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.

OligoR: A Native HDX/MS Data Processing Application Dedicated to Oligonucleotides.

Hydrogen-deuterium exchange mass spectrometry (HDX/MS) is increasingly used to study the dynamics of protein conformation. Coupled to native MS, HDX can also characterize the conformations of oligonucleotides and their binding to cations, small molecules, and proteins. Data processing and visualization of native HDX/MS of oligonucleotides requires dedicated software solutions. OligoR is a web-browser-based application that addresses the specific needs of DNA HDX/MS and native MS experiments from raw data in an open format to visualization and export of results. Whole experiments spanning many time points can be processed in minutes for several mass-separated species. To access valuable folding dynamics information, we have developed a simple and robust approach to deconvolute bimodal isotope distributions, even when they are highly overlapping. This approach is based on modeling physically possible isotope distributions determined from chemical formulae and could be extended to any type of analyte (proteins, peptides, sugars, and small molecules). All results are presented in interactive data tables, and publication-quality figures can be generated, customized, and exported.

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).

Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases.

Six 1',5'-anhydrohexitol uridine triphosphates were synthesized with aromatic substitutions appended via a carboxamide linker to the 5-position of their bases. An improved method for obtaining such 5-substituted hexitol nucleosides and nucleotides is described. The incorporation profile of the nucleotide analogues into a DNA duplex overhang using recently evolved XNA polymerases is compared. Long, mixed HNA sequences featuring the base modifications are generated. The apparent binding affinity of four of the nucleotides to the enzyme, the rate of the chemical step and of product release, plus the specificity constant for the incorporation of these modified nucleotides into a DNA duplex overhang using the HNA polymerase T6G12_I521L are determined via pre-steady-state kinetics. HNA polymers displaying aromatic functional groups could have significant impact on the isolation of stable and high-affinity binders and catalysts, or on the design of nanomaterials.

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.

Unexpected Position-Dependent Effects of Ribose G-Quartets in G-Quadruplexes.

To understand the role of ribose G-quartets and how they affect the properties of G-quadruplex structures, we studied three systems in which one, two, three, or four deoxyribose G-quartets were substituted with ribose G-quartets. These systems were a parallel DNA intramolecular G-quadruplex, d(TTGGGTGGGTTGGGTGGGTT), and two tetramolecular G-quadruplexes, d(TGGGT) and d(TGGGGT). Thermal denaturation experiments revealed that ribose G-quartets have position-dependent and cumulative effects on G-quadruplex stability. An unexpected destabilization was observed when rG quartets were presented at the 5'-end of the G stack. This observation challenges the general belief that RNA residues stabilize G-quadruplexes. Furthermore, in contrast to past proposals, hydration is not the main factor determining the stability of our RNA/DNA chimeric G-quadruplexes. Interestingly, the presence of rG residues in a central G-quartet facilitated the formation of additional tetramolecular G-quadruplex topologies showing positive circular dichroism signals at 295 nm. 2D NMR analysis of the tetramolecular TGgGGT (lowercase letter indicates ribose) indicates that Gs in the 5'-most G-quartet adopt the syn conformation. These analyses highlight several new aspects of the role of ribose G-quartets on G-quadruplex structure and stability, and demonstrate that the positions of ribose residues are critical for tuning G-quadruplex properties.

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.

Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism.

In recent years, an increasing number of reports have been focused on the structure and biological role of non-canonical nucleic acid secondary structures. Many of these studies involve the use of oligonucleotides that can often adopt a variety of structures depending on the experimental conditions, and hence change the outcome of an assay. The knowledge of the structure(s) formed by oligonucleotides is thus critical to correctly interpret the results, and gain insight into the biological role of these particular sequences. Herein we demonstrate that size-exclusion HPLC (SE-HPLC) is a simple yet surprisingly powerful tool to quickly and effortlessly assess the secondary structure(s) formed by oligonucleotides. For the first time, an extensive calibration and validation of the use of SE-HPLC to confidently detect the presence of different species displaying various structure and/or molecularity, involving >110 oligonucleotides forming a variety of secondary structures (antiparallel, parallel, A-tract bent and mismatched duplexes, triplexes, G-quadruplexes and i-motifs, RNA stem loops), is performed. Moreover, we introduce simple metrics that allow the use of SE-HPLC without the need for a tedious calibration work. We show that the remarkable versatility of the method allows to quickly establish the influence of a number of experimental parameters on nucleic acid structuration and to operate on a wide range of oligonucleotide concentrations. Case studies are provided to clearly illustrate the all-terrain capabilities of SE-HPLC for oligonucleotide secondary structure analysis. Finally, this manuscript features a number of important observations contributing to a better understanding of nucleic acid structural polymorphism.

A pyrimidopyrimidine Janus-AT nucleoside with improved base-pairing properties to both A and T within a DNA duplex: the stabilizing effect of a second endocyclic ring nitrogen.

Janus bases are heterocyclic nucleic acid base analogs that present two different faces able to simultaneously hydrogen bond to nucleosides that form Watson-Crick base pairs. The synthesis of a Janus-AT nucleotide analogue, (N)JAT , that has an additional endocyclic ring nitrogen and is thus more capable of efficiently discriminating T/A over G/C bases when base-pairing in a standard duplex-DNA context is described. Conversion to a phosphoramidite ultimately afforded incorporation into an oligonucleotide. In contrast to the first generation of carbocyclic Janus heterocycles, it remains in its unprotonated state at physiological pH and, therefore, forms very stable Watson-Crick base pairs with either A or T bases. Biophysical and computational methods indicate that (N)JAT is an improved candidate for sequence-specific genome targeting.

Base-pairing behavior of a carbocyclic Janus-AT nucleoside analogue capable of recognizing A and T within a DNA duplex.

Janus-type nucleosides are heterocycles with two faces, each of which is designed to complement the H-bonding interactions of natural nucleosides comprising a canonical Watson-Crick base pair. By intercepting all of the hydrogen bonds contained within the base pair, oligomeric Janus nucleosides are expected to achieve sequence-specific DNA recognition through the formation of J-loops that will be more stable than D-loops, which simply replaces one base-pair with another. Herein, we report the synthesis of a novel Janus-AT nucleoside analogue, JAT , affixed on a carbocyclic analogue of deoxyribose that was converted to the corresponding phosphoramidite. A single JAT was successfully incorporated into a DNA strand by solid phase for targeting both A and T bases, and characterized through biophysical and computational methods. Experimental UV-melting and circular dichroism data demonstrated that within the context of a standard duplex, JAT associates preferentially with T over A, and much more poorly with C and G. Density functional theory calculations confirm that the JAT structure is well suited to associate only with A and T thereby highlighting the importance of the electronic structure in terms of H-bonding. Finally, molecular dynamics simulations validated the observation that JAT can substitute more effectively as an A-analogue than as a T-analogue without substantial distortion of the B-helix. Overall, this new Janus nucleotide is a promising tool for the targeting of A-T base pairs in DNA, and will lead to the development of oligo-Janus-nucleotide strands for sequence-specific DNA recognition.

Visualizing the quadruplex: from fluorescent ligands to light-up probes.

Detection of quadruplex structures by visual methods is a major challenge of the quadruplex nucleic acid research area. Consequently, considerable efforts are under way for the discovery of quadruplex specific agents endowed with fluorescence properties. In this review chapter we propose a comprehensive and critical overview of the diverse molecular design and strategies that have been described to identify quadruplex-selective fluorescent probes. Innovative compounds as well as classical DNA dyes are reviewed. The compounds have been divided into three classes: (1) "light-up" probes that display a strong enhancement upon G4 binding, (2) "light-off" probes that display a decreased fluorescence upon binding, and (3) permanent probes ("tagged" G4-binders) that exhibit no variation of fluorescence but display quadruplex binding specificity. The labeling performances of probes in various analytical contexts (in solution, in gel, at the level of chromosomes, and in fixed cells) are also reported and commented on when available. Finally we address the strengths and weaknesses of each probe class and highlight the critical features that must be addressed in developing a practicable quadruplex-specific labeling agent.

Asymmetric distyrylpyridinium dyes as red-emitting fluorescent probes for quadruplex DNA.

The interactions of three cationic distyryl dyes, namely 2,4-bis(4-dimethylaminostyryl)-1-methylpyridinium (1a), its derivative with a quaternary aminoalkyl chain (1b), and the symmetric 2,6-bis(4-dimethylaminostyryl)-1-methylpyridinium (2a), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA-binding and DNA-probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4-disubstituted) dyes 1a and 1b bind to quadruplex DNA structures with a near-micromolar affinity and a fair selectivity with respect to double-stranded (ds) DNA [K(a)(G4)/K(a)(ds)=2.5-8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80-100-fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double-stranded DNA. This optical selectivity allows these dyes to be used as quadruplex-DNA-selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2a displays a strong binding preference for double-stranded DNA [K(a) (ds)/K(a) (G4)=40-100), presumably due to binding in the minor groove. In addition, 2a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20-50-fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA-recognition properties and fluorimetric response of organic dyes.

A streptavidin paramagnetic-particle based competition assay for the evaluation of the optical selectivity of quadruplex nucleic acid fluorescent probes.

Although quadruplex nucleic acids are thought to be involved in many biological processes, they are massively overwhelmed by duplex DNA in the cell. Small molecules, able to probe quadruplex nucleic acids with high optical selectivity, could possibly achieve the visualization of these processes. The aim of the method described herein is to evaluate quickly the optical selectivity of quadruplex nucleic acid probes, in isothermal conditions, using widely available materials, small quantities of oligonucleotides and virtually any kind and quantity of biological competitor. The assay relies on the use of streptavidin-coated paramagnetic particles and biotinylated quadruplex forming oligonucleotides, allowing a quick and easy separation of the quadruplex target from the competitor. In the present study, two quadruplex nucleic acids (the DNA and RNA human telomeric repeats) have been used as targets while a duplex DNA oligonucleotide, total DNA, total RNA, another quadruplex nucleic acid and a protein have been used as competitors. The optical selectivity of various probes, displaying different photophysical properties and binding selectivities, has been successfully examined, allowing the identification of a best candidate for further cell microscopy experiments. This assay allows a quick and reliable assessment of the labeling properties of a quadruplex binder in cellular environment conditions. It is an interesting alternative to gel electrophoresis experiments since it is performed in solution, has a well-resolved separation system and allows easy quantifications.

New 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazoline and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinoline Derivatives: Synthesis and Biological Evaluation as Novel Anticancer Agents by Targeting G-Quadruplex.

The syntheses of novel 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazolines 12 and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinolines 13 are reported here in six steps starting from various halogeno-quinazoline-2,4-(1H,3H)-diones or substituted anilines. The antiproliferative activities of the products were determined in vitro against a panel of breast (MCF-7 and MDA-MB-231), human adherent cervical (HeLa and SiHa), and ovarian (A2780) cell lines. Disubstituted 6- and 7-phenyl-bis(3-dimethylaminopropyl)aminomethylphenyl-quinazolines 12b, 12f, and 12i displayed the most interesting antiproliferative activities against six human cancer cell lines. In the series of quinoline derivatives, 6-phenyl-bis(3-dimethylaminopropyl)aminomethylphenylquinoline 13a proved to be the most active. G-quadruplexes (G4) stacked non-canonical nucleic acid structures found in specific G-rich DNA, or RNA sequences in the human genome are considered as potential targets for the development of anticancer agents. Then, as small aza-organic heterocyclic derivatives are well known to target and stabilize G4 structures, their ability to bind G4 structures have been determined through FRET melting, circular dichroism, and native mass spectrometry assays. Finally, telomerase inhibition ability has been also assessed using the MCF-7 cell line.

Cationic pentaheteroaryls as selective G-quadruplex ligands by solvent-free microwave-assisted synthesis.

We report herein a solvent-free and microwaved-assisted synthesis of several water soluble acyclic pentaheteroaryls containing 1,2,4-oxadiazole moieties (1-7). Their binding interactions with DNA quadruplex structures were thoroughly investigated by FRET melting, fluorescent intercalator displacement assay (G4-FID) and CD spectroscopy. Among the G-quadruplexes considered, attention was focused on telomeric repeats together with the proto-oncogenic c-kit sequences and the c-myc oncogene promoter. Compound 1, and to a lesser extent 2 and 5, preferentially stabilise an antiparallel structure of the telomeric DNA motif, and exhibit an opposite binding behaviour to structurally related polyoxazole (TOxaPy), and do not bind duplex DNA. The efficiency and selectivity of the binding process was remarkably controlled by the structure of the solubilising moieties.

Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae.

G-quadruplexes are nucleic acid secondary structures for which many biological roles have been proposed but whose existence in vivo has remained elusive. To assess their formation, highly specific G-quadruplex ligands are needed. Here, we tested Phen-DC(3) and Phen-DC(6), two recently released ligands of the bisquinolinium class. In vitro, both compounds exhibit high affinity for the G4 formed by the human minisatellite CEB1 and inhibit efficiently their unwinding by the yeast Pif1 helicase. In vivo, both compounds rapidly induced recombination-dependent rearrangements of CEB1 inserted in the Saccharomyces cerevisiae genome, but did not affect the stability of other tandem repeats lacking G-quadruplex forming sequences. The rearrangements yielded simple-deletion, double-deletion or complex reshuffling of the polymorphic motif units, mimicking the phenotype of the Pif1 inactivation. Treatment of Pif1-deficient cells with the Phen-DC compounds further increased CEB1 instability, revealing additional G4 formation per cell. In sharp contrast, the commonly used N-methyl-mesoporphyrin IX G-quadruplex ligand did not affect CEB1 stability. Altogether, these results demonstrate that the Phen-DC bisquinolinium compounds are potent molecular tools for probing the formation of G-quadruplexes in vivo, interfere with their processing and elucidate their biological roles.