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.

Application in a biomonitoring context of three-spined stickleback immunomarker reference ranges.

The relevance of a biomarker for biomonitoring programs was influenced both by the knowledge on biomarker natural inter-individual and site variabilities and by the sensitivity of the biomarker towards environmental perturbations. To minimize data misinterpretation, robustness reference values for biomarkers were important in biomonitoring programs. Specific three-spined stickleback, Gasterosteus aculeatus, immune reference ranges for field studies had been determined based on laboratory data and one reference station (Contentieuse river at Houdancourt). In this study, data obtained in one uncontaminated and three contaminated sites were compared to these reference ranges as a validation step before considering them for larger scale biomonitoring programs. When the field reference range were compared to data from the uncontaminated station (Béronelle), only few deviations were shown. In this way, data coming from uncontaminated station (Béronelle) was integrated in the field reference ranges to improve the evaluation of site variability. The new field reference ranges provided better discrimination of sites and spanned a larger range of fish lengths than the initial reference ranges. Furthermore, the results suggest lysosomal presence during several months and phagocytosis capacity in autumn may be the most relevant immunomarkers towards identifying contaminated sites. In the future, combining this reference value approach with active biomonitoring could facilitate the obtention of data in multiple stream conditions.

Novel Insight into Proximal DNA Domain Interactions from Temperature-Controlled Electrospray Ionization Mass Spectrometry.

Quadruplexes are non-canonical nucleic acid structures essential for many cellular processes. Hybrid quadruplex-duplex oligonucleotide assemblies comprised of multiple domains are challenging to study with conventional biophysical methods due to their structural complexity. Here, we introduce a novel method based on native mass spectrometry (MS) coupled with a custom-built temperature-controlled nanoelectrospray ionization (TCnESI) source designed to investigate interactions between proximal DNA domains. Thermal denaturation experiments were aimed to study unfolding of multi-stranded oligonucleotide constructs derived from biologically relevant structures and to identify unfolding intermediates. Using the TCnESI MS, we observed changes in Tm and thermodynamic characteristics of proximal DNA domains depending on the number of domains, their position, and order in a single experiment.

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.

Probing ligand and cation binding sites in G-quadruplex nucleic acids by mass spectrometry and electron photodetachment dissociation sequencing.

Mass spectrometry provides exquisite details on ligand and cation binding stoichiometries with a DNA target. The next important step is to develop reliable methods to determine the cation and ligand binding sites in each complex separated by using a mass spectrometer. To circumvent the caveat of ligand derivatization for cross-linking, which may alter the ligand binding mode, we explored a tandem mass spectrometry (MS/MS) method that does not require ligand derivatization, and is therefore also applicable to localize metal cations. By putting more negative charge states on the complexes using supercharging agents, and by creating radical ions by electron photodetachment, oligonucleotide bonds become weaker than the DNA-cation or DNA-ligand noncovalent bonds upon collision-induced dissociation of the radicals. This electron photodetachment (EPD) method allows one to locate the binding regions of cations and ligands by top-down sequencing of the oligonucleotide target. The very potent G-quadruplex ligands 360A and PhenDC3 were found to replace a potassium cation and bind close to the central loop of 4-repeat human telomeric sequences.

Impact of confinement and food access restriction on the three-spined stickleback (Gasterosteus aculeatus, L.) during caging: a multi-biomarker approach.

Caging is an active biomonitoring strategy that employs a sentinel species, sometimes a species naturally absent from the studied site, in the surveillance of water bodies to verify whether biota may be at risk. The main advantage of caging is the possibility to standardize several biotic and abiotic parameters. However, little knowledge is available about the effects of confinement on physiology and metabolism of caged organisms. The aim of this study is to characterize confinement and food access restriction effects, induced via caging experiments using a multi-biomarker approach (biometric data, immunity, antioxidant, metabolic detoxication, and digestive enzymes). The study has been undertaken using the same experiment conducted in ecosystem conditions using three-spined stickleback (Gasterosteus aculeatus) during two different periods: one in April, corresponding to breeding season, and the other in October, outside breeding season. Fifteen fish were maintained for 21 days in different conditions (caged or uncaged and with or without food supply). The main result was that confinement stress had little impact on the biological markers of sticklebacks. However, the stressors seemed to increase the negative effects of food restriction on these biomarkers, when sticklebacks needed more energy, that is, during their breeding period. Outside breeding period, most investigated biomarkers were not impacted by caging. This study showed a way to specify the conditions of application and interpretation of biomarkers during active monitoring to ensure an effective, reliable diagnosis of water body quality.

Thermal Denaturation of DNA G-Quadruplexes and Their Complexes with Ligands: Thermodynamic Analysis of the Multiple States Revealed by Mass Spectrometry.

Designing ligands targeting G-quadruplex nucleic acid structures and affecting cellular processes is complicated because there are multiple target sequences and some are polymorphic. Further, structure alone does not reveal the driving forces for ligand binding. To know why a ligand binds, the thermodynamics of binding must be characterized. Electrospray mass spectrometry enables one to detect and quantify each specific stoichiometry (number of strands, cations, and ligands) and thus to simultaneously determine the equilibrium constants for each complex. Using a temperature-controlled nanoelectrospray source, we determined the temperature dependence of the equilibrium constants, and thus the enthalpic and entropic contributions to the formation of each stoichiometry. Enthalpy drives the formation of each quartet-K+-quartet unit, whereas entropy drives the formation of quartet-K+-triplet units. Consequently, slip-stranded structures can become more abundant as the temperature increases. In the presence of ligands (Phen-DC3, TrisQ, TMPyP4, Cu-ttpy), we observed that, even when only a 1:1 (ligand/quadruplex) complex is observed at room temperature, new states are populated at intermediate temperatures, including 2:1 complexes. In most cases, ligand-G4-quadruplex binding is entropically driven, and we discuss that this may have resulted from biases when ranking ligand potency using melting experiments. Other thermodynamic profiles could be linked to topology changes in terms of number of G-quartets (reflected in the number of specific K+ ions in the complex). The thermodynamics of ligand binding to each form, one ligand at a time, provides unprecedented detail on the interplay between ligand binding and topology changes in terms of number of G-quartets.

Analysis of Interactions between Telomeric i-Motif DNA and a Cyclic Tetraoxazole Compound.

The interaction of a macrocyclic tetraoxazole compound, L2H2-4OTD (1), with two aminoalkyl side chains and telomeric i-motif, was investigated by means of electrophoretic mobility shift assay, circular dichroism spectroscopy, mass spectrometry and NMR spectroscopy analyses. The results indicate that 1 interacts with the i-motif structure at two preferred binding sites.

Folding and misfolding pathways of G-quadruplex DNA.

G-quadruplexes adopt various folding topologies, but information on their folding pathways remains scarce. Here, we used electrospray mass spectrometry to detect and quantify the specifically bound potassium ions, and circular dichroism to characterize the stacking topology of each ensemble. For human telomeric (hTel) sequences containing the d((GGGTTA)3GGG) core, K+ binding affinity and cooperativity strongly depends on the chosen construct. The shortest sequences bind only one K+ at low KCl concentration, and this 2-quartet G-quadruplex is antiparallel. Flanking bases increase the K+ binding cooperativity. To decipher the folding pathways, we investigated the kinetics of K+ binding to telomeric (hybrid) and c-myc (parallel) G-quadruplexes. G-quadruplexes fold via branched pathways with multiple parallel reactions. Up to six states (one ensemble without K+, two ensembles with 1-K+ and three ensembles with 2-K+) are separated based on their formation rates and ion mobility spectrometry. All G-quadruplexes first form long-lived misfolded structures (off-pathway compared to the most stable structures) containing one K+ and two quartets in an antiparallel stacking arrangement. The results highlight the particular ruggedness of G-quadruplex nucleic acid folding landscapes. Misfolded structures can play important roles for designing artificial G-quadruplex based structures, and for conformational selection by ligands or proteins in a biological context.

Drift Tube Ion Mobility: How to Reconstruct Collision Cross Section Distributions from Arrival Time Distributions?

Ion mobility spectrometry allows one to determine ion collision cross sections, which are related to ion size and shape. Collision cross sections (CCS) are usually discussed based on the peak center, yet the width of each peak contains further information on the distribution of collision cross sections of each conformational ensemble. Here, we analyze how to convert arrival time distributions (ATD) to CCS distributions (CCSD). With a calibration curve taking into account the CCS dependence of the time spent outside the mobility region, one can reconstruct CCS distributions with correct peak center values. However, the peak widths are incorrectly rendered because ion diffusion, which affects the peak width in the time domain, is irrelevant to collision cross sections. For drift tube ion mobility, we describe a new method, coined "FWHMstep", using a step-field experiment and processing the peak's full width at half-maximum to reconstruct CCSDs. The width of the CCS distribution helps to characterize the analyte's structural heterogeneity, and/or its flexibility (i.e., the variety of ways the analyte ions can rearrange following electrospray into kinetically stable gas-phase conformations).

What stoichiometries determined by mass spectrometry reveal about the ligand binding mode to G-quadruplex nucleic acids.

G-quadruplexes (G4s) have become important drug targets to regulate gene expression and telomere maintenance. Many studies on G4 ligand binding focus on determining the ligand binding affinities and selectivities. Ligands, however, can also affect the G4 conformation. Here we explain how to use electrospray ionization mass spectrometry (ESI-MS) to monitor simultaneously ligand binding and cation binding stoichiometries. The changes in potassium binding stoichiometry upon ligand binding hint at ligand-induced conformational changes involving a modification of the number of G-quartets. We investigated the interaction of three quadruplex ligands (PhenDC3, 360A and Pyridostatin) with a variety of G4s. Electrospray mass spectrometry makes it easy to detect K+ displacement (interpreted as quartet disruption) upon ligand binding, and to determine how many ligand molecules must be bound for the quartet opening to occur. The reasons for ligand-induced conversion to antiparallel structures with fewer quartets are discussed. Conversely, K+ intake (hence quartet formation) was detected upon ligand binding to G-rich sequences that did not form quadruplexes in 1mM K+ alone. This demonstrates the value of mass spectrometry for assessing not only ligand binding, but also ligand-induced rearrangements in the target sequence. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Evaluation of chlorpyrifos effects, alone and combined with lipopolysaccharide stress, on DNA integrity and immune responses of the three-spined stickleback, Gasterosteus aculeatus.

Organism immune defences might be weakened by pollutants, largely detected in aquatic ecosystems, leading to the facilitation for opportunistic pathogens to infect organisms. In this context, destabilization of fish non-specific immune parameters and erythrocyte DNA integrity was tested, on a model fish species, the three-spined stickleback (Gasterosteus aculeatus), after exposure to chlorpyrifos (CPF). Alone, pesticide exposure induced a genotoxic potential (chlorpyrifos at 1.75 and 0.88µg/L) in addition to a decrease in phagocytosis capacity and a stimulation of respiratory burst. Then, to mimic pathogenic infection, fish exposure to chlorpyrifos was combined with lipopolysaccharides (LPS) stress. In this second experiment, an increase of DNA damage was observed in fish exposed to a lower concentration of chlorpyrifos and LPS. Moreover, at the higher concentration of chlorpyrifos, an early destabilization of innate immunity was observed as suggested by the absence of an increase of lysosomal presence in fish injected with LPS. This study highlighted the usefulness of stress on stress responses to better understand the impact of contaminants on the organism's health.

Quadruplex Turncoats: Cation-Dependent Folding and Stability of Quadruplex-DNA Double Switches.

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.

Selective and Cooperative Ligand Binding to Antiparallel Human Telomeric DNA G-Quadruplexes.

The quest for ligands that specifically bind to particular G-quadruplex nucleic acid structures is particularly important to conceive molecules with specific effects on gene expression or telomere maintenance, or conceive structure-specific molecular probes. Using electrospray mass spectrometry in native conditions, we reveal a highly cooperative and selective 2:1 binding of Cu(II) -tolylterpyridine complexes to human telomeric G-quadruplexes. Circular dichroism and comparisons of affinities for different sequences reveal a marked preference for antiparallel structures with diagonal loops and/or wide-medium-narrow-medium groove-width order. The cooperativity is attributed to conformational changes in the polymorphic telomeric G-quadruplex sequences, which convert preferably into an antiparallel three-quartet topology upon binding of two ligands.

Ligand-induced conformational changes with cation ejection upon binding to human telomeric DNA G-quadruplexes.

The rational design of ligands targeting human telomeric DNA G-quadruplexes is a complex problem due to the structural polymorphism that these sequences can adopt in physiological conditions. Moreover, the ability of ligands to switch conformational equilibria between different G-quadruplex structures is often overlooked in docking approaches. Here, we demonstrate that three of the most potent G-quadruplex ligands (360A, Phen-DC3, and pyridostatin) induce conformational changes of telomeric DNA G-quadruplexes to an antiparallel structure (as determined by circular dichroism) containing only one specifically coordinated K(+) (as determined by electrospray mass spectrometry) and, hence, presumably only two consecutive G-quartets. Control ligands TrisQ, known to bind preferentially to hybrid than to antiparallel structures, and L2H2-6M(2)OTD, known not to disrupt the hybrid-1 structure, did not show such K(+) removal. Instead, binding of the cyclic oxazole L2H2-6M(2)OTD was accompanied by the uptake of one additional K(+). Also contrasting with telomeric G-quadruplexes, the parallel-stranded Pu24-myc G-quadruplex, to which Phen-DC3 is known to bind by end-stacking, did not undergo cation removal upon ligand binding. Our study therefore evidences that very affine ligands can induce conformational switching of the human telomeric G-quadruplexes to an antiparallel structure and that this conformational change is accompanied by removal of one interquartet cation.

Mass spectrometry and ion mobility spectrometry of G-quadruplexes. A study of solvent effects on dimer formation and structural transitions in the telomeric DNA sequence d(TAGGGTTAGGGT).

We survey here state of the art mass spectrometry methodologies for investigating G-quadruplexes, and will illustrate them with a new study on a simple model system: the dimeric G-quadruplex of the 12-mer telomeric DNA sequence d(TAGGGTTAGGGT), which can adopt either a parallel or an antiparallel structure. We will discuss the solution conditions compatible with electrospray ionisation, the quantification of complexes using ESI-MS, the interpretation of ammonium ion preservation in the complexes in the gas phase, and the use of ion mobility spectrometry to resolve ambiguities regarding the strand stoichiometry, or separate and characterise different structural isomers. We also describe that adding electrospray-compatible organic co-solvents (methanol, ethanol, isopropanol or acetonitrile) to aqueous ammonium acetate increases the stability and rate of formation of dimeric G-quadruplexes, and causes structural transitions to parallel structures. Structural changes were probed by circular dichroism and ion mobility spectrometry, and the excellent correlation between the two techniques validates the use of ion mobility to investigate G-quadruplex folding. We also demonstrate that parallel G-quadruplex structures are easier to preserve in the gas phase than antiparallel structures.

Unprecedented hour-long residence time of a cation in a left-handed G-quadruplex.

Cations are critical for the folding and assembly of nucleic acids. In G-quadruplex structures, cations can bind between stacked G-tetrads and coordinate with negatively charged guanine carbonyl oxygens. They usually exchange between binding sites and with the bulk in solution with time constants ranging from sub-millisecond to seconds. Here we report the first observation of extremely long-lived K+ and NH4 + ions, with an exchange time constant on the order of an hour, when coordinated at the center of a left-handed G-quadruplex DNA. A single-base mutation, that switched one half of the structure from left- to right-handed conformation resulting in a right-left hybrid G-quadruplex, was shown to remove this long-lived behaviour of the central cation.

Studying biomolecular folding and binding using temperature-jump mass spectrometry.

Characterizing folding and complex formation of biomolecules provides a view into their thermodynamics, kinetics and folding pathways. Deciphering kinetic intermediates is particularly important because they can often be targeted by drugs. The key advantage of native mass spectrometry over conventional methods that monitor a single observable is its ability to identify and quantify coexisting species. Here, we show the design of a temperature-jump electrospray source for mass spectrometry that allows one to perform fast kinetics experiments (0.16-32 s) at different temperatures (10-90 °C). The setup allows recording of both folding and unfolding kinetics by using temperature jumps from high to low, and low to high, temperatures. Six biological systems, ranging from peptides to proteins to DNA complexes, exemplify the use of this device. Using temperature-dependent experiments, the folding and unfolding of a DNA triplex are studied, providing detailed information on its thermodynamics and kinetics.