Solution and Gas-Phase Stability of DNA Junctions from Temperature-Controlled Electrospray Ionization and Surface-Induced Dissociation.

DNA three-way junction (TWJ) structures transiently form during key cellular processes such as transcription, replication, and DNA repair. Despite their significance, the thermodynamics of TWJs, including the influence of strand length, base pair composition, and ligand binding on TWJ stability and dissociation mechanisms, are poorly understood. To address these questions, we interfaced temperature-controlled nanoelectrospray ionization mass spectrometry (TC-nESI-MS) with a cyclic ion mobility spectrometry (cIMS) instrument that was also equipped with a surface-induced dissociation (SID) stage. This novel combination allowed us to investigate the structural intermediates of three TWJ complexes and examine the effects of GC base pairs on their dissociation pathways. We found that two TWJ-specific ligands, 2,7-tris-naphthalene (2,7-TrisNP) and tris-phenoxybenzene (TrisPOB), lead to TWJ stabilization, revealed by an increase in the melting temperature (Tm) by 13 or 26 °C, respectively. To gain insights into conformational changes in the gas phase, we employed cIMS and SID to analyze TWJs and their complexes with ligands. Analysis of IM arrival distributions suggested a single-step dissociation of TWJs and their intermediates for the three studied TWJ complexes. Upon ligand binding, a higher SID energy by 3 V (2,7-TrisNP) and 5 V (TrisPOB) was required to induce 50% dissociation of TWJ, compared to 38 V in the absence of ligands. Our results demonstrate the power of utilizing TC-nESI-MS in combination with cIMS and SID for thermodynamic characterization of TWJ complexes and investigation of ligand binding. These techniques are essential for the TWJ design and development as drug targets, aptamers, and structural units for functional biomaterials.

Exploring Gas-Phase MS Methodologies for Structural Elucidation of Branched N-Glycan Isomers.

Structural isomers of N-glycans that are identical in mass and atomic composition provide a great challenge to conventional mass spectrometry (MS). This study employs additional dimensions of structural elucidation including ion mobility (IM) spectroscopy coupled to hydrogen/deuterium exchange (HDX) and electron capture dissociation (ECD) to characterize three main A2 N-glycans and their conformers. A series of IM-MS experiments were able to separate the low abundance N-glycans and their linkage-based isomers (α1-3 and α1-6 for A2G1). HDX-IM-MS data indicated the presence of multiple gas-phase structures for each N-glycan including the isomers of A2G1. Identification of A2G1 isomers by their collision cross section was complicated due to the preferential collapse of sugars in the gas phase, but it was possible by further ECD fragmentation. The cyclic IM-ECD approach was capable of assigning and identifying each isomer to its IM peak. Two unique cross-ring fragments were identified for each isomer: m/z = 624.21 for α1-6 and m/z = 462.16 for α1-3. Based on these key fragments, the first IM peak, indicating a more compact conformation, was assigned to α1-3 and the second IM peak, a more extended conformer, was assigned to α1-6.

Competition of Ligands and the 18-mer Binding Domain of the RHAU Helicase for G-Quadruplexes: Orthosteric or Allosteric Binding Mechanism?

Stabilizing the DNA and RNA structures known as G-quadruplexes (G4s) using specific ligands is a strategy that has been proposed to fight cancer. However, although G-quadruplex:ligand (G4:L) interactions have often been investigated, whether or not ligands are able to disrupt G-quadruplex:protein (G4:P) interactions remains poorly studied. In this study, using native mass spectrometry, we have investigated ternary G4:L:P complexes formed by G4s, some of the highest affinity ligands, and the binding domain of the RHAU helicase. Our results suggest that RHAU binds not only preferentially to parallel G4s, but also to free external G-quartets. We also found that, depending on the G4, ligands could prevent the binding of the peptide, either by direct competition for the binding sites (orthosteric inhibition) or by inducing conformational changes (allosteric inhibition). Notably, the ligand Cu-ttpy (ttpy=4'-tolyl-2,2':6',2''-terpyridine) induced a conformational change that increased the binding of the peptide. This study illustrates that it is important to not only characterize drug-target interactions, but also how the binding to other partners is affected.

A Modified Traveling Wave Ion Mobility Mass Spectrometer as a Versatile Platform for Gas-Phase Ion-Molecule Reactions.

Taken individually, chemical labeling and mass spectrometry are two well-established tools for the structural characterization of biomolecular complexes. A way to combine their respective advantages is to perform gas-phase ion-molecule reactions (IMRs) inside the mass spectrometer. This is, however, not so well developed because of the limited range of usable chemicals and the lack of commercially available IMR devices. Here, we modified a traveling wave ion mobility mass spectrometer to enable IMRs in the trapping region of the instrument. Only one minor hardware modification is needed to allow vapors of a variety of liquid reagents to be leaked into the trap traveling wave ion guide of the instrument. A diverse set of IMRs can then readily be performed without any loss in instrument performance. We demonstrate the advantages of implementing IMR capabilities in general, and to this quadrupole-ion mobility-time-of-flight (Q-IM-TOF) mass spectrometer in particular, by exploiting the full functionality of the instrument, including mass selection, ion mobility separation, and post-mobility fragmentation. The potential to carry out gas-phase IMR kinetics experiments is also illustrated. We demonstrate the versatility of the setup using gas-phase IMRs of established utility for biological mass spectrometry, including hydrogen-deuterium exchange, ion-molecule proton transfer reactions, and covalent modification of DNA anions using trimethylsilyl chloride.

Modelling the effect of season, sex, and body size on the three-spined stickleback, Gasterosteus aculeatus, cellular innate immunomarkers: A proposition of laboratory reference ranges.

Innate immunomarkers reflect both environmental contamination and fish health status, providing useful information in environmental risk assessment studies. Nevertheless, the lack of knowledge about the effect of confounding factors can lead to data misinterpretation and false diagnoses. The aim of this study was to evaluate the impact of three confounding factors (season, sex and body size) on three-spined stickleback innate immunomarkers in laboratory conditions. Results shown strong seasonal variations in stickleback innate immunomarkers, with higher immune capacities in late winter-early spring and a disturbance during the spawning period in late spring-summer. Sex and body size had a season dependant effect on almost all tested immunomarkers. Reference ranges were established in laboratory-controlled conditions (i.e. laboratory reference ranges) and compared with data obtained from in vivo chemical expositions. The predictive power of the statistical model depended on the immunomarker, but the control data of the in vivo experiments, realized in same laboratory conditions, were globally well include in the laboratory reference ranges. Moreover, some statistical effects of the in vivo exposures were correlated with an augmentation of values outside the reference ranges, indicating a possible harmful effect for the organisms. As confounding factors influence is a major limit to integrate immunomarkers in biomonitoring programs, modelling their influence on studied parameter may help to better evaluated environmental contaminations.

Design, Synthesis, and Evaluation of 2,9-Bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline Derivatives as G-Quadruplex Ligands.

Genomic 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.