to clearly illustrate the all-terrain capabilities of SE-HPLC for oligonucleotide secondary structure analysis. Finally, this manuscript features a number of important observations contributing to a better understanding of nucleic acid structural polymorphism.
Detection of quadruplex structures by visual methods is a major challenge of the quadruplex nucleic acid research area. Consequently, considerable efforts are under way for the discovery of quadruplex specific agents endowed with fluorescence properties. In this review chapter we propose a comprehensive and critical overview of the diverse molecular design and strategies that have been described to identify quadruplex-selective fluorescent probes. Innovative compounds as well as classical DNA dyes are reviewed. The compounds have been divided into three classes: (1) "light-up" probes that display a strong enhancement upon G4 binding, (2) "light-off" probes that display a decreased fluorescence upon binding, and (3) permanent probes ("tagged" G4-binders) that exhibit no variation of fluorescence but display quadruplex binding specificity. The labeling performances of probes in various analytical contexts (in solution, in gel, at the level of chromosomes, and in fixed cells) are also reported and commented on when available. Finally we address the strengths and weaknesses of each probe class and highlight the critical features that must be addressed in developing a practicable quadruplex-specific labeling agent.
Nucleic acids have been among the first targets for antitumor drugs and antibiotics, and with the unveiling of new biological roles in regulation of gene expression, specific DNA and RNA structures have become very attractive targets, especially when the corresponding proteins are undruggable. Biophysical assays to test target structure and ligand binding stoichiometry, affinity, specificity and binding modes are part of the drug development pipeline. Mass spectrometry offers unique advantages as a biophysical method due to its ability to distinguish each stoichiometry present in a mixture. In addition, advanced mass spectrometry approaches (reactive probing, fragmentation techniques, ion mobility spectrometry, ion spectroscopy) provide more detailed information on the complexes. Here we review the fundamentals of mass spectrometry and all its particularities when studying non-covalent nucleic acid structures, and then review what has been learned thanks to mass spectrometry on nucleic acid structures, self-assemblies (e.g., duplexes or G-quadruplexes), and their complexes with ligands. 47
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