The Effect of DNA Sequence Directionality on G‐Quadruplex Folding

Sequence inversion in G‐rich DNA from 5′→3′ to 3′→5′ exerts a substantial effect on the number of structures formed, while the type of G‐quadruplex fold is in fact determined by the presence of K⁺ or Na⁺ ions. The melting temperatures of G‐quadruplexes adopted by oligonucleotides with sequences in the 5′→3′ direction are higher than those of their 3′→5′ counterparts with both KCl and NaCl. CD, UV, and NMR spectroscopy demonstrates the importance of primary sequence for the structural diversity of G‐quadruplexes. The changes introduced by mere sequence reversal of the G‐rich DNA segment have a substantial impact on the polymorphic nature of the resulting G‐quadruplexes and their potential physiological roles. The insights resulting from this study should enable extension of the empirical rules for the prediction of G‐quadruplex topology.     

Competitive binding exchange between alkali metal ions (K+, Rb+, and Cs+) and Na+ ions bound to the dimeric quadruplex [d(G4T4G4)]2: a 23Na and 1H NMR study

A comparative study of the competitive cation exchange between the alkali metal ions K⁺, Rb⁺, and Cs⁺ and the Na⁺ ions bound to the dimeric quadruplex [d(G₄T₄G₄)]₂ was performed in aqueous solution by a combined use of the ²³Na and ¹H NMR spectroscopy. The titration data confirm the different binding affinities of these ions for the G‐quadruplex and, in particular, major differences in the behavior of Cs⁺ as compared to the other ions were found. Accordingly, Cs⁺ competes with Na⁺ only for the binding sites at the quadruplex surface (primarily phosphate groups), while K⁺ and Rb⁺ are also able to replace sodium ions located inside the quadruplex. Furthermore, the ¹H NMR results relative to the CsCl titration evidence a close approach of Cs⁺ ions to the phosphate groups in the narrow groove of [d(G₄T₄G₄)]₂. Based on a three‐site exchange model, the ²³Na NMR relaxation data lead to an estimate of the relative binding affinity of Cs⁺ versus Na⁺ for the quadruplex surface of 0.5 at 298 K. Comparing this value to those reported in the literature for the surface of the G‐quadruplex formed by 5′‐guanosinemonophosphate and for the surface of double‐helical DNA suggests that topology factors may have an important influence on the cation affinity for the phosphate groups on DNA. Copyright © 2009 John Wiley & Sons, Ltd.     

Binding Behaviors for Different Types of DNA G‐Quadruplexes: Enantiomers of [Ru(bpy)2(L)]2+ (L=dppz,dppz‐idzo)

Polymorphic DNA G‐quadruplex recognition has attracted great interest in recent years. The strong binding affinity and potential enantioselectivity of chiral [Ru(bpy)₂(L)]²⁺ (L=dipyrido[3,2‐a:2′,3′‐c]phenazine, dppz‐10,11‐imidazolone; bpy=2,2′‐bipyridine) prompted this investigation as to whether the two enantiomers, Δ and Λ, can show different effects on diverse structures with a range of parallel, antiparallel and mixed parallel/antiparallel G‐quadruplexes. These studies provide a striking example of chiral‐selective recognition of DNA G‐quadruplexes. As for antiparallel (tel‐Na⁺) basket G‐quadruplex, the Λ enantiomers bind stronger than the Δ enantiomers. Moreover, the behavior reported here for both enantiomers stands in sharp contrast to B‐DNA binding. The chiral selectivity toward mixed parallel/antiparallel (tel‐K⁺) G‐quadruplex of both compounds is weak. Different loop arrangements can change chiral complex selectivity for both antiparallel and mixed parallel/antiparallel G‐quadruplex. Whereas both Δ and Λ isomers bind to parallel G‐quadruplexes with comparable affinity, no appreciable stereoselective G‐quadruplex binding of the isomers was observed. In addition, different binding stoichiometries and binding modes for Δ and Λ enantiomers were confirmed. The results presented here indicate that chiral selective G‐quadruplex binding is not only related to G‐quadruplex topology, but also to the sequence and the loop constitution.     

Ion‐Responsive Hemin–G‐Quadruplexes for Switchable DNAzyme and Enzyme Functions

Programmed nucleic acid sequences undergo K⁺ ion‐induced self‐assembly into G‐quadruplexes and separation of the supramolecular structures by the elimination of K⁺ ions by crown ether or cryptand ion‐receptors. This process allows the switchable formation and dissociation of the respective G‐quadruplexes. The different G‐quadruplex structures bind hemin, and the resulting hemin–G‐quadruplex structures reveal horseradish peroxidase DNAzyme catalytic activities. The following K⁺ ion/receptor switchable systems are described: 1) The K⁺‐induced self‐assembly of the Mg²⁺‐dependent DNAzyme subunits into a catalytic nanostructure using the assembly of G‐quadruplexes as bridging unit. 2) The K⁺‐induced stabilization of the anti‐thrombin G‐quadruplex nanostructure that inhibits the hydrolytic functions of thrombin. 3) The K⁺‐induced opening of DNA tweezers through the stabilization of G‐quadruplexes on the “tweezers’ arms" and the release of a strand bridging the tweezers into a closed structure. In all of the systems reversible, switchable, functions are demonstrated. For all systems two different signals are used to follow the switchable functions (fluorescence and the catalytic functions of the derived hemin–G‐quadruplex DNAzyme).     

Elongated Thrombin Binding Aptamer: A G‐Quadruplex Cation‐Sensitive Conformational Switch

Aptamer‐based biosensors offer promising perspectives for high performance, specific detection of proteins. The thrombin binding aptamer (TBA) is a G‐quadruplex‐forming DNA sequence, which is frequently elongated at one end to increase its analytical performances in a biosensor configuration. Herein, we investigate how the elongation of TBA at its 5′ end affects its structure and stability. Circular dichroism spectroscopy shows that TBA folds in an antiparallel G‐quadruplex conformation with all studied cations (Ba²⁺, Ca²⁺, K⁺, Mg²⁺, Na⁺, NH₄⁺, Sr²⁺ and the [Ru(NH₃)₆]^2+/3+ redox marker) whereas other structures are adopted by the elongated aptamers in the presence of some of these cations. The stability of each structure is evaluated on the basis of UV spectroscopy melting curves. Thermal difference spectra confirm the quadruplex character of all conformations. The elongated sequences can adopt a parallel or an antiparallel structure, depending on the nature of the cation; this can potentially confer an ion‐sensitive switch behavior. This switch property is demonstrated with the frequently employed redox complex [Ru(NH₃)₆]³⁺, which induces the parallel conformation at very low concentrations (10 equiv per strand). The addition of large amounts of K⁺ reverts the conformation to the antiparallel form, and opens interesting perspectives for electrochemical biosensing or redox‐active responsive devices.     

Dinuclear Ruthenium(II) Complexes That Induce and Stabilise G‐Quadruplex DNA

A series of dinuclear ruthenium(II) complexes were synthesised, and the complexes were determined to be new highly selective compounds for binding to telomeric G‐quadruplex DNA. The interactions of these complexes with telomeric G‐quadruplex DNA were studied by using circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assays, isothermal titration calorimetry (ITC) and molecular modelling. The results showed that the complexes 1 , 2 and 4 induced and stabilised the formation of antiparallel G‐quadruplexes of telomeric DNA in the absence of salt or in the presence of 100 mM K⁺‐containing buffer. Furthermore, complexes 1 and 2 strongly bind to and effectively stabilise the telomeric G‐quadruplex structure and have significant selectivity for G‐quadruplex over duplex DNA. In comparison, complex 3 had a much lesser effect on the G‐quadruplex, suggesting that possession of a suitably sized plane for good π–π stacking with the G‐quadruplets is essential for the interaction of the dinuclear ruthenium(II) complexes with the G‐quadruplex. Moreover, telomerase inhibition by the four complexes and their cellular effects were studied, and complex 1 was determined to be the most promising inhibitor of both telomerase and HeLa cell proliferation.     

A New Pathway of DNA G‐Quadruplex Formation

A new folding intermediate of Oxytricha nova telomeric Oxy‐1.5 G‐quadruplex was characterized in aqueous solution using NMR spectroscopy, native gel electrophoresis, thermal differential spectra (TDS), CD spectroscopy, and differential scanning calorimetry (DSC). NMR experiments have revealed that this intermediate (i‐Oxy‐1.5) exists in two symmetric bimolecular forms in which all guanine bases are involved in GG N1‐carbonyl symmetric base pairs. Kinetic analysis of K⁺‐induced structural transitions shows that folding of Oxy‐1.5 G‐quadruplex from i‐Oxy‐1.5 is much faster and proceeds through less intermediates than folding from single strands. Therefore, a new folding pathway of Oxy‐1.5 G‐quadruplex is proposed. This study provides evidence that G‐rich DNA sequences can self‐assemble into specific pre‐organized DNA structures that are predisposed to fold into G‐quadruplex when interacting with cations such as potassium ions.     

Recognizing Hybrid/Mixed G-quadruplex in Human Telomeres by Using a Cyanine Dye Supramolecule with Confocal Laser Scanning Microscopy

Single‐stranded telomeric DNA tends to form a four‐base‐paired planar structure termed G‐quadruplex. Although kinds of G‐quadruplex structures in vitro have been documented in the presence of potassium or sodium, recognition of these DNA motifs (both in vitro and in vivo) is still an important issue in understanding the biological function of the G‐quadruplex structures in telomeres as well as developing anticancer agents. Herein we address this important question through the distinctive properties of a supramolecular system of cyanine dye 3,3′‐di(3‐sulfopropyl)‐4,5,4′,5′‐dibenzo‐9‐methyl‐thiacarbocyanine triethylammonium salt (MTC) upon binding to different DNA motifs. Interaction of MTC with hybrid/mixed G‐quadruplex results in a set of unique spectrophotometric signatures which are completely different from those arising from binding to other DNA motifs. Furthermore, such feature could be extended to map the locations of DNAs on interface. Linear duplex and mixed G‐quadruplex in human telomeres assembled on Au film and stained by MTC were directly recognized by confocal laser scanning microscopy (CLSM). All results suggested that MTC supramolecular system may be a good probe of specific G‐quadruplex structure.     

Molecular Recognition of the Hybrid‐2 Human Telomeric G‐Quadruplex by Epiberberine: Insights into Conversion of Telomeric G‐Quadruplex Structures

Human telomeres can form DNA G‐quadruplex (G4), an attractive target for anticancer drugs. Human telomeric G4s bear inherent structure polymorphism, challenging for understanding specific recognition by ligands or proteins. Protoberberines are medicinal natural‐products known to stabilize telomeric G4s and inhibit telomerase. Here we report epiberberine (EPI) specifically recognizes the hybrid‐2 telomeric G4 predominant in physiologically relevant K⁺ solution and converts other telomeric G4 forms to hybrid‐2, the first such example reported. Our NMR structure in K⁺ solution shows EPI binding induces extensive rearrangement of the previously disordered 5′‐flanking and loop segments to form an unprecedented four‐layer binding pocket specific to the hybrid‐2 telomeric G4; EPI recruits the (?1) adenine to form a “quasi‐triad” intercalated between the external tetrad and a T:T:A triad, capped by a T:T base pair. Our study provides structural basis for small‐molecule drug design targeting the human telomeric G4.     

A dinuclear ruthenium(II) complex as an inducer and potential luminescent switch-on probe for G-quadruplex DNA

Given that recognition and regulation of G-quadruplex nucleic acid structures is an important goal for the development of chemical tools and medicinal agents, a dinuclear ruthenium complex [Ru₂(bpy)₄(bip-phenol)](ClO₄)₄ {bpy?=?2,2′-bipyridine, bip-phenol?=?2,4-bis(1H-imidazo[4,5-f] [1,10] phenanthroline-2-yl)phenol} has been synthesized and characterized, and its interactions with telomeric G-quadruplex DNA have been explored by photophysical and biophysical methods. This complex can induce and stabilize the formation of an antiparallel G-quadruplex of telomeric DNA in the absence of salt, or in the presence of K⁺/Na⁺-containing buffer. The complex binds strongly to the telomeric G-quadruplex, with a binding constant K_b?>?10⁶ and a 2:1 [complex]/[quadruplex] binding ratio. Fluorescence titrations revealed that the complex behaves as a promising photophysical “light switch” for G-quadruplex DNA, with 8.6- and 8.4-fold fluorescence enhancements in Na⁺ and K⁺ buffers, respectively.     

Fluorescent Bioprobes: Structural Matching in the Docking Processes of Aggregation‐Induced Emission Fluorogens on DNA Surfaces

Whereas most conventional DNA probes are flat disklike aromatic molecules, we explored the possibility of developing quadruplex sensors with nonplanar conformations, in particular, the propeller‐shaped tetraphenylethene (TPE) salts with aggregation‐induced emission (AIE) characteristics. 1,1,2,2‐Tetrakis[4‐(2‐triethylammonioethoxy)phenyl]ethene tetrabromide (TPE‐ 1 ) was found to show a specific affinity to a particular quadruplex structure formed by a human telomeric DNA strand in the presence of K⁺ ions, as indicated by the enhanced and bathochromically shifted emission of the AIE fluorogen. Steady‐state and time‐resolved spectral analyses revealed that the specific binding stems from a structural matching between the AIE fluorogen and the DNA strand in the folding process. Computational modeling suggests that the AIE molecule docks on the grooves of the quadruplex surface with the aid of electrostatic attraction. The binding preference of TPE‐ 1 enables it to serve as a bioprobe for direct monitoring of cation‐driven conformational transitions between the quadruplexes of various conformations, a job unachievable by the traditional G‐quadruplex biosensors. Methyl thiazolyl tetrazolium (MTT) assays reveal that TPE‐ 1 is cytocompatible, posing no toxicity to living cells.     

Multivariate investigation of interaction between porphyrin ligands and human telomeric DNA

G-quadruplexes are formed by association of DNA strands containing multiple contiguous guanines. The capability of drugs to induce formation or stabilize G-quadruplexes is an active area of cancer therapy investigation. We evaluated interaction between two cationic tetrapyridinoporphyrazines with Na⁺ and K⁺ forms of human telomeric G-quadruplex DNA by chemometrics method. An antiparallel quadruplex structure was found to be stabilized more greatly by these two isomers in the presence of K⁺ and Na⁺ ions. Equilibrium model of a ligand binding with DNA oligomer has been considered as a process of small molecule adsorption on to a lattice of multiple binding sites. In multivariate analysis methods, it is accounted this assertion that during saturation of the macromolecule by a ligand should expect effect of cooperativity due to changes in DNA conformation or the mutual influence between bound ligands. Such phenomenon cannot be entirely described by the classical stepwise complex formation model. From the results of absorption and circular dichroism measurements, the unique site for the ligand binding is suggested to be the intercalating in guanine tetrad plane quadruplex. We found a 2:1 binding stoichiometry for both ligands and Tel22.     

Using Diffusion NMR To Characterize Guanosine Self‐Association: Insights into Structure and Mechanism

This paper presents results from a series of pulsed field gradient (PFG) NMR studies on lipophilic guanosine nucleosides that undergo cation‐templated assembly in organic solvents. The use of PFG‐NMR to measure diffusion coefficients for the different aggregates allowed us to observe the influences of cation, solvent and anion on the self‐assembly process. Three case studies are presented. In the first study, diffusion NMR confirmed formation of a hexadecameric G‐quadruplex [G 1 ]₁₆ ? 4 K⁺ ? 4 pic^? in CD₃CN. Furthermore, hexadecamer formation from 5′‐TBDMS‐2′,3′‐isopropylidene G 1 and K⁺ picrate was shown to be a cooperative process in CD₃CN. In the second study, diffusion NMR studies on 5′‐(3,5‐bis(methoxy)benzoyl)‐2′,3′‐isopropylidene G 4 showed that hierarchical self‐association of G₈‐octamers is controlled by the K⁺ cation. Evidence for formation of both discrete G₈‐octamers and G₁₆‐hexadecamers in CD₂Cl₂ was obtained. The position of this octamer–hexadecamer equilibrium was shown to depend on the K⁺ concentration. In the third case, diffusion NMR was used to determine the size of a guanosine self‐assembly where NMR signal integration was ambiguous. Thus, both diffusion NMR and ESI‐MS show that 5′‐O‐acetyl‐2′,3′‐O‐isopropylidene G 7 and Na⁺ picrate form a doubly charged octamer [G 7 ]₈ ? 2 Na⁺ ? 2 pic^? 9 in CD₂Cl₂. The anion's role in stabilizing this particular complex is discussed. In all three cases the information gained from the diffusion NMR technique enabled us to better understand the self‐assembly processes, especially regarding the roles of cation, anion and solvent.     

DNA Triplexes‐Guided Assembly of G‐Quadruplexes for Constructing Label‐free Fluorescent Logic Gates

Assembly of G‐quadruplexes guided by DNA triplexes in a controlled manner is achieved for the first time. The folding of triplex sequences in acidic conditions brings two separated guanine‐rich sequences together and subsequently a G‐quadruplex structure is formed in the presence of K⁺. Based on this novel platform, label‐free fluorescent logic gates, such as AND, INHIBIT, and NOR, are constructed with ions as input and the fluorescence of a G‐quadruplex‐specific fluorescent probe NMM as output.     

Effect of the Ancillary Ligands on the Spectral Properties and G‐Quadruplexes DNA Binding Behavior: A Combined Experimental and Theoretical Study

In an effort to explore the effect of ancillary ligands on the spectral properties and overall G‐quadruplex DNA binding behavior, two new ruthenium(II) complexes [Ru(phen)₂(dppzi)]²⁺ ( 1 ) and [Ru(dmp)₂(dppzi)]²⁺ ( 2 ) (phen=1,10‐phenanthroline, dmp=2,9‐dimethyl‐1,10‐phenanthroline, dppzi=dipyrido[3,2‐a:2′,3′‐c]phenazine‐10,11‐imidazole) were prepared. Complex 1 can emit luminescence in the absence and presence of G‐quadruplexes DNA. However, with ?CH₃ substituent on the 2‐ and 9‐positions of the phen ancillary ligand, no detectable luminescence is observed for complex 2 in any organic solvent or in the absence and/or presence of G‐quadruplex DNA. Experimental and molecular docking studies indicated that both complexes interacted with the human telomeric repeat AG3(T2AG3)3 (22AG) G‐quadruplex with the stoichiometric ratio of 1:1, but the two complexes showed different G‐quadruplex DNA binding affinity. Complex 1 binds to the G‐quadruplexes DNA more tightly than complex 2 does. Our results demonstrate that methyl groups on the phen ancillary ligand significantly affect the spectral properties and the overall DNA binding behavior of the complexes. Such difference in spectral properties and DNA binding affinities of these two complexes can be reasonably explained by DFT/TD‐DFT calculations. This work provides guidance not only on exploring the G‐quadruplexes DNA binding behavior of complexes, but also understanding the unique luminescence mechanism.     

Involvement of Long‐Lived Intermediate States in the Complex Folding Pathway of the Human Telomeric G‐Quadruplex

The energy landscapes of human telomeric G‐quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real‐time NMR spectroscopy, we investigated the K⁺‐induced folding of the human telomeric DNA sequence 5′‐TTGGG(TTAGGG)₃A‐3′. Three long‐lived states were detected during folding: a major conformation (hybrid‐1), a previously structurally uncharacterized minor conformation (hybrid‐2), and a partially unfolded state. The minor hybrid‐2 conformation is formed faster than the more stable hybrid‐1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin‐like structures.     

G‐Quadruplex‐Linked Supersandwich DNA Structure for Electrochemical Amplified Detection of Thrombin

In this paper, a novel strategy of electrochemical amplified detection of thrombin based on G‐quadruplex‐linked supersandwich structure was described. In the presence of K⁺ and hemin, the original hairpin DNA sequence activated an autonomous cross‐opening process to build up hemin/G‐quadruplex structure and can hybridize to form supersandwich structure containing multiple signal labels. With the addition of thrombin, it conjugated with its aptamer, leading to a remarkably descended signal. The supersandwich‐amplified electrochemical sensor system was highly sensitive in the concentration range from 10^?6 to 10^?10 M with a detection limit of 10 pM and also demonstrated excellent selectivity. The amplifying supersandwich structure with multiple labels can be implemented as a versatile sensing platform for analyzing other DNA in the presence of the appropriate probe.     

Structure‐Based Design of Platinum(II) Complexes as c‐myc Oncogene Down‐Regulators and Luminescent Probes for G‐Quadruplex DNA

A series of platinum(II) complexes with tridentate ligands was synthesized and their interactions with G‐quadruplex DNA within the c‐myc gene promoter were evaluated. Complex 1 , which has a flat planar 2,6‐bis(benzimidazol‐2‐yl)pyridine (bzimpy) scaffold, was found to stabilize the c‐myc G‐quadruplex structure in a cell‐free system. An in silico G‐quadruplex DNA model has been constructed for structure‐based virtual screening to develop new Pt^II‐based complexes with superior inhibitory activities. By using complex 1 as the initial structure for hit‐to‐lead optimization, bzimpy and related 2,6‐bis(pyrazol‐3‐yl)pyridine (dPzPy) scaffolds containing amine side‐chains emerge as the top candidates. Six of the top‐scoring complexes were synthesized and their interactions with c‐myc G‐quadruplex DNA have been investigated. The results revealed that all of the complexes have the ability to stabilize the c‐myc G‐quadruplex. Complex 3 a ([Pt^II L2R ] ⁺ ; L2 =2,6‐bis[1‐(3‐piperidinepropyl)‐1H‐enzo[d]imidazol‐2‐yl]pyridine, R =Cl) displayed the strongest inhibition in a cell‐free system (IC₅₀=2.2 μM ) and was 3.3‐fold more potent than that of 1 . Complexes 3 a and 4 a ([Pt^II L3R ]⁺; L3 =2,6‐bis[1‐(3‐morpholinopropyl)‐1H‐pyrazol‐3‐yl]pyridine, R =Cl) were found to effectively inhibit c‐myc gene expression in human hepatocarcinoma cells with IC₅₀ values of ≈17 μM , whereas initial hit 1 displayed no significant effect on gene expression at concentrations up to 50 μM . Complexes 3 a and 4 a have a strong preference for G‐quadruplex DNA over duplex DNA, as revealed by competition dialysis experiments and absorption titration; 3 a and 4 a bind G‐quadruplex DNA with binding constants (K) of approximately 10⁶–10⁷ dm³ mol^?1, which are at least an order of magnitude higher than the K values for duplex DNA. NMR spectroscopic titration experiments and molecular modeling showed that 4 a binds c‐myc G‐quadruplex DNA through an external end‐stacking mode at the 3′‐terminal face of the G‐quadruplex. Intriguingly, binding of c‐myc G‐quadruplex DNA by 3 b is accompanied by an increase of up to 38‐fold in photoluminescence intensity at λ_max=622 nm.     

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 ( 1 a ), its derivative with a quaternary aminoalkyl chain ( 1 b ), and the symmetric 2,6‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 2 a ), 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 1 a and 1 b 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 2 a 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, 2 a 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.