Interaction Energies and Dynamics of Alkali and Alkaline-Earth Cations in Quadruplex-DNA-Structures

We have investigated the nonbonded interaction energies and dynamical properties of different types of cations in quadruplex DNA structures using the GROMOS force field [1]. Quadruplex structures consist of planar guanine-quartets stacking together and causing the formation of a channel, large enought to enclose several cations (Figure 1). In recent years many experimental studies have indicated a prefered formation of this unusually stabel complexes with K⁺-ions. However, the high selectivity of this cation has not yet been understood [2].To determine the most stable coordination sites and the mobility of cations, we have calculated the pair potential energy of alkali and alkaline-earth cations along the helical axis of a model quadruplex structure (Figure 2). Our force field calculations indicate that small ions like Li⁺, Na⁺, Mg²⁺ and Ca²⁺ are free to move throught the channel. In contrast, for K⁺ and larger ions a high potential barrier appears, located in the plane of the tetramer unit. These findings are in agreement with data from X-ray crystallography, indicating that K⁺ cations are located between two planes while Na⁺ ions also can occupy coordination sites in the G-quartet plane.Considering solvent atoms in our calculations leads to the observation that a cation at the end of the quadruplex strongly interacts with one water molecule located near the entrance of the cage. Snapshots taken at different times of the MD simulation provide configurations which differ mainly in the position of this complexing water molecule. Moving away from the entrance of the cage causes a significant decrease of the potential barrier for K⁺ and smaller cage cations (Figure 3). For the larger ions the potential barrier is much higher than the thermal energy (not shown), preventing the cations from leaving and entering the cage.This conclusion is in agreement with results from our MD simulations. We followed the dynamics of different cations. While K⁺ is able to leave as well as to re-migrate into the channel (movie I), this was not observed for other types of cations. Figure 4 shows the time history of the positional fluctuations of potassium along the helical axis, and in Figure 5 we have monitored the distance between the O6-oxygen atoms of the outer G-quartet. It becomes clearly evident that the cation movement through the planes is correlated with the dynamic behaviour of the tetrameric planes. When the K⁺-ion penetrates the tetrameric unit to enter the quadruplex, the O6-O6-distance - a measure of size of the hole of the plane - increases. After a while the cation has reached the cage position and the G-quartet contracts to the initial value. That means the tetrameric planes perform a kind of breathing motion.For lithium ions we find a much higher mobility of the cation within the quadruplex channel. Two of three ions are leaving the cage instantaniously (not shown).Another indication of the experimentally observed much weaker complexation tendency of quadruplexes with lithium is the change in the distances of planes (a measure for the cage size) with time. While in the case of potassium the distance of planes is nearly the same for all three cages, for lithium the central occupied cage is much smaller than the cage in the starting structure, indicating that the DNA structure has to adjust its conformation to the cation size. On the other side the outer unoccupied cages are much greater and less stable. Due to this cation induced quadruplex deformation we observe an unwinding of the DNA-structure in the presens of lithium ions at longer simulation periods (400 ps).     

Solid-state 23Na NMR determination of the number and coordination of sodium cations bound to Oxytricha nova telomere repeat d(G4T4G4)

We report a solid-state (23)Na NMR study of the bound sodium cations in a G-quadruplex formed by Oxytricha nova telomere DNA repeat, d(G(4)T(4)G(4)) (Oxy-1.5). Using a 2D multiple-quantum magic-angle spinning (23)Na NMR method, we observed three sodium cations residing inside the quadruplex channel of the Na(+) form of Oxy-1.5. Each of these sodium cations is sandwiched between two G-quartets. We found no evidence for sodium cations in the T(4) loop region. For comparison, solid-state (15)N MAS NMR spectra were also obtained for the (15)NH(4)(+) form of Oxy-1.5. The insufficient resolution in the (15)N MAS NMR spectra did not permit determination of the number of NH(4)(+) ions inside the quadruplex channel. The solid-state (23)Na and (15)N NMR spectra for Oxy-1.5 were also compared with those obtained for guanosine 5'-monophosphate.     

Interaction of pyrrolobenzodiazepine (PBD) ligands with parallel intermolecular G-quadruplex complex using spectroscopy and ESI-MS

Studies on ligand interaction with quadruplex DNA, and their role in stabilizing the complex at concentration prevailing under physiological condition, has attained high interest. Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the interaction of PBD and TMPyP4 ligands, stoichiometry and selectivity to G-quadruplex DNA. Two synthetic ligands from PBD family, namely pyrene-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrid (PBD1), mixed imine-amide pyrrolobenzodiazepine dimer (PBD2) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) were studied. G-rich single-stranded oligonucleotide d(5'GGGGTTGGGG3') designated as d(T(2)G(8)), from the telomeric region of Tetrahymena Glaucoma, was considered for the interaction with ligands. ESI-MS and spectroscopic methods viz., circular dichroism (CD), UV-Visible, and fluorescence were employed to investigate the G-quadruplex structures formed by d(T(2)G(8)) sequence and its interaction with PBD and TMPyP4 ligands. From ESI-MS spectra, it is evident that the majority of quadruplexes exist as d(T(2)G(8))(2) and d(T(2)G(8))(4) forms possessing two to ten cations in the centre, thereby stabilizing the complex. CD band of PBD1 and PBD2 showed hypo and hyperchromicity, on interaction with quadruplex DNA, indicating unfolding and stabilization of quadruplex DNA complex, respectively. UV-Visible and fluorescence experiments suggest that PBD1 bind externally where as PBD2 intercalate moderately and bind externally to G-quadruplex DNA. Further, melting experiments using SYBR Green indicate that PBD1 unfolds and PBD2 stabilizes the G-quadruplex complex. ITC experiments using d(T(2)G(8)) quadruplex with PBD ligands reveal that PBD1 and PBD2 prefer external/loop binding and external/intercalative binding to quadruplex DNA, respectively. From experimental results it is clear that the interaction of PBD2 and TMPyP4 impart higher stability to the quadruplex complex.     

Molecular dynamics of DNA quadruplex molecules containing inosine, 6-thioguanine and 6-thiopurine

The ability of the four-stranded guanine (G)-DNA motif to incorporate nonstandard guanine analogue bases 6-oxopurine (inosine, I), 6-thioguanine (tG), and 6-thiopurine (tI) has been investigated using large-scale molecular dynamics simulations. The simulations suggest that a G-DNA stem can incorporate inosines without any marked effect on its structure and dynamics. The all-inosine quadruplex stem d(IIII)(4) shows identical dynamical properties as d(GGGG)(4) on the nanosecond time scale, with both molecular assemblies being stabilized by monovalent cations residing in the channel of the stem. However, simulations carried out in the absence of these cations show dramatic differences in the behavior of d(GGGG)(4) and d(IIII)(4). Whereas vacant d(GGGG)(4) shows large fluctuations but does not disintegrate, vacant d(IIII)(4) is completely disrupted within the first nanosecond. This is a consequence of the lack of the H-bonds involving the N2 amino group that is not present in inosine. This indicates that formation of the inosine quadruplex could involve entirely different intermediate structures than formation of the guanosine quadruplex, and early association of cations in this process appears to be inevitable. In the simulations, the incorporation of 6-thioguanine and 6-thiopurine sharply destabilizes four-stranded G-DNA structures, in close agreement with experimental data. The main reason is the size of the thiogroup leading to considerable steric conflicts and expelling the cations out of the channel of the quadruplex stem. The G-DNA stem can accommodate a single thioguanine base with minor perturbations. Incorporation of a thioguanine quartet layer is associated with a large destabilization of the G-DNA stem whereas the all-thioguanine quadruplex immediately collapses.     

Divalent transition metal cations counteract potassium-induced quadruplex assembly of oligo(dG) sequences.

Nucleic acids containing tracts of contiguous guanines tend to self-associate into four-stranded (quadruplex) structures, based on reciprocal non-Watson-Crick (G*G*G*G) hydrogen bonds. The quadruplex structure is induced/stabilized by monovalent cations, particularly potassium. Using circular dichroism, we have determined that the induction/stabilization of quadruplex structure by K+is specifically counteracted by low concentrations of Mn2+(4-10 mM), Co2+(0.3-2 mM) or Ni2+(0.3-0.8 mM). G-Tract-containing single strands are also capable of sequence-specific non-Watson-Crick interaction with d(G. C)-tract-containing (target) sequences within double-stranded DNA. The assembly of these G*G.C-based triple helical structures is supported by magnesium, but is potently inhibited by potassium due to sequestration of the G-tract single strand into quadruplex structure. We have used DNase I protection assays to demonstrate that competition between quadruplex self-association and triplex assembly is altered in the presence of Mn2+, Co2+or Ni2+. By specifically counteracting the induction/stabilization of quadruplex structure by potassium, these divalent transition metal cations allow triplex formation in the presence of K+and shift the position of equilibrium so that a very high proportion of triplex target sites are bound. Thus, variation of the cation environment can differentially promote the assembly of multistranded nucleic acid structural alternatives.     

Mix and measure fluorescence screening for selective quadruplex binders

The human genome contains thousands of regions, including that of the telomere, that have the potential to form quadruplex structures. Many of these regions are potential targets for therapeutic intervention. There are many different folding patterns for quadruplex DNAs and the loops exhibit much more variation than do the quartets. The successful targeting of a particular quadruplex structure requires distinguishing that structure from all of the other quadruplex structures that may be present. A mix and measure fluorescent screening method has been developed, that utilizes multiple reporter molecules that bind to different features of quadruplex DNA. The reporter molecules are used in combination with DNAs that have a variety of quadruplex structures. The screening is based on observing the increase or decrease in the fluorescence of the reporter molecules. The selectivity of a set of test molecules has been determined by this approach.     

Effect of rubidium and cesium ions on the dimeric quaduplex formed by the Oxytricha nova telomeric repeat oligonucleotide d(GGGGTTTTGGGG)

The DNA sequence d(GGGGTTTTGGGG) consists of 1.5 units of the repeat in telomeres of Oxytricha nova. It has been shown by NMR and x-ray crystallographic analysis that it is capable to form a dimeric quadruplex structure and that a variety of cations, namely K(+), Na(+), and NH(4)(+), are able to interact with this complex with different affinity, leading to complexes characterized by different local conformations. Thus, in order to improve the knowledge of this kind of molecule, and in particular to provide further insight into the role of monovalent cations in the G-quadruplex folding and conformation, we have investigated by (1)H-NMR the effect of the addition of Rb(+) and Cs(+) to the quadruplex formed by the oligonucleotide d(GGGGTTTTGGGG).     

A nanosecond molecular dynamics study of antiparallel d(G)7 quadruplex structures: effect of the coordinated cations

Nanosecond scale molecular dynamics simulations have been performed on antiparallel Greek key type d(G7) quadruplex structures with different coordinated ions, namely Na+ and K+ ion, water and Na+ counter ions, using the AMBER force field and Particle Mesh Ewald technique for electrostatic interactions. Antiparallel structures are stable during the simulation, with root mean square deviation values of approximately 1.5 A from the initial structures. Hydrogen bonding patterns within the G-tetrads depend on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate different cations. However, alternating syn-anti arrangement of bases along a chain as well as in a quartet is maintained through out the MD simulation. Coordinated Na+ ions, within the quadruplex cavity are quite mobile within the central channel and can even enter or exit from the quadruplex core, whereas coordinated K+ ions are quite immobile. MD studies at 400K indicate that K+ ion cannot come out from the quadruplex core without breaking the terminal G-tetrads. Smaller grooves in antiparallel structures are better binding sites for hydrated counter ions, while a string of hydrogen bonded water molecules are observed within both the small and large grooves. The hydration free energy for the K+ ion coordinated structure is more favourable than that for the Na+ ion coordinated antiparallel quadruplex structure.     

Oxidative damage generated by an oxo-metalloporphyrin onto the human telomeric sequence

The cationic metalloporphyrin Mn-TMPyP activated by KHSO(5) has been used as cleaver of an oligonucleotide containing the four human telomere repeats of 5'-GGGTTA. This oligonucleotide formed an intramolecular quadruplex DNA under 200 mM KCl as probed by DMS footprinting and could fold into different quadruplex structures under 200 mM NaCl. We found that the oxo-metalloporphyrin was able to mediate efficient oxidative cleavage of the quadruplex. The location of damage showed that the metalloporphyrin was able to bind to the last G-tetrad of the quadruplex structure via an external interaction. This metalloporphyrin-G-tetrad interaction needs a relatively high flexibility of the single-stranded linker regions to allow the partial stacking of the metalloporphyrin with the last G-tetrad planar structure. The oxidative damage consisted of guanine oxidation within the interacting G-tetrad together with an 1'-carbon hydroxylation of deoxyribose residues of the thymidine residues located on the neighboring single-stranded loop. So the high-valent oxo-metalloporphyrin is able to mediate both electron-abstraction or H-abstraction on G or T residues, respectively, within the DNA quadruplex target.     

Relative stability of G‐quadruplex structures: Interactions between the human Bcl2 promoter region and derivatives of carbazole and diphenylamine

The bcl2 promoter region forms a G‐quadruplex structure, which is a crucial target for anticancer drug development. In this study, we provide theoretical predictions of the stability of different G‐quadruplex folds of the 23‐mer bcl2 promoter region and G‐quadruplex ligand. We take into account the whole G‐quadruplex structure, including bound‐cations and solvent effects, in order to compute the ligand binding free energy using molecular dynamics simulation. Two series of the carbazole and diphenylamine derivatives are used to screen for the most potent drug in terms of stabilization. The energy analysis identifies the predominant energy components affecting the stability of the various different G‐quadruplex folds. The energy associated with the stability of the G‐quadruplex‐K⁺ structures obtained displays good correlation with experimental T_m measurements. We found that loop orientation has an intrinsic influence on G‐quadruplex stability and that the basket structure is the most stable. Furthermore, parallel loops are the most effective drug binding site. Our studies also demonstrate that rigidity and planarity are the key structural elements of a drug that stabilizes the G‐quadruplex structure. BMVC‐4 is the most potential G‐quadruplex ligand. This approach demonstrates significant promise and should benefit drug design. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1038–1050, 2014.     

Sr2+ induces unusually stable d(GGGTGGGTGGGTGGG) quadruplex dimers

Guanine‐rich sequences are able to form quadruplexes consisting of G‐quartet structural units. Quadruplexes play an important role in the regulation of gene expression and have therapeutic and biotechnological potential. The HIV‐1 integrase inhibitor, (GGGT)₄, and its variants demonstrate unusually high thermal stability. This property has been exploited in the use of quadruplex formation to drive various endergonic reactions of nucleic acids such as isothermal DNA amplification. Quadruplex stability is mainly determined by cations, which specifically bind into the inner core of the structure. In the present work, we report a systematic study of a variant of the HIV‐1 integrase inhibitor, GGGTGGGTGGGTGGG (G3T), in the presence of alkali and alkaline‐earth cations. We show that Sr²⁺‐G3T is characterized by the highest thermal stability and that quadruplex formation requires only one Sr²⁺ ion that binds with low micromolar affinity. These concentrations are sufficient to drive robust isothermal quadruplex priming DNA amplification reaction. The Sr²⁺‐quadruplexes are also able to form unusually stable dimers through end‐to‐end stacking. The multimerization can be induced by a combination of quadruplex forming cations (i.e., K⁺ or Sr²⁺) and non‐specific Mg²⁺.     

Effect of Coordinated Ions on Structure and Flexibility of Parallel G-quadruplexes: A Molecular Dynamics Study

Abstract Single tract guanine residues can associate to form stable parallel quadruplex structures in the presence of certain cations. Nanosecond scale molecular dynamics simulations have been performed on fully solvated fibre model of parallel d(G(7)) quadruplex structures with Na(+) or K(+) ions coordinated in the cavity formed by the O6 atoms of the guanine bases. The AMBER 4.1 force field and Particle Mesh Ewald technique for electrostatic interactions have been used in all simulations. These quadruplex structures are stable during the simulation, with the middle four base tetrads showing root mean square deviation values between 0.5 to 0.8 ? from the initial structure as well the high resolution crystal structure. Even in the absence of any coordinated ion in the initial structure, the G-quadruplex structure remains intact throughout the simulation. During the 1.1 ns MD simulation, one Na(+) counter ion from the solvent as well as several water molecules enter the central cavity to occupy the empty coordination sites within the parallel quadruplex and help stabilize the structure. Hydrogen bonding pattern depends on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate cations of different sizes. In the absence of any coordinated ion, due to strong mutual repulsion, O6 atoms within G-tetrad are forced farther apart from each other, which leads to a considerably different hydrogen bonding scheme within the G-tetrads and very favourable interaction energy between the guanine bases constituting a G-tetrad. However, a coordinated ion between G-tetrads provides extra stacking energy for the G-tetrads and makes the quadruplex structure more rigid. Na(+) ions, within the quadruplex cavity, are more mobile than coordinated K(+) ions. A number of hydrogen bonded water molecules are observed within the grooves of all quadruplex structures.     

Effect of coordinated ions on structure and flexiblity of parallel G-quandruplexes: a molecular dynamics study

Single tract guanine residues can associate to form stable parallel quadruplex structures in the presence of certain cations. Nanosecond scale molecular dynamics simulations have been performed on fully solvated fibre model of parallel d(G7) quadruplex structures with Na+ or K+ ions coordinated in the cavity formed by the 06 atoms of the guanine bases. The AMBER 4.1 force field and Particle Mesh Ewald technique for electrostatic interactions have been used in all simulations. These quadruplex structures are stable during the simulation, with the middle four base tetrads showing root mean square deviation values between 0.5 to 0.8 A from the initial structure as well the high resolution crystal structure. Even in the absence of any coordinated ion in the initial structure, the G-quadruplex structure remains intact throughout the simulation. During the 1.1 ns MD simulation, one Na+ counter ion from the solvent as well as several water molecules enter the central cavity to occupy the empty coordination sites within the parallel quadruplex and help stabilize the structure. Hydrogen bonding pattern depends on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate cations of different sizes. In the absence of any coordinated ion, due to strong mutual repulsion, 06 atoms within G-tetrad are forced farther apart from each other, which leads to a considerably different hydrogen bonding scheme within the G-tetrads and very favourable interaction energy between the guanine bases constituting a G-tetrad. However, a coordinated ion between G-tetrads provides extra stacking energy for the G-tetrads and makes the quadruplex structure more rigid. Na+ ions, within the quadruplex cavity, are more mobile than coordinated K+ ions. A number of hydrogen bonded water molecules are observed within the grooves of all quadruplex structures.     

Biophysical probing of the binding properties of a Cu(II) complex with G‐quadruplex DNA: an experimental and computational study

Telomerase inhibition through G‐quadruplex stabilization by small molecules is of great interest as a novel anticancer therapeutic strategy. Here, we show that newly synthesized Cu‐complex binds to G‐quadruplex DNA and induces changes in its stability. This biophysical interaction was investigated in vitro using spectroscopic, voltammetric and computational techniques. The binding constant for this complex to G‐quadruplex using spectroscopic and electrochemical methods is in the order of 10⁵. The binding stoichiometry was investigated using spectroscopic techniques and corresponded to a ratio of 1: 1. Fluorescence titration results reveal that Cu‐complex is quenched in the presence of G‐quadruplex DNA. Analysis of the fluorescence emission at different temperatures shows that ΔH° > 0, ΔS° > 0 and ΔG° < 0, and indicates that hydrophobic interactions played a major role in the binding processes. MD simulation results suggested that this ligand could stabilize the G‐quadruplex structure. An optimized docked model of the G‐quadruplex–ligand mixture confirmed the experimental results. Based on the results, we conclude that Cu‐complex as an anticancer candidate can bind and stabilize the G‐quadruplex DNA structure. Copyright © 2015 John Wiley & Sons, Ltd.     

A parallel stranded G‐quadruplex composed of threose nucleic acid (TNA)

G‐rich sequences can adopt four‐stranded helical structures, called G‐quadruplexes, that self‐assemble around monovalent cations like sodium (Na⁺) and potassium (K⁺). Whether similar structures can be formed from xeno‐nucleic acid (XNA) polymers with a shorter backbone repeat unit is an unanswered question with significant implications on the fold space of functional XNA polymers. Here, we examine the potential for TNA (α‐l ‐threofuranosyl nucleic acid) to adopt a four‐stranded helical structure based on a planar G‐quartet motif. Using native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) and solution‐state nuclear magnetic resonance (NMR) spectroscopy, we show that despite a backbone repeat unit that is one atom shorter than the backbone repeat unit found in DNA and RNA, TNA can self‐assemble into stable G‐quadruplex structures that are similar in thermal stability to equivalent DNA structures. However, unlike DNA, TNA does not appear to discriminate between Na⁺ and K⁺ ions, as G‐quadruplex structures form equally well in the presence of either ion. Together, these findings demonstrate that despite a shorter backbone repeat unit, TNA is capable of self‐assembling into stable G‐quadruplex structures.     

An exonuclease I hydrolysis assay for evaluating G-quadruplex stabilization by small molecules

Telomere length homeostasis is a prerequisite for the generation and growth of cancer. In >85% cancer cells, telomere length is maintained by telomerase that add telomere repeats to the end of telomere DNA. Because the G-rich strand of telomere DNA can fold into G-quadruplex that inhibits telomerase activity, stabilizing telomere quadruplex by small molecules is emerging as a potential therapeutic strategy against cancer. In these applications, the specificity of small molecules toward quadruplex over other forms of DNA is an important property to ensure no processes other than telomere elongation are interrupted. The evaluating assays currently available more or less have difficulty identifying or distinguishing quadruplex-irrelevant effect from quadruplex stabilization. Here, we describe an exonuclease I hydrolysis assay that evaluates quadruplex stabilization by DNA-interacting compounds, discriminates inhibitory effect from different sources and helps determine the optimal compound concentration.     

Structural and functional study of a simple,rapid, and label‐free DNAzyme‐based DNA biosensor for optimization activity

Peroxidase‐mimicking DNAzyme has a potential to self‐assemble into a G‐quadruplex and shows peroxidase activity. In comparison to proteins, peroxidase‐mimicking DNAzyme is less expensive and more stable. Herein, it is used in fabricating non‐labeling biosensors. This paper investigates the structural and functional properties of a DNA biosensor based on split DNAzyme with a detection limit in nM range (9.48 nM). Two halves of DNAzyme were linked by a complementary sequence of DNA target. Hybridization of the DNA target pulled two DNAzyme halves apart and peroxidase activity decreased. This study can be divided into 3 stages. First, the characteristics of DNAzyme were studied by Circular Dichroism technique and UV–Vis spectroscopy to find out DNAzyme's optimum activity. It is worth to note that some divalent cations were used to form G‐quadruplex, in addition to common monovalent cations. Furthermore, the hemin incubation was also optimized. Secondly, the structural and functional properties of two types of split DNAzyme were compared with DNAzyme. Thirdly, the hybridization of DNA target was monitored. The results revealed that peroxidase activities of split types decreased by half without any specific conformational changes. Interestingly, the catalytic activities of split DNAzymes could be promoted by adding Mg²⁺. Besides, it was demonstrated that the structure, peroxidation reaction, and DNA target hybridization of 2:2 and 3:1 split modes were almost alike. It was also illustrated that magnesium promoted the possibility of hybridization.     

Photoisomerizable arylstilbazolium ligands recognize parallel and antiparallel structures of G-quadruplexes

The photoisomerization and DNA interaction studies of three arylstilbazolium derivatives with various samples of nucleic acids (duplexes, triplexes and tetraplexes) are reported. The equilibrium dialysis study revealed high binding affinities of ligands to tetraplex structures. The quadruplex-binding affinity could be switched by light, e.g., the E,E and E,Z isomers of 1,4-bis(vinylquinolinium)benzene (1) interacted with parallel and antiparallel tetraplexes exhibiting different binding selectivity. The E,Z-1 showed higher binding preference for c-myc DNA (a propeller-type quadruplex), whereas the E,E-1 favorably interacted with telomeric DNA (a basket-type quadruplex). The presence of quadruplex DNA hampered photoisomerization of quadruplex-bound ligand.     

Targeting human telomeric G‐quadruplex DNA with antitumour natural alkaloid aristololactam‐β‐D‐glucoside and its comparison with daunomycin

Study on anticancer agents that act via stabilization of telomeric G‐quadruplex DNA has emerged as novel and exciting field for anticancer drug discovery. The interaction of carbohydrate containing anticancer alkaloid aristololactam‐β‐D‐glucoside (ADG) with human telomeric G‐quadruplex DNA sequence was characterized by different biophysical techniques. The binding parameters were compared with daunomycin (DAN), a well‐known chemotherapeutic drug. The Scatchard binding isotherms revealed noncooperative binding for both with the binding affinity values of (1.01 ± 0.05) × 10⁶ and (1.78 ± 0.18) × 10⁶ M⁻¹ for ADG and DAN, respectively. Circular dichroism, ferrocyanide quenching study, anisotropy study, thiazole orange displacement, optical melting, differential scanning calorimetry study, and molecular docking study suggest significant stacking and stabilizing efficiency of ADG with comparison to DAN. The energetics of the interaction for ADG and DAN revealed that both reactions were predominantly entropy driven. Negative heat capacity values were obtained from the temperature dependence of the enthalpy change. The standard molar Gibbs energy change exhibited only marginal alterations with temperature suggesting the occurrence of enthalpy‐entropy compensation. These findings indicate that ADG can act as a stabilizer of telomeric G‐quadruplex DNA and thereby can be considered as a potential telomerase inhibitor.     

Effect of Coordinated Ions on Structure and Flexibility of Parallel G-quadruplexes: A Molecular Dynamics Study

Abstract

Single tract guanine residues can associate to form stable parallel quadruplex structures in the presence of certain cations. Nanosecond scale molecular dynamics simulations have been performed on fully solvated fibre model of parallel d(G₇) quadruplex structures with Na⁺ or K⁺ ions coordinated in the cavity formed by the O6 atoms of the guanine bases. The AMBER 4.1 force field and Particle Mesh Ewald technique for electrostatic interactions have been used in all simulations. These quadruplex structures are stable during the simulation, with the middle four base tetrads showing root mean square deviation values between 0.5 to 0.8 Å from the initial structure as well the high resolution crystal structure. Even in the absence of any coordinated ion in the initial structure, the G-quadruplex structure remains intact throughout the simulation. During the 1.1 ns MD simulation, one Na⁺ counter ion from the solvent as well as several water molecules enter the central cavity to occupy the empty coordination sites within the parallel quadruplex and help stabilize the structure. Hydrogen bonding pattern depends on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate cations of different sizes. In the absence of any coordinated ion, due to strong mutual repulsion, O6 atoms within G-tetrad are forced farther apart from each other, which leads to a considerably different hydrogen bonding scheme within the G-tetrads and very favourable interaction energy between the guanine bases constituting a G-tetrad. However, a coordinated ion between G-tetrads provides extra stacking energy for the G-tetrads and makes the quadruplex structure more rigid. Na⁺ ions, within the quadruplex cavity, are more mobile than coordinated K⁺ ions. A number of hydrogen bonded water molecules are observed within the grooves of all quadruplex structures.