G-Quadruplexes as Sensors of Intracellular Na+/K+ Ratio: Potential Role in Regulation of Transcription and Translation.

Data on the structure of G-quadruplexes, noncanonical nucleic acid forms, supporting an idea of their potential participation in regulation of gene expression in response to the change in intracellular Na+i/K+i ratio are considered in the review. Structural variety of G-quadruplexes, role of monovalent cations in formation of this structure, and thermodynamic stability of G-quadruplexes are described. Data on the methods of their identification in the cells and biological functions of these structures are presented. Analysis of information about specific interactions of G-quadruplexes with some proteins was conducted, and their potential participation in the development of some pathological conditions, in particular, cancer and neurodegenerative diseases, is considered. Special attention is given to the plausible role of G-quadruplexes as sensors of intracellular Na+i/K+i ratio, because alteration of this parameter affects folding of G-quadruplexes changing their stability and, thereby, organization of the regulatory elements of nucleic acids. The data presented in the conclusion section demonstrate significant change in the expression of some early response genes under certain physiological conditions of cells and tissues depending on the intracellular Na+i/K+i ratio.

Effects of monovalent and divalent cations on the rheology of entangled DNA.

In this paper we investigate the effects of varying cation valency and concentration on the rheology of entangled λDNA solutions. We show that monovalent cations moderately increase the viscoelasticty of the solutions mainly by stabilising linear concatenation of λDNA "monomers" via hybridisation of their sticky ends. On the contrary, divalent cations have a far more complex and dramatic effect on the rheology of the solution and we observe evidence of inter-molecular DNA-DNA bridging by Mg2+. We argue that these results may be interesting in the context of dense solutions of single and double stranded DNA, e.g. in vivo or in biotechnology applications such as DNA origami and DNA hydrogels.

Flanking AT base pairs affect the localization of monovalent cations in DNA A-tracts.

Capillary electrophoresis has been used to measure the free solution mobilities of a series of 26-base pair (bp) DNA oligomers containing two phased A4T1in-tracts embedded in flanking sequences containing 0 to 11 additional AT bps. A random-sequence 26-bp oligomer with 12 isolated AT bps was used as the reference. Mobility ratios (A-tract/reference) were measured in background electrolytes (BGEs) containing mixtures of small monovalent cations and tetrabutylammonium (TBA+ ) or tetrapropylammonium (TPA+ ) ions. The mobility ratios observed in 0.3 M TBA+ were >1.00, suggesting that the TBA+ ions had formed electrostatic contact pairs with the AT bp in the reference and in the A-tract flanking sequences, decreasing the mobilities of both oligomers. The TBA-AT pairing interactions could be eliminated by increasing the concentration of small monovalent cations in the BGE. In 0.3 M TPA+ , electrostatic contact pairs were formed with the AT bps in the flanking sequences and in the A-tracts. Interestingly, the shapes of the mobility ratio profiles observed for the A4T1in-tract oligomers depended on the total number of A + T residues in the oligomer.

New Hydrophilic Derivatives of Lasalocid and Their Complexes with Selected Metal Cations.

Two new esters of lasalocid, that are more hydrophilic, with glucose (LasGlu) and xylitol (LasX), have been synthesized, and their complexation of monovalent cations has been studied by various spectrometric and spectroscopic methods, such as ESI mass spectrometry, 1H, 13C NMR and FT-IR. Analyses of the results confirmed the synthesis of new esters with good yields. In order to carry out further studies, it was necessary to purify them using "flash" liquid chromatography. It was confirmed that the newly obtained molecules, as well as their complexes with lithium, sodium and potassium cations, were stabilized by a strong system of intramolecular hydrogen bonds. It was found that the hydroxyl groups of esters derived from xylitol and glucose were also involved in the complexation of cations. The results of the PM6 semiempirical calculations permitted determination of the heat of formation (HOF), and visualization of the structure of the new esters and their complexes with the cations studied. All computation results are in agreement with the spectroscopic data.

Intricate 3D architecture of a DNA mimic of GFP.

Numerous studies have shown how RNA molecules can adopt elaborate three-dimensional (3D) architectures1-3. By contrast, whether DNA can self-assemble into complex 3D folds capable of sophisticated biochemistry, independent of protein or RNA partners, has remained mysterious. Lettuce is an in vitro-evolved DNA molecule that binds and activates4 conditional fluorophores derived from GFP. To extend previous structural studies5,6 of fluorogenic RNAs, GFP and other fluorescent proteins7 to DNA, we characterize Lettuce-fluorophore complexes by X-ray crystallography and cryogenic electron microscopy. The results reveal that the 53-nucleotide DNA adopts a four-way junction (4WJ) fold. Instead of the canonical L-shaped or H-shaped structures commonly seen8 in 4WJ RNAs, the four stems of Lettuce form two coaxial stacks that pack co-linearly to form a central G-quadruplex in which the fluorophore binds. This fold is stabilized by stacking, extensive nucleobase hydrogen bonding-including through unusual diagonally stacked bases that bridge successive tiers of the main coaxial stacks of the DNA-and coordination of monovalent and divalent cations. Overall, the structure is more compact than many RNAs of comparable size. Lettuce demonstrates how DNA can form elaborate 3D structures without using RNA-like tertiary interactions and suggests that new principles of nucleic acid organization will be forthcoming from the analysis of complex DNAs.

Two Opposing Effects of Monovalent Cations on the Stability of i-Motif Structure.

At acidic pH, cytosine-rich single-stranded DNA can be folded into a tetraplex structure called i-motif (iM). In recent studies, the effect of monovalent cations on the stability of iM structure has been addressed, but a consensus about the issue has not been reached yet. Thus, we investigated the effects of various factors on the stability of iM structure using fluorescence resonance energy transfer (FRET)-based analysis for three types of iM derived from human telomere sequences. We confirmed that the protonated cytosine-cytosine (C:C+) base pair is destabilized as the concentration of monovalent cations (Li+, Na+, K+) increases and that Li+ has the greatest tendency of destabilization. Intriguingly, monovalent cations would play an ambivalent role in iM formation by making single-stranded DNA flexible and pliant for an iM structure. In particular, we found that Li+ has a notably greater flexibilizing effect than Na+ and K+. All taken together, we conclude that the stability of iM structure is controlled by the subtle balance of the two counteractive effects of monovalent cations: electrostatic screening and disruption of cytosine base pairing.

Cation valence dependence of hydrogen bond and stacking potentials in DNA mesoscopic models.

Monovalent and divalent cations play a crucial role in living cells and for molecular techniques such as PCR. Here we evaluate DNA melting temperatures in magnesium (Mg2+) and magnesium­potassium (Mg2++ K+) buffers with a mesoscopic model that allows us to estimate hydrogen bonds and stacking interaction potentials. The Mg2+ and Mg2++ K+ results are compared to previous calculations for sodium ions (Na+), in terms of equivalent sodium concentration and ionic strength. Morse potentials, related to hydrogen bonding, were found to be essentially constant and unaffected by cation conditions. However, for stacking interactions we find a clear dependence with ionic strength and cation valence. The highest ionic strength variations, for both hydrogen bonds and stacking interactions, was found at the sequence terminals. This suggests that end-to-end interactions in DNA will be strongly dependent on cation valence and ionic strength.

The influence of pH and monovalent/divalent cations on the structural and physicochemical properties of myofibrillar protein from silver carp.

The present study aimed to investigate effects of pH and monovalent (Na+ and K+)/divalent (Ca2+ and Mg2+) cations on the structural and physicochemical properties of myofibrillar protein (MP) from silver carp. MP treated with divalent cation had lesser change for the structure than that treated with monovalent cation. Ca2+-ATPase activity of MP treated with monovalent cation was increased firstly and then decreased, while that treated with divalent cation was decreased with increasing ionic strength. Surface hydrophobicity and Z-average of MP treated with divalent cations was lower than that with monovalent cations, while they decreased and then increased with the pH shifting from 3.0 to 9.0. Zeta potential of MP was increased and then decreased with increasing the pH but decreased and then increased with increasing ionic strength. In general, the pH and monovalent/divalent cations could cause various hydrophobic and electrostatic interactions, resulting in changes of the physicochemical properties of MP.

Carboxylate binding prefers two cations to one.

Almost all studies of specific ion binding by carboxylates (-COO-) have considered only a single cation, but clustering of ions and ligands is a common phenomenon. We apply density functional theory to investigate how variations in the number of acetate ligands in binding to two monovalent cations affects ion binding preferences. We study a series of monovalent (Li+, Na+, K+, Cs+) ions relevant to experimental work on many topics, including ion channels, battery storage, water purification and solar cells. We find that the preferred optimal structure has 3 acetates except for Cs+, which has 2 acetates. The optimal coordination of the cation by the carboxylate O atoms is 4 for both Na+ and K+, and 3 for Li+ and Cs+. There is a 4-fold coordination minimum just a few kcal mol-1 higher than the optimal 3-fold structure for Li+. For two cations, multiple minima occur in the vicinity of the lowest free energy state. We find that, for Li, Na and K, the preferred optimal structure with two cations is favored over a mixture of single cation complexes, providing a basis for understanding ionic cluster formation that is relevant for engineering proteins and other materials for rapid, selective ion transport.

Sodium Ions as Regulators of Transcription in Mammalian Cells.

The maintenance of an uneven distribution of Na+ and K+ ions between the cytoplasm and extracellular medium is the basis for the functioning of any animal cell. Changes in the intracellular ratio of these cations occur in response to numerous stimuli and are important for the cell activity regulation. Numerous experimental data have shown that gene transcription in mammalian cells can be regulated by changes in the intracellular [Na+]i/[K+]i ratio. Here, we discuss possible mechanisms of such regulation in various cell types, with special attention to the [Ca2+]-independent signaling pathways that suggest the presence of an intracellular sensor of monovalent cations. As such sensor, we propose the secondary structures of nucleic acids called G-quadruplexes. They are widely represented in mammalian genomes and are often found in the promoters of genes encoding transcription factors.

Diffusion NMR-based comparison of electrostatic influences of DNA on various monovalent cations.

Counterions are important constituents for the structure and function of nucleic acids. Using 7Li and 133Cs nuclear magnetic resonance (NMR) spectroscopy, we investigated how ionic radii affect the behavior of counterions around DNA through diffusion measurements of Li+ and Cs+ ions around a 15-bp DNA duplex. Together with our previous data on 23Na+ and 15NH4+ ions around the same DNA under the same conditions, we were able to compare the dynamics of four different monovalent ions around DNA. From the apparent diffusion coefficients at varied concentrations of DNA, we determined the diffusion coefficients of these cations inside and outside the ion atmosphere around DNA (Db and Df, respectively). We also analyzed ionic competition with K+ ions for the ion atmosphere and assessed the relative affinities of these cations for DNA. Interestingly, all cations (i.e., Li+, Na+, NH4+, and Cs+) analyzed by diffusion NMR spectroscopy exhibited nearly identical Db/Df ratios despite the differences in their ionic radii, relative affinities, and diffusion coefficients. These results, along with the theoretical relationship between diffusion and entropy, suggest that the entropy change due to the release of counterions from the ion atmosphere around DNA is also similar regardless of the monovalent ion types. These findings and the experimental diffusion data on the monovalent ions are useful for examination of computational models for electrostatic interactions or ion solvation.

A DFT/PCM Study on the Affinity of Salinomycin to Bind Monovalent Metal Cations.

The affinity of the polyether ionophore salinomycin to bind IA/IB metal ions was accessed using the Gibbs free energy of the competition reaction between SalNa (taken as a reference) and its rival ions: [M+-solution] + [SalNa] → [SalM] + [Na+-solution] (M = Li, K, Rb, Cs, Cu, Ag, Au). The DFT/PCM computations revealed that the ionic radius, charge density and accepting ability of the competing metal cations, as well as the dielectric properties of the solvent, have an influence upon the selectivity of salinomycin. The optimized structures of the monovalent metal complexes demonstrate the flexibility of the ionophore, allowing the coordination of one or two water ligands in SalM-W1 and SalM-W2, respectively. The metal cations are responsible for the inner coordination sphere geometry, with coordination numbers spread between 2 (Au+), 4 (Li+ and Cu+), 5/6 (Na+, K+, Ag+), 6/7 (Rb+) and 7/8 (Cs+). The metals' affinity to salinomycin in low-polarity media follows the order of Li+ > Cu+ > Na+ > K+ > Au+ > Ag+ > Rb+ > Cs+, whereas some derangement takes place in high-dielectric environment: Li+ ≥ Na+ > K+ > Cu+ > Au+ > Ag+ > Rb+ > Cs+.

Guanine-Based G-Quadruplexes Templated by Various Cations toward Potential Use as Single-Ion Conductors.

Inspired by the atomic-sized, shape-regulated features of G-quadruplexes comprising guanine motifs with a monovalent metal cation, the G-quadruplex-forming ability, and properties of a guanine-based π-conjugated Y2 molecule containing bithiophene and peripheral dodecyl chain units in the presence of various cation salts (Li+ , Na+ , K+ , and Mg2+ ) were exploited. A series of structural characterization revealed that Y2 yielded desirable G-quadruplexes with all the tested cations as a consequence of the combination of a hydrogen-bonded cyclic G-quartet, π-stacking, and cation-dipole interactions. The radius and nature of the coordinating cations crucially affected the structural characteristics of G-quadruplexes, leading to variations in the ion migration ability inside the cavity of the G-quadruplex (Li+ >Na+ >K+ >Mg2+ ), as characterized through theoretical and experimental investigations. These results not only improve the understanding of G-quadruplex self-assemblies based on guanine but also provide an impetus for their diverse potential applications, especially in the field of Li batteries.

Engineering potassium activation into biosynthetic thiolase.

Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation-π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation-π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.

pH-dependent sedimentation of DNA in the presence of divalent, but not monovalent, metal ions.

Precipitation of DNA is performed frequently in molecular biology laboratories for the purpose of purification and concentration of samples and also for transfer of DNA into cells. Metal ions are used to facilitate these processes, though their precise functions are not well characterized. In the current study we have investigated the precipitation of double-stranded DNA by group 1 and group 2 metal ions. Double-stranded DNAs were not sedimented efficiently by metals alone, even at high concentrations. Increasing the pH to 11 or higher caused strong DNA precipitation in the presence of the divalent group 2 metals magnesium, calcium, strontium and barium, but not group 1 metals. Group 2 sedimentation profiles were distinctly different from that of the transition metal zinc, which caused precipitation at pH 8. Analysis of DNAs recovered from precipitates formed with calcium revealed that structural integrity was retained and that sedimentation efficiency was largely size-independent above 400 bp. Several tests supported a model whereby single-stranded DNA regions formed by denaturation at high pH became bound by the divalent metal cations. Neutralization of negative surface charges reduced the repulsive forces between molecules, leading to formation of insoluble aggregates that could be further stabilized by cation bridging (ionic crosslinking).

Influence of the Nucleophilic Ligand on the Reactivity of Carbonyl Rhenium(I) Complexes towards Methyl Propiolate: A Computational Chemistry Perspective.

A comparative theoretical study on the reactivity of the complexes [ReY(CO)3(bipy)] (Y = NH2, NHMe, NHpTol, OH, OMe, OPh, PH2, PHMe, PMe2, PHPh, PPh2, PMePh, SH, SMe, SPh; bipy = 2,2'-bipyridine) towards methyl propiolate was carried out to analyze the influence of both the heteroatom (N, O, P, S) and the alkyl and/or aryl substituents of the Y ligand on the nature of the product obtained. The methyl substituent tends to accelerate the reactions. However, an aromatic ring bonded to N and O makes the reaction more difficult, whereas its linkage to P and S favour it. On the whole, ligands with O and S heteroatoms seem to disfavour these processes more than ligands with N and P heteroatoms, respectively. Phosphido and thiolato ligands tend to yield a coupling product with the bipy ligand, which is not the general case for hydroxo, alcoxo or amido ligands. When the Y ligand has an O/N and an H atom the most likely product is the one containing a coupling with the carbonyl ligand, which is not always obtained when Y contains P/S. Only for OMe and OPh, the product resulting from formal insertion into the Re-Y bond is the preferred.

Ultrafast excited state dynamics of silver ion-mediated cytosine-cytosine base pairs in metallo-DNA.

To better understand the nexus between structure and photophysics in metallo-DNA assemblies, the parallel-stranded duplex formed by the all-cytosine oligonucleotide, dC20, and silver nitrate was studied by circular dichroism (CD), femtosecond transient absorption spectroscopy, and time-dependent-density functional theory calculations. Silver(I) ions mediate Cytosine-Cytosine (CC) base pairs by coordinating to the N3 atoms of two cytosines. Although these silver(I) mediated CC base pairs resemble the proton-mediated CC base pairs found in i-motif DNA at first glance, a comparison of experimental and calculated CD spectra reveals that silver ion-mediated i-motif structures do not form. Instead, the parallel-stranded duplex formed between dC20 and silver ions is proposed to contain consecutive silver-mediated base pairs with high propeller twist-like ones seen in a recent crystal structure of an emissive, DNA-templated silver cluster. Femtosecond transient absorption measurements with broadband probing from the near UV to the near IR reveal an unusually long-lived (>10 ns) excited state in the dC20 silver ion complex that is not seen in dC20 in single-stranded or i-motif forms. This state is also absent in a concentrated solution of cytosine-silver ion complexes that are thought to assemble into planar ribbons or sheets that lack stacked silver(I) mediated CC base pairs. The large propeller twist angle present in metal-mediated base pairs may promote the formation of long-lived charged separated or triplet states in this metallo-DNA.

Cytotoxicity of Exogenous Acetoacetate in Lithium Salt Form Is Mediated by Lithium and Not Acetoacetate.

BACKGROUND/AIM: The ketogenic diet has recently gained interest as potential adjuvant therapy for cancer. Many researchers have endeavored to support this claim in vitro. One common model utilizes treatment with exogenous acetoacetate in lithium salt form (LiAcAc). We aimed to determine whether the effects of treatment with LiAcAc on cell viability, as reported in the literature, accurately reflect the influence of acetoacetate. MATERIALS AND METHODS: Breast cancer and normal cell lines were treated with acetoacetate, in lithium and sodium salt forms, and cell viability was assessed. RESULTS: The effect of LiAcAc on cells was mediated by Li ions. Our results showed that the cytotoxic effects of LiAcAc treatment were significantly similar to those caused by LiCl, and also treatment with NaAcAc did not cause any significant cytotoxic effect. CONCLUSION: Treatment of cells with LiAcAc is not a convincing in vitro model for studying ketogenic diet. These findings are highly important for interpreting previously published results, and for designing new experiments to study the ketogenic diet in vitro.

Metal ions confinement defines the architecture of G-quartet, G-quadruplex fibrils and their assembly into nematic tactoids.

G-quadruplex, assembled from a square array of guanine (G) molecules, is an important structure with crucial biological roles in vivo but also a versatile template for ordered functional materials. Although the understanding of G-quadruplex structures is the focus of numerous studies, little is known regarding the control of G-quartet stacking modes and the spontaneous orientation of G-quadruplex fibrils. Here, the effects of different metal ions and their concentrations on stacking modes of G-quartets are elucidated. Monovalent cations (typically K+) facilitate the formation of G-quadruplex hydrogels with both heteropolar and homopolar stacking modes, showing weak mechanical strength. In contrast, divalent metal ions (Ca2+, Sr2+, and Ba2+) at given concentrations can control G-quartet stacking modes and increase the mechanical rigidity of the resulting hydrogels through ionic bridge effects between divalent ions and borate. We show that for Ca2+ and Ba2+ at suitable concentrations, the assembly of G-quadruplexes results in the establishment of a mesoscopic chirality of the fibrils with a regular left-handed twist. Finally, we report the discovery of nematic tactoids self-assembled from G-quadruplex fibrils characterized by homeotropic fibril alignment with respect to the interface. We use the Frank-Oseen elastic energy and the Rapini-Papoular anisotropic surface energy to rationalize two different configurations of the tactoids. These results deepen our understanding of G-quadruplex structures and G-quadruplex fibrils, paving the way for their use in self-assembly and biomaterials.

d-Alanine-d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations.

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine-d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl-d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+ Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.