We describe here various ion activation experiments realized in the Omnitrap platform integrated on the timsOmni mass spectrometer for the analysis of oligonucleotides in the negative ion mode. The activation methods include resonance collision-induced dissociation (RCID), electron detachment dissociation (EDD), infrared laser multiple-photon activation (IRMPD) and UV laser photodissociation (UVPD) at 266 nm. Special emphasis is given to EDD, either as a standalone technique or in conjunction with vibrational reactivation of the ion radicals. We describe EDD on standard 6-mer DNA sequences that have been extensively characterized on other instruments, followed by a comparison of several activation approaches for the phosphorothioate-based oligonucleotide therapeutics Fomivirsen, and conclude with the fragmentation analysis of 46-mer DNA and RNA. EDD alone already provides excellent sequence information on short oligonucleotides such as Fomivirsen, but MS3 combinations such as EDD-RCID or EDD-IRMPD proved even more effective, including for the 46-mer DNA (less prone to fragmentation than RNA) at a relatively low charge states. The diversity of ion activation combinations available on the Omnitrap platform is demonstrated by MS4 experiments, which experimentally validate the formation of d ions from a• radical fragments, and w ions from z• radical fragments produced by EDD.

G-quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine-rich sequences that assemble into stacked guanine tetrads. These unusual RNA or DNA structures are implicated in essential regulatory processes in both human and viral genomes. In recent years, compounds that bind and stabilise G4s have emerged as promising antiviral agents against several viruses such as HIV-1, HCV or HPV. We report the design, synthesis, and biophysical characterization of a novel class of G4-stabilizing ligands based on a positively charged dibenzoacridinium (DBA) core. A combination of FRET-melting assays, fluorescence quenching assay (FQA), circular dichroism spectroscopy, native mass spectrometry, NMR titration, and molecular dynamics simulations confirmed that DBA ligands bind selectively to G4s, with thermal stabilization (ΔT m) values reaching +18 °C and dissociation constants (K d) as low as 0.14 μM. In cellular assays, DBA4 and DBA5 compounds exhibited antiviral activity against HIV-1, with IC50 values of 1 μM and 3 μM, respectively. These results identify DBAs as a new class of G-quadruplex ligands with anti-HIV properties.

Understanding how biomolecules acquire their charge and retain their solution conformation during electrospray ionization (ESI) is crucial for native mass spectrometry (native MS) interpretation. Here, we examine the charging and gas phase conformation of nucleic acid constructs comprising folded G-quadruplex "beads" linked by unstructured polythymine regions. Under physiological ionic strength, these oligonucleotides exhibit a multimodal charge-state and collision cross section distribution, revealing multiple conformational ensembles, in contrast to the unimodal profiles typically observed for shorter oligonucleotides. Native MS observations for intermediate charge states are compatible with ion production via the recently proposed bead-ejection scenario, in addition to the charge residue scenario for low charge states and chain ejection for the highest charge states or for sequences with thymine overhangs on both ends. The preservation of the local structures in ions charged above the Rayleigh limit helps to infer the presence of folded subunits. The position of the G-quadruplex subunit and ionic strength govern the charging and retention of G-quadruplex folded regions. Our findings broaden the existing conceptual framework underpinning nucleic acid ionization.

G-quadruplexes (G4s) are non-canonical DNA structures that form in guanine-rich genomic regions and play key roles in the regulation of gene expression. These structures are frequently located within promoter regions and often overlap CpG islands, making them particularly sensitive to epigenetic modifications that can influence their stability and biological function. The promoter of the oncogene c-KIT contains three G4-forming sequences; among them, c-KIT1 was selected as a model system because it includes cytosines positioned near guanines involved in G-tetrad formation, providing an ideal context to probe how local epigenetic changes influence G4 architecture. The aim of this study was to determine how specific cytosine modifications affect G4 structure, thermodynamic stability, and ligand interactions. To this end, c-KIT1 and six related variants bearing epigenetic modifications, specifically 5-methylcytosine or 5-carboxylcytosine at defined positions, were investigated. Mass spectrometry experiments confirmed that all modified sequences maintain a parallel G4 topology. Circular Dichroism Spectroscopy (CD) and Differential Scanning Calorimetry (DSC) were then employed to evaluate G4 formation and the thermodynamic stability of all investigated G4s. To determine whether epigenetic cytosine modifications influence G4-ligand interactions, CD melting experiments were also performed in the presence of a panel of HDAC inhibitors. Among the tested compounds, one ligand produced the most pronounced increase in G4 thermal stability, identifying it as a promising candidate for selective G4 targeting.

Elucidating how biomolecules acquire charge and retain their solution conformation during electrospray ionization (ESI) is crucial for native MS analysis. Here, we examine the charging and gas phase conformation of nucleic acid constructs comprising folded G-quadruplex “beads” linked by unstructured polythymine regions. Under physiological ionic strength, these oligonucleotides exhibit a multimodal charge-state and collision cross section distribution, revealing multiple conformational ensembles, in contrast to the unimodal profiles typically observed for shorter oligonucleotides. The ion mobility results for intermediate charge states are compatible with ion production via the recently proposed bead ejection mechanism (BEM), in addition to the charge residue model (CRM) for low charge states and chain ejection model (CEM) for the highest charge states or for sequences with thymine overhangs on both ends. The position of the G-quadruplex domain and ionic strength governs the charging and retention of G-quadruplex folded regions. Our findings broaden the existing mechanistic framework underpinning nucleic acid ionization. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, ANR-18-CE29-0013-POLYnESI Conseil Régional de Nouvelle-Aquitaine, PROMIS

[This corrects the article DOI: 10.1021/jacsau.3c00550.].

BackgroundThe use of simple and hybrid fragmentation techniques for the identification of molecules in tandem mass spectrometry provides different and complementary information on the structure of molecules. Nevertheless, these techniques have not been as widely explored for oligonucleotides as for peptides or proteins. The analysis of microRNAs (miRNAs) warrants special attention, given their regulatory role and their relationship with several diseases. The application of different fragmentation techniques will be very interesting for their identification.ResultsFour synthetic miRNAs and a DNA sequence were fragmented in an ESI-FT-ICR mass spectrometer using both simple and hybrid fragmentation techniques: CID, nETD followed by CID, IRMPD, and, for the first time, nETD in combination with IRMPD. The main fragmentation channel was base loss. The use of nETD-IRMPD resulted in d/z, a/w, and c/y ions at higher intensities. Moreover, nETD-IRMPD provided high sequence coverage and low internal fragmentation. Native MS analysis revealed that only miR159 and the DNA sequence formed stable dimers under physiological ionic strength. The use of organic co-solvents or additives resulted in a lower sequence coverage due to lesser overall ionization efficiency.NoveltyThis work demonstrates that the combination of nETD and IRMPD for miRNA fragmentation constitutes a suitable alternative to common fragmentation methods. This strategy resulted in efficient fragmentation of [miRNA]5- using low irradiation times and fewer internal fragments while ensuring a high sequence coverage. Moreover, given that such low charge states predominate upon spraying in physiological-like conditions, native MS can be applied for obtaining structural information at the same time.

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched alpha 2,3-disialylated core 2 O-glycans: a selected conformation of the 3 ' SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections.

Native ion mobility mass spectrometry is potentially useful for the biophysical characterization of proteins, as the electrospray charge state distribution and the collision cross section distribution depend on their solution conformation. We examine here the charging and gas-phase conformation of multi-domain therapeutic proteins comprising globular domains tethered by disordered linkers. The charge and collision cross section distributions are multimodal, suggesting several conformations in solution, as confirmed by solution hydrogen/deuterium exchange. The most intriguing question is the ionization mechanism of these structures: a fraction of the population does not follow the charged residue mechanism but cannot ionize by pure chain ejection either. We deduce that a hybrid mechanism is possible, wherein globular domains are ejected one at a time from a parent droplet. The charge vs solvent accessible surface area correlations of denatured and intrinsically disordered proteins are also compatible with this "bead ejection mechanism", which we propose as a general tenet of biomolecule electrospray.

We prepared a series of water-soluble aromatic oligoamide sequences all composed of a segment prone to form a single helix and a segment prone to dimerize into a double helix. These sequences exclusively assemble as antiparallel duplexes. The modification of the duplex inner rim by varying the nature of the substituents borne by the aromatic monomers allowed us to identify sequences that can hybridize by combining two chemically different strands, with high affinity and complete selectivity in water. X-ray crystallography confirmed the expected antiparallel configuration of the duplexes whereas NMR spectroscopy and mass spectrometry allowed us to assess precisely the extent of the hybridization. The hybridization kinetics of the aromatic strands was shown to depend on both the nature of the substituents responsible for strand complementarity and the length of the aromatic strand. These results highlight the great potential of aromatic hetero-duplex as a tool to construct non-symmetrical dynamic supramolecular assemblies.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Eight [Ru(bpy)2L]2+ and three [Ru(phen)2L]2+complexes (where bpy = 2,2'-bipyridine and phen = 1,10-phenanthroline are ancillary ligands, and L = a polypyridyl experimental ligand) were investigated for their G-quadruplex binding abilities. Fluorescence resonance energy transfer melting assays were used to screen these complexes for their ability to selectively stabilize human telomeric DNA variant, Tel22. The best G-quadruplex stabilizers were further characterized for their binding properties (binding constant and stoichiometry) using UV-vis, fluorescence spectroscopy, and mass spectrometry. The ligands' ability to alter the structure of Tel22 was determined via circular dichroism and PAGE studies. We identified me2allox as the experimental ligand capable of conferring excellent stabilizing ability and good selectivity to polypyridyl Ru(II) complexes. Replacing bpy by phen did not significantly impact interactions with Tel22, suggesting that binding involves mostly the experimental ligand. However, using a particular ancillary ligand can help fine-tune G-quadruplex-binding properties of Ru(II) complexes. Finally, the fluorescence "light switch" behavior of all Ru(II) complexes in the presence of Tel22 G-quadruplex was explored. All Ru(II) complexes displayed "light switch" properties, especially [Ru(bpy)2(diamino)]2+, [Ru(bpy)2(dppz)]2+, and [Ru(bpy)2(aap)]2+. Current work sheds light on how Ru(II) polypyridyl complexes interact with human telomeric DNA with possible application in cancer therapy or G-quadruplex sensing.

Amyloid aggregation of the intrinsically disordered protein (IDP) tau is involved in several diseases, called tauopathies. Some tauopathies can be inherited due to mutations in the gene encoding tau, which might favor the formation of tau amyloid fibrils. This work aims at deciphering the mechanisms through which the disease-associated single-point mutations promote amyloid formation. We combined biochemical and biophysical characterization, notably, small-angle X-ray scattering (SAXS), to study six different FTDP-17 derived mutations. We found that the mutations promote aggregation to different degrees and can modulate tau conformational ensembles, intermolecular interactions, and liquid-liquid phase separation propensity. In particular, we found a good correlation between the aggregation lag time of the mutants and their radii of gyration. We show that mutations disfavor intramolecular protein interactions, which in turn favor extended conformations and promote amyloid aggregation. This work proposes a new connection between the structural features of tau monomers and their propensity to aggregate, providing a novel assay to evaluate the aggregation propensity of IDPs.

When sprayed from physiological ionic strength, nucleic acids typically end up with low levels of charging and in compact conformations. Increasing the electrospray negative charging of nucleic acids while preserving the native non-covalent interactions can help distinguish solution folds by ion mobility mass spectrometry. To get fundamental insight into the supercharging mechanisms of nucleic acids in the negative mode, we studied model G-quadruplex structures and single strand controls in 100 mM ammonium acetate. We found that adding 0.4% propylene carbonate, 0.4% sulfolane or 0.1% m -NBA induces native supercharging through the charged residue mechanism. However, although 0.4% m -NBA shows the highest supercharging ability, it induces unwanted unfolding of solution-folded G-quadruplexes. The supercharging effect resembles the effect of lowering the ionic strength, which could be explained by partial neutralization of the ampholytes when droplets become more concentrated in their non-aqueous components. The supercharging ability ranks: PC < sulfolane < m -NBA. m -NBA adducts to G-quadruplexes with high charge states confirms that the supercharging agent interacts directly with DNA. Surprisingly, in presence of supercharging agents, more negative charge states also bear more alkali metal ion adducts. This suggests that native supercharging results from larger droplets evaporating to the charged residue, leading to higher concentration of both the supercharging agent and of alkali counterions. However, when negative charge carriers from the electrolyte become too rare, chain ejection accompanied by denaturation, and hence non-native supercharging, can become predominant.

Amphipathic water-soluble helices formed from synthetic peptides or foldamers are promising building blocks for the creation of self-assembled architectures with non-natural shapes and functions. While rationally designed artificial quaternary structures such as helix bundles have been shown to contain preformed cavities suitable for guest binding, there are no examples of adaptive binding of guest molecules by such assemblies in aqueous conditions. We have previously reported a foldamer 6-helix bundle that contains an internal nonpolar cavity able to bind primary alcohols as guest molecules. Here, we show that this 6-helix bundle can also interact with larger, more complex guests such as n-alkyl glycosides. X-ray diffraction analysis of co-crystals using a diverse set of guests together with solution and gas-phase studies reveals an adaptive binding mode whereby the apo form of the 6-helix bundle undergoes substantial conformational change to accommodate the hydrocarbon chain in a manner reminiscent of glycolipid transfer proteins in which the cavity forms upon lipid uptake. The dynamic nature of the self-assembling and molecular recognition processes reported here marks a step forward in the design of functional proteomimetic molecular assemblies.

Glycosyl conjugation to drugs is a strategy being used to take advantage of glucose transporters (GLUT) overexpression in cancer cells in comparison with non-cancerous cells. Its extension to the conjugation of drugs to thiosugars tries to exploit their higher biostability when compared to O-glycosides. Here, we have synthesized a series of thiosugar naphthalene diimide conjugates as G-quadruplex ligands and have explored modifications of the amino sidechain comparing dimethyl amino and morpholino groups. Then, we studied their antiproliferative activity in colon cancer cells, and their antiparasitic activity in T. brucei and L. major parasites, together with their ability to bind quadruplexes and their cellular uptake and location. We observed higher toxicity for the sugar-NDI-NMe2 derivatives than for the sugar-NDI-morph compounds, both in mammalian cells and in parasites. Our experiments indicate that a less efficient binding to quadruplexes and a worse cellular uptake of the carb-NDI-morph derivatives could be the reasons for these differences. We found small variations in cytotoxicity between O-carb-NDIs and S-carb-NDIs, except against non-cancerous human fibroblasts MRC-5, where thiosugar-NDIs tend to be less toxic. This leads to a notable selectivity for β-thiomaltosyl-NDI-NMe2 12 (9.8 fold), with an IC50 of 0.3 μM against HT-29 cells. Finally, the antiparasitic activity observed for the carb-NDI-NMe2 derivatives against T. brucei was in the nanomolar range with a good selectivity index in the range of 30- to 69- fold.

When sprayed from physiological ionic strength, nucleic acids typically end up with low levels of charging and in compact conformations. Increasing the electrospray negative charging of nucleic acids while preserving the native noncovalent interactions can help distinguish solution folds by ion mobility mass spectrometry. To get fundamental insight into the supercharging mechanisms of nucleic acids in the negative mode, we studied model G-quadruplex structures and single-strand controls in 100 mM ammonium acetate. We found that adding 0.4% of propylene carbonate, 0.4% of sulfolane, or 0.1% of m-NBA induces native supercharging. However, although 0.4% of m-NBA shows the highest supercharging ability, it induces unwanted unfolding of solution-folded G-quadruplexes. The supercharging effect resembles the effect of lowering the ionic strength, and this could be explained by partial neutralization of the ampholytes when droplets become more concentrated in their nonaqueous components. The supercharging ability ranks PC < sulfolane < m-NBA. m-NBA adducts to G-quadruplexes with high-charge states confirm that the supercharging agent interacts directly with DNA. Surprisingly, in the presence of supercharging agents, the most negatively-charged states also bear more alkali metal ion adducts. Larger droplets are known to result in more counterion adduction, so our results are consistent with native supercharging conditions producing larger droplets evaporating to a charged residue. However, when negative charge carriers from the electrolyte become too rare, chain ejection accompanied by denaturation, and hence non-native supercharging, can become predominant.

Nucleic acids have been among the first targets for antitumor drugs and antibiotics. 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 assess target structure as well as ligand binding stoichiometry, affinity, specificity, and binding modes are part of the drug development process. Mass spectrometry offers unique advantages as a biophysical method owing 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 noncovalent 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.

Drift tube ion mobility spectrometry (DTIMS) coupled with mass spectrometry was used to determine the collision cross-sections (DTCCS) of polyoxometalate anions in helium and nitrogen. As the geometry of the ion, more than its mass, determines the collision cross-section with a given drift gas molecule, we found that both Lindqvist ions Mo6O192- and W6O192- had a DTCCSHe value of 103 ± 2 Å2, and both Keggin ions PMo12O403- and PW12O403- had a DTCCSHe value of 170 ± 2 Å2. Similarly, ion mobility experiments in N2 led to DTCCSN2 values of 223 ± 2 Å2 and 339 ± 4 Å2 for Lindqvist and Keggin anions, respectively. Using optimized structures and partial charges determined from density functional theory calculations, followed by CCS calculations via the trajectory method, we determined Lennard-Jones 6-12 potential parameters ε, σ of 5.60 meV, 3.50 Å and 3.75 meV, 4.40 Å for both Mo and W atoms interacting with He and N2, respectively. These parameters reproduced the CCS of polyoxometalates within 2% accuracy.

The combination between ion mobility mass spectrometry and molecular dynamics simulations is demonstrated for the first time to afford valuable information on structural changes undergone by dendriplexes containing ds-DNA and low-generation dendrimers when transferred from the solution to the gas phase. Dendriplex ions presenting 1:1 and 2:1 stoichiometries are identified using mass spectrometry experiments, and the collision cross sections (CCS) of the 1:1 ions are measured using drift time ion mobility experiments. Structural predictions using Molecular Dynamics (MD) simulations showed that gas-phase relevant structures, i.e., with a good match between the experimental and theoretical CCS, are generated when the global electrospray process is simulated, including the solvent molecule evaporation, rather than abruptly transferring the ions from the solution to the gas phase. The progressive migration of ammonium groups (either NH4+ from the buffer or protonated amines of the dendrimer) into the minor and major grooves of DNA all along the evaporation processes is shown to compact the DNA structure by electrostatic and hydrogen-bond interactions. The subsequent proton transfer from the ammonium (NH4+ or protonated amino groups) to the DNA phosphate groups allows creation of protonated phosphate/phosphate hydrogen bonds within the compact structures. MD simulations showed major structural differences between the dendriplexes in solution and in the gas phase, not only due to the loss of the solvent but also due to the proton transfers and the huge difference between the solution and gas-phase charge states.