Solution-State Structure of a Long-Loop G-Quadruplex Formed Within Promoters of Plasmodium falciparum B var Genes.

We report the high-resolution NMR solution-state structure of an intramolecular G-quadruplex with a diagonal loop of ten nucleotides. The G-quadruplex is formed by a 34-nt DNA sequence, d[CAG3T2A2G3TATA2CT3AG4T2AG3T2], named UpsB-Q-1. This sequence is found within promoters of the var genes of Plasmodium falciparum, which play a key role in malaria pathogenesis and evasion of the immune system. The [3+1]-hybrid G-quadruplex formed under physiologically relevant conditions exhibits a unique equilibrium between two structures, both stabilized by base stacking and non-canonical hydrogen bonding. Unique equilibrium of the two closely related 3D structures originates from a North-South repuckering of deoxyribose moiety of residue T27 in the lateral loop. Besides the 12 guanines involved in three G-quartets, most residues in loop regions are involved in interactions at both G-quartet-loop interfaces.

Mass Spectrometry of Nucleic Acid Noncovalent Complexes.

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.

A General Framework to Interpret Hydrogen-Deuterium Exchange Native Mass Spectrometry of G-Quadruplex DNA.

G-quadruplexes (G4s) are secondary structures formed by guanine-rich oligonucleotides involved in various biological processes. However, characterizing G4s is challenging, because of their structural polymorphism. Here, we establish how hydrogen-deuterium exchange native mass spectrometry (HDX/MS) can help to characterize the G4 structures and dynamics in solution. We correlated the time range of G4 exchange to the number of guanines involved in the inner and outer tetrads. We also established relationships among exchange rates, numbers of tetrads and bound cations, and stability. The use of HDX/native MS allows for the determination of tetrads formed and assessment of G4 stability at a constant temperature. A key finding is that stable G4s exchange through local fluctuations (EX2 exchange), whereas less stable G4s also undergo exchange through partial or complete unfolding (EX1 exchange). Deconvolution of the bimodal isotope distributions resulting from EX1 exchange provides valuable insight into the kinetics of folding and unfolding processes and allows one to detect and characterize transiently unfolded intermediates, even if scarcely populated. HDX/native MS thus represents a powerful tool for a more comprehensive exploration of the folding landscapes of G4s.

Native Electrospray Ionization of Multi-Domain Proteins via a Bead Ejection Mechanism.

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.

Optimization of G-Quadruplex Ligands through a SAR Study Combining Parallel Synthesis and Screening of Cationic Bis(acylhydrazones).

G-quadruplexes (G4s), secondary structures adopted by guanine-rich DNA and RNA sequences, are implicated in numerous biological processes and have been suggested as potential drug targets. Accordingly, there is an increasing interest in developing high-throughput methods that allow the generation of congeneric series of G4-targeting molecules ("ligands") and investigating their interactions with the targets. We have developed an operationally simple method of parallel synthesis to generate "ready-to-screen" libraries of cationic acylhydrazones, a motif that we have previously identified as a promising scaffold for potent, biologically active G4 ligands. Combined with well-established screening techniques, such as fluorescence melting, this method enables the rapid synthesis and screening of combinatorial libraries of potential G4 ligands. Following this protocol, we synthesized a combinatorial library of 90 bis(acylhydrazones) and screened it against five different nucleic acid structures. This way, we were able to analyze the structure-activity relationships within this series of G4 ligands, and identified three novel promising ligands whose interactions with G4-DNAs of different topologies were studied in detail by a combination of several biophysical techniques, including native mass spectrometry, and molecular modeling.

DNA G-quadruplexes for native mass spectrometry in potassium: a database of validated structures in electrospray-compatible conditions.

G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually ∼100 mM) potassium, and report here their circular dichroism (CD), melting temperature (Tm), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM trimethylammonium acetate. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).

Adaptive Binding of Alkyl Glycosides by Nonpeptidic Helix Bundles in Water: Toward Artificial Glycolipid Binding Proteins.

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.

Negative Electrospray Supercharging Mechanisms of Nucleic Acid Structures.

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.

Native Mass Spectrometry and Nucleic Acid G-Quadruplex Biophysics: Advancing Hand in Hand.

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 noncovalent interactions between antitumor drugs and their DNA target. Twenty years ago, our attention turned toward specific DNA structures such as the G-quadruplex (a structure formed by guanine-rich strands). Mass spectrometry allows one to discern which molecules interact with one another by measuring the masses of the complexes, and quantify 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 also derived complete thermodynamic and kinetic description of G-quadruplex folding pathways by measuring cation binding, one at a time. Getting quantitative information requires accounting for nonspecific adduct formation and for the response factors of the different molecular forms. With these caveats in mind, the approach is now 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 approach 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 spectrometry. 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 molecules 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.

Lennard-Jones interaction parameters of Mo and W in He and N2 from collision cross-sections of Lindqvist and Keggin polyoxometalate anions.

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.

Phen-DC3 Induces Refolding of Human Telomeric DNA into a Chair-Type Antiparallel G-Quadruplex through Ligand Intercalation.

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.

Quadruplex DNA-guided ligand selection from dynamic combinatorial libraries of acylhydrazones.

Dynamic combinatorial libraries of acylhydrazones were prepared from diacylhydrazides and several cationic or neutral aldehydes in the presence of 5-methoxyanthranilic acid catalyst. Pull-down experiments with magnetic beads functionalized with a G-quadruplex (G4)-forming oligonucleotide led to the identification of putative ligands, which were resynthesized or emulated by close structural analogues. G4-binding properties of novel derivatives were assessed by fluorimetric titrations, mass spectrometry and thermal denaturation experiments, giving evidence of strong binding (Kd < 10 nM) for two compounds.

Design, synthesis, and antiproliferative effect of 2,9-bis[4-(pyridinylalkylaminomethyl)phenyl]-1,10-phenanthroline derivatives on human leukemic cells by targeting G-quadruplex.

Current multiagent chemotherapy regimens have improved the cure rate in acute leukemia patients, but they are highly toxic and poorly efficient in relapsed patients. To improve the treatment approaches, new specific molecules are needed. The G-quadruplexes (G4s), which are noncanonical nucleic acid structures found in specific guanine-rich DNA or RNA, are involved in many cellular events, including control of gene expression. G4s are considered as targets for the development of anticancer agents. Heterocyclic molecules are well known to target and stabilize G4 structures. Thus, a new series of 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives (1a-i) was designed, synthesized, and evaluated against five human myeloid leukemia cell lines (K562, KU812, MV4-11, HL60, and U937). Their ability to stabilize various oncogene promoter G4 structures (c-MYC, BCL-2, and K-RAS) as well as the telomeric G4 was also determined through the fluorescence resonance energy transfer melting assay and native mass spectrometry. In addition, the more bioactive ligands 1g-i were tested for telomerase activity in HuT78 and MV4-11 protein extracts.

Native Hydrogen/Deuterium Exchange Mass Spectrometry of Structured DNA Oligonucleotides.

Although solution hydrogen-deuterium exchange mass spectrometry (HDX/MS) is well-established for the analysis of the structure and dynamics of proteins, it is currently not exploited for nucleic acids. Here we used DNA G-quadruplex structures as model systems to demonstrate that DNA oligonucleotides are amenable to in-solution HDX/MS in native conditions. In trimethylammonium acetate solutions and in soft source conditions, the protonated phosphate groups are fully back-exchanged in the source, while the exchanged nucleobases remain labeled without detectable back-exchange. As a result, the exchange rates depend strongly on the secondary structure (hydrogen bonding status) of the oligonucleotides, but neither on their charge state nor on the presence of nonspecific adducts. We show that native mass spectrometry methods can measure these exchange rates on the second to the day time scale with high precision. Such combination of HDX with native MS opens promising avenues for the analysis of the structural and biophysical properties of oligonucleotides and their complexes.

Effective Temperature and Structural Rearrangement in Trapped Ion Mobility Spectrometry.

Modern ion mobility instrumentation is typically operated above the low field limit, which may activate the ions and cause structural rearrangement or fragmentation during analysis. Here, we quantitatively assessed the internal heating experienced by ions during trapped ion mobility spectrometry (TIMS) experiments. To this end, the fragmentation yields of fragile benzylpyridinium "thermometer" ions were monitored during both the accumulation and analysis steps inside the TIMS tunnel. The corresponding fragmentation rate constants were translated into a vibrational effective temperature Teff,vib. Our results demonstrate significant fragmentation upstream and inside the TIMS tunnel that corresponds to Teff,vib ≈ 510 K during both the accumulation and analysis steps. Broadening our scope to cytochrome c and lysozyme, we showed that although compact "native" folds can be preserved, the collision cross section distributions are highly sensitive to the transmission voltages and the analysis time scale. Our results are discussed with regard to Teff,vib data previously acquired on traveling-wave (TWIMS) ion mobility in the context of native mass spectrometry and conformational landscape exploration.

Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology.

Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic-to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here.

Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data.

The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.

Recommendations for reporting ion mobility Mass Spectrometry measurements.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

More is not always better: finding the right trade-off between affinity and selectivity of a G-quadruplex ligand.

Guanine-rich nucleic acid sequences can fold into four-stranded G-quadruplex (G4) structures. Despite growing evidence for their biological significance, considerable work still needs to be done to detail their cellular occurrence and functions. Herein, we describe an optimized core-extended naphthalene diimide (cex-NDI) to be exploited as a G4 light-up sensor. The sensing mechanism relies on the shift of the aggregate-monomer equilibrium towards the bright monomeric state upon G4 binding. In contrast with the majority of other ligands, this novel cex-NDI is able to discriminate among G4s with different topologies, with a remarkable fluorescent response for the parallel ones. We investigate this sensing by means of biophysical methods, comparing the lead compound to a non-selective analogue. We demonstrate that mitigating the affinity of the binding core for G4s results in an increased selectivity and sensitivity of the fluorescent response. This is achieved by replacing positively charged substituents with diethylene glycol (DEG) side chains. Remarkably, the limit of detection values obtained for parallel G4s are more than one order of magnitude lower than those of the parallel-selective ligand N-methyl mesoporphyrin IX (NMM). Interestingly, the classical fluorescent intercalator displacement (FID) assay failed to reveal binding of cex-NDI to G4 because of the presence a ternary complex (G4-TO-cex-NDI) revealed by electrospray-MS. Our study thus provides a rational basis to design or modify existent scaffolds to redirect the binding preference of G4 ligands.

Electronic spectroscopy of isolated DNA polyanions.

In solution, UV-vis spectroscopy is often used to investigate structural changes in biomolecules (e.g., nucleic acids), owing to changes in the environment of their chromophores (e.g., the nucleobases). Here we address whether action spectroscopy could achieve the same for gas-phase ions, while taking advantage of the additional spectrometric separation of complex mixtures. We 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 can 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 photon absorption and internal conversion. In contrast, the action spectra reconstructed from the electron photodetachment (ePD) efficiency reveal interesting phenomena. First, ePD depends on the charge state because it depends on electron binding energies. We illustrate with the G-quadruplex [dTG4T]4 that the ePD action spectrum shifts with the charge state, pointing to possible caveats when comparing the spectra of systems having different charge densities to deduce structural parameters. Second, ePD is particularly efficient for purines but not pyrimidines. ePD thus reflects not only absorption, but also particular relaxation pathways of the electronic excited states. As these pathways lead to photo-oxidation, their investigation in model gas-phase systems may prove useful to elucidating mechanisms of photo-oxidative damage, which are linked to mutations and cancers.