Search

Human telomeric G-quadruplex DNA structures are attractive anticancer drug targets, but the target's polymorphism complicates the drug design: different ligands prefer different folds, and very few complexes have been solved at high resolution. Here we report that Phen-DC3, one of the most prominent G-quadruplex ligands in terms of high binding affinity and selectivity, causes dTAGGG(TTAGGG)3 to completely change its fold in KCl solution from a hybrid-1 to an antiparallel chair-type structure, wherein the ligand intercalates between a two-quartet unit and a pseudo-quartet, thereby ejecting one potassium ion. This unprecedented high-resolution NMR structure shows for the first time a true ligand intercalation into an intramolecular G-quadruplex.

While studying nucleic acids to reveal the weak interactions responsible for their three-dimensional structure and for their interactions with drugs, we also contributed to the field of biomolecular mass spectrometry, both in terms of fundamental understanding and with new methodological developments. A first goal was to develop mass spectrometry approaches to detect non-covalent interactions between antitumor drugs and their DNA target. Twenty years ago, our attention turned towards specific DNA structures such as the G-quadruplex (a structure formed by guanine-rich strands). Mass spectrometry allows to discern which molecules interact with one another by measuring the masses of the complexes, and quantity the affinities by measuring their abundance. The most important findings came from unexpected masses. For example, we showed the formation of higher- or lower-order structures by G-quadruplexes used in traditional biophysical assays. We can also derive complete thermodynamic and kinetic description of G-quadruplex folding pathways by measuring cation binding, one at a time. Getting quantitative information required accounting for nonspecific adduct formation and for the response factors of the different molecular forms. But with these caveats in mind, the approach is mature enough for routine biophysical characterization of nucleic acids. A second goal is to obtain more detailed structural information on each of the complexes separated by the mass spectrometer. One such approaches is ion mobility spectrometry, and even today the challenge lies in the structural interpretation of the measurements. We showed that, although structures such as G-quadruplexes are well-preserved in the MS conditions, double helices actually get more compact in the gas phase. These major rearrangements forced us to challenge comfortable assumptions. Further work is still needed to generalize how to deduce structures in solution from ion mobility spectrometry data, and in particular how to account for the electrospray charging mechanisms and for ion internal energy effects. These studies also called for complementary approaches to ion mobility. Recently, we applied isotope exchange labeling mass spectrometry to characterize nucleic acid structures for the first time, and we reported the first ever circular dichroism ion spectroscopy measurement on mass-selected trapped ions. Circular dichroism plays a key role in assigning the stacking topology, and our new method now opens the door to characterizing a wide variety of chiral molecule by mass spectrometry. In summary, advanced mass spectrometry approaches to characterize gas-phase structures work well for G-quadruplexes because they are stiffened by inner cations. The next objective will be to generalize these methodologies to a wider range of nucleic acid structures.

DNA and proteins are chiral: their three-dimensional structure cannot be superimposed with its mirror image. Circular dichroism spectroscopy is widely used to characterize chiral compounds, but data interpretation is difficult in the case of mixtures. We recorded for the first time the electronic circular dichroism spectra of DNA helices separated in a mass spectrometer. We electrosprayed guanine-rich strands having various secondary structures as negative ions, irradiated them with a laser, and measured the difference in electron photodetachment efficiency between left and right circularly polarized light. The reconstructed circular dichroism ion spectra resemble the solution ones, thereby allowing us to assign the DNA helical topology. The ability to measure circular dichroism directly on biomolecular ions expands the capabilities of mass spectrometry for structural analysis., Comment: main text (7 pages, 2 figures) + supporting information (20 pages, 11 figures)

In solution, UV-vis spectroscopy is often used to investigate structural changes in biomolecules (i.e., nucleic acids), owing to changes in the environment of their chromophores (i.e., the nucleobases). Here we address whether action spectroscopy could achieve the same for gas-phase ions, while taking the advantage of additional spectrometric separation of complex mixtures. We therefore systematically studied the action spectroscopy of homo-base 6-mer DNA strands (dG6, dA6, dC6, dT6) and discuss the results in light of gas-phase structures validated by ion mobility spectrometry and infrared ion spectroscopy, of electron binding energies measured by photoelectron spectroscopy, and of calculated electronic photo-absorption spectra. When UV photons interact with oligonucleotide polyanions, two main actions may take place: (1) fragmentation and (2) electron detachment. The action spectra reconstructed from fragmentation follow the absorption spectra well, and result from multiple cycles of absorption and internal conversion. The action spectra reconstructed from the electron photodetachment (ePD)

Phosphorylation is one of the most important protein modifications, and electron-transfer dissociation tandem mass spectrometry (ETD-MS/MS) is a potentially useful method for the sequencing of phosphopeptides, including determination of the phosphorylation site. Notably, ETD-MS/MS typically provides useful information when the precursor contains more than three positive charges. It is not yet used as an analysis method for large-scale phosphopeptide production due to difficulties occurring in the production of acidic phosphopeptides having more than three positive charges. To increase the charge state of phosphopeptides, we used dinuclear metal complexes, which selectively bind to the phosphate group in phosphopeptides with the addition of positive charge(s). Dinuclear copper, zinc, and gallium complexes were tested and it was found that the type of metal present in the complex strongly affected the affinity of the phosphorylated compounds and their ETD fragmentation. The dinuclear copper complex interacted weakly with the phosphate groups and ETD-induced peptide fragmentation was largely suppressed by the presence of Cu2+, which worked as an electron trap. The dinuclear gallium complex was strongly bound to a phosphate group. However, the ligand binding to gallium acted as an electron trap and the presence of dinuclear gallium complex in the precursor for ETD-MS/MS hampered the sequencing of the phosphopeptides, as in the case of dinuclear copper complexes. In contrast, dinuclear zinc complexes efficiently bind to phosphopeptides with an increase in the charge state, facilitating phosphopeptide sequencing by ETD-MS/MS. The fragmentation of the ligand and peptide backbone in the dinuclear zinc--phosphopeptide complex were competitively induced by ETD. These processes are influenced by the ligand structure and so the detailed ETD fragmentation pathways were investigated using density functional theory calculations.

Hydrogen bonding between nucleobases produces diverse DNA structural motifs, including canonical duplexes, guanine (G) quadruplexes and cytosine (C) i-motifs. Incorporating metal-mediated base pairs into nucleic acid structures can introduce new functionalities and enhanced stabilities. Here we demonstrate, using mass spectrometry (MS), ion mobility spectrometry (IMS) and fluorescence resonance energy transfer (FRET), that parallel-stranded structures consisting of up to 20 G-Ag(I)-G contiguous base pairs are formed when natural DNA sequences are mixed with silver cations in aqueous solution. FRET indicates that duplexes formed by poly(cytosine) strands with 20 contiguous C-Ag(I)-C base pairs are also parallel. Silver-mediated G-duplexes form preferentially over G-quadruplexes, and the ability of Ag+ to convert G-quadruplexes into silver-paired duplexes may provide a new route to manipulating these biologically relevant structures. IMS indicates that G-duplexes are linear and more rigid than B-DNA. DFT calculations were used to propose structures compatible with the IMS experiments. Such inexpensive, defect-free and soluble DNA-based nanowires open new directions in the design of novel metal-mediated DNA nanotechnology., Comment: 42 pages (12 pages manuscript, 30 pages supporting information). Changes in version 3: various minor revisions in text; Figure 1 replaced

The meaningful comparison of ion mobility (IM) results and of collision cross section (CCS) values on different platforms is a prerequisite for using CCS for identification or structural assignment. The amount of internal energy imparted to the ions prior to the ion mobility cell is a source of experimental variation. Here we investigated the effects of virtually all tuning parameters of the Agilent 6560 IM-Q-TOF on the arrival time distributions of Ubiquitin7+, and found conditions in which the native state prevails. We will discuss the effects of solvent evaporation conditions in the source, in the entire pre-IM DC voltage gradient, and with the funnel RF amplitudes, and will also report on ubiquitin7+ conformations in different solvents, including native supercharging conditions. Collision-induced unfolding (CIU) can be conveniently provoked in two distinct regions: behind the source capillary (by changing the fragmentor voltage) and in the trapping funnel (by changing the trap entrance grid delta voltage). The softness of the instrumental conditions were then optimized with the benchmark DNA G-quadruplex [(dG4T4G4)2.(NH4+)3-8H]5-, for which ion activation results in ammonia loss. To reduce the ion internal energy and obtain the intact 3-NH4+ complex, we reduced the post-IM voltage gradient, but this resulted in a lower IM resolving power due to increased diffusion behind the drift tube. The article thus describes the various trade-offs between ion activation, ion transmission, and ion mobility performance for native MS of very fragile structures., Comment: 25 pages including 12 figures

Fundamental questions in ion mobility spectrometry have practical implications for analytical applications in general, and omics in particular, in three respects. (1) Understanding how ion mobility and collision cross section values depend on the collision gas, on the electric field and on temperature is crucial to ascertain their transferability across instrumental platforms. (2) Predicting collision cross section values for new analytes is necessary to exploit the full potential of ion mobility in discovery workflows. (3) Finally, understanding the fate of ion structures in the gas phase is essential to infer meaningful information on solution structures based on gas-phase ion mobility measurements. We review here the most recent advances in ion mobility fundamentals, relevant to these three aspects., Comment: 22 pages, 3 figures and 3 tables. Compared to the published version and to the previous arXiv version, an error in Eq. (1) and an error in the equation two lines below have been corrected