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.
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).
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.
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.
The amount of internal energy imparted to the ions prior to the ion mobility cell influences the ion structure and thus the collision cross section. Non-covalent complexes with few internal degrees ...of freedom and/or high charge densities are particularly sensitive to collisional activation. Here, we investigated the effects of virtually all tuning parameters of the Agilent 6560 IM-Q-TOF on the arrival time distributions of ubiquitin
7+
and found conditions in which the native state prevails. We discuss the effects of solvent evaporation conditions in the source, of the entire pre-IM DC voltage gradient, of the funnel RF amplitudes. We also report on ubiquitin
7+
conformations in different solvents, including native supercharging conditions. Collision-induced unfolding (CIU) can be conveniently provoked either behind the source capillary or in the trapping funnel. The softness of the instrumental conditions behind the mobility cell was further optimized with the DNA G-quadruplex (dG
4
T
4
G
4
)
2
·(NH
4
+
)
3
-8H
5−
, for which ion activation results in ammonia loss. To reduce the ion internal energy and obtain the intact 3-NH
4
+
complex, we reduce the post-IM voltage gradient, but this results in a lower IM resolving power due to increased diffusion behind the drift tube. The article describes the various trade-offs between ion activation, ion transmission, and ion mobility performance for native MS of very fragile structures.
Graphical Abstract
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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.
Stabilization of G-quadruplex DNA structures by small molecules has emerged as a promising strategy for the development of anticancer drugs. Since G-quadruplex structures can adopt various ...topologies, attaining specific stabilization of a G-quadruplex topology to halt a particular biological process is daunting. To achieve this, we have designed and synthesized simple structural scaffolds based on an indolylmethyleneindanone pharmacophore, which can specifically stabilize the parallel topology of promoter quadruplex DNAs (c-MYC, c-KIT1, and c-KIT2), when compared to various topologies of telomeric and duplex DNAs. The lead ligands (InEt2 and InPr2) are water-soluble and meet a number of desirable criteria for a small molecule drug. Highly specific induction and stabilization of the c-MYC and c-KIT quadruplex DNAs (ΔT 1/2 up to 24 °C) over telomeric and duplex DNAs (ΔT 1/2 ∼ 3.2 °C) by these ligands were further validated by isothermal titration calorimetry and electrospray ionization mass spectrometry experiments (K a ∼ 105 to 106 M–1). Low IC50 (∼2 μM) values were emerged for these ligands from a Taq DNA polymerase stop assay with the c-MYC quadruplex forming template, whereas the telomeric DNA template showed IC50 values >120 μM. Molecular modeling and dynamics studies demonstrated the 5′- and 3′-end stacking modes for these ligands. Overall, these results demonstrate that among the >1000 quadruplex stabilizing ligands reported so far, the indolylmethyleneindanone scaffolds stand out in terms of target specificity and structural simplicity and therefore offer a new paradigm in topology specific G-quadruplex targeting for potential therapeutic and diagnostic applications.
We report on the fate of nucleic acids conformation in the gas phase as sampled using native mass spectrometry coupled to ion mobility spectrometry. On the basis of several successful reports for ...proteins and their complexes, the technique has become popular in structural biology, and the conformation survival becomes more and more taken for granted. Surprisingly, we found that DNA and RNA duplexes, at the electrospray charge states naturally obtained from native solution conditions (≥100 mM aqueous NH4OAc), are significantly more compact in the gas phase compared to the canonical solution structures. The compaction is observed for all duplex sizes (gas-phase structures are more compact than canonical B-helices by ∼20% for 12-bp, and by up to ∼30% for 36-bp duplexes), and for DNA and RNA alike. Molecular modeling (density functional calculations on small helices, semiempirical calculations on up to 12-bp, and molecular dynamics on up to 36-bp duplexes) demonstrates that the compaction is due to phosphate group self-solvation prevailing over Coulomb repulsion. Molecular dynamics simulations starting from solution structures do not reproduce the experimental compaction. To be experimentally relevant, molecular dynamics sampling should reflect the progressive structural rearrangements occurring during desolvation. For nucleic acid duplexes, the compaction observed for low charge states results from novel phosphate–phosphate hydrogen bonds formed across both grooves at the very late stages of electrospray.
The design and construction of biomimetic self-assembling systems is a challenging yet potentially highly rewarding endeavour that contributes to the development of new biomaterials, catalysts, ...drug-delivery systems and tools for the manipulation of biological processes. Significant progress has been achieved by engineering self-assembling DNA-, protein- and peptide-based building units. However, the design of entirely new, completely non-natural folded architectures that resemble biopolymers ('foldamers') and have the ability to self-assemble into atomically precise nanostructures in aqueous conditions has proved exceptionally challenging. Here we report the modular design, formation and structural elucidation at the atomic level of a series of diverse quaternary arrangements formed by the self-assembly of short amphiphilic α-helicomimetic foldamers that bear proteinaceous side chains. We show that the final quaternary assembly can be controlled at the sequence level, which permits the programmed formation of either discrete helical bundles that contain isolated cavities or pH-responsive water-filled channels with controllable pore diameters.
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