Effects of Six‐Membered Carbohydrate Rings on Structure,Stability, and Kinetics of G‐Quadruplexes

We have evaluated the conformational, thermal, and kinetic properties of d(TGGGGT) analogues with one or five of the ribose nucleotides replaced with the carbohydrate residues hexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA), or altritol nucleic acid (ANA). All of the modified oligonucleotides formed G‐quadruplexes, but substitution with the six‐membered rings resulted in a mixture of G‐quadruplex structures. UV and CD melting analyses showed that the structure formed by d(TGGGGT) modified with HNA was stabilized whereas that modified with CeNA was destabilized, relative to the structure formed by the unmodified oligonucleotide. Substitution at the fourth base of the G‐tract with ANA resulted in a greater stabilization effect than substitution at the first G residue; substitution with five ANA residues resulted in significant stabilization of the G‐quadruplex. A single substitution with CeNA at the first base of the G‐tract or five substitutions with HNA resulted in striking deceleration or acceleration of G‐quadruplex formation, respectively. Our results shed light on the effect of the sugar moiety on the properties of G‐quadruplex structures.     

Tuning the Stereoselectivity of a DNA‐Catalyzed Michael Addition through Covalent Modification

Complexes of G‐quadruplex DNA and Cu^II ions have previously been applied as catalysts in asymmetric reactions, but the largely unspecific and noncovalent nature of the interaction has impeded understanding of the structural basis of catalysis. To better control the formation of a catalytically competent species, DNA quadruplexes were derivatized with linker‐bpy‐Cu^II complexes in a site‐specific manner and applied in asymmetric aqueous Michael additions. These modified quadruplexes exhibited high rate acceleration and stereoselectivity. Different factors were found to be important for the catalytic performance of the modified G‐quadruplexes, among them, the position of modification, the topology of the quadruplex, the nature of the ligand, and the length of the linker between the ligand and DNA. Moving the same ligand by just two nucleotides inverted the stereochemical outcome: quadruplexes modified at position 10 formed the (?)‐enantiomer with up to 92 % ee, while DNA derivatized at position 12 formed the (+)‐enantiomer with up to 75 % ee. This stereopreference was maintained when applied to structurally different Michael acceptors. This work demonstrates a new and simple way to tune the stereoselectivity in DNA‐based asymmetric catalysis.     

Carbohydrate–DNA Interactions at G‐Quadruplexes: Folding and Stability Changes by Attaching Sugars at the 5′‐End

Quadruplex DNA structures are attracting an enormous interest in many areas of chemistry, ranging from chemical biology, supramolecular chemistry to nanoscience. We have prepared carbohydrate–DNA conjugates containing the oligonucleotide sequences of G‐quadruplexes (thrombin binding aptamer (TBA) and human telomere (TEL)), measured their thermal stability and studied their structure in solution by using NMR and molecular dynamics. The solution structure of a fucose–TBA conjugate shows stacking interactions between the carbohydrate and the DNA G‐tetrad in addition to hydrogen bonding and hydrophobic contacts. We have also shown that attaching carbohydrates at the 5′‐end of a quadruplex telomeric sequence can alter its folding topology. These results suggest the possibility of modulating the folding of the G‐quadruplex by linking carbohydrates and have clear implications in molecular recognition and the design of new G‐quadruplex ligands.     

A Flexible Terpyridine Derivative Interacts Specifically with G‐Quadruplexes

G‐quadruplexes formed by nucleic acids are implicated in pathologies ranging from cancers to neurodegenerative diseases. We evaluated interactions of 29 bi‐ and terpyridine derivatives with G‐quadruplexes and duplexes. FRET‐melting, circular dichroism, and ¹H NMR spectroscopy showed that one terpyridine derivative interacted very selectively with G‐quadruplexes. This G‐quadruplex ligand inhibited helicase activity and should influence G‐quadruplex‐related biological processes.     

Pyridyl‐Substituted Corrole Isomers: Synthesis and their Regulation to G‐quadruplex Structures

G‐quadruplex DNA plays an important role in the potential therapeutic target for the design and development of anticancer drugs. As various G‐quadruplex sequences in the promoter regions or telomeres can form different secondary structural modes and display a diversity of biology functions, variant G‐quadruplex interactive agents may be necessary to cure different disease by differentiating variant types of G‐quadruplexes. We synthesize five cationic methylpyridylium corroles and compare the interactions of corroles with different types of G‐quadruplexes such as cmyc, htelo, and bcl2 by using surface plasmon resonance. Because of the importance of human telomere G‐quadruplex DNA, we focus on the biological properties of the interactions between human telomere G‐quadruplex DNA and corrole isomers using CD, T_m, PCR‐stop (PCR= polymerase chain reaction), and polymerase‐stop assay, which demonstrate the excellent ability of the corrole to induce and stabilize the G‐quadruplex. This study provides the first experimental insight into how selectivity might be achieved for different G‐quadruplexes by a single group of methylpyridylium corrole isomers that may be optimized for potential selective cancer therapy.     

Photocrosslinking of G‐Quadruplex‐Forming Sequences found in Human Promoters

While is it well known that human telomeric DNA sequences can adopt G‐quadruplex structures, some promoters sequences have also been found to form G‐quadruplexes, and over 40% of promoters contain putative G‐quadruplex‐forming sequences. Because UV light has been shown to crosslink human telomeric G‐quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G‐quadruplexes in promoters. To investigate this possibility, 15 potentially UV‐crosslinkable G‐quadruplex‐forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G‐quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non‐B DNA structures, such as G‐quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.     

Quantitative Detection of G‐Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination

G‐quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G‐quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE , which showed distinguishable fluorescence lifetime responses between G‐quadruplexes and other DNA topologies, and fluorescence quantum yield (Φ_f) enhancement upon G‐quadruplex binding. We determined two NBTE ‐G‐quadruplex complex structures with high Φ_f values by NMR spectroscopy. The structures indicated NBTE interacted with G‐quadruplexes using three arms through π–π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φ_f values and lifetime response of NBTE upon G‐quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G‐quadruplex DNA in live cells with NBTE and found G‐quadruplex DNA content in cancer cells is 4‐fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.     

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.     

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).     

Inverting the G‐Tetrad Polarity of a G‐Quadruplex by Using Xanthine and 8‐Oxoguanine

G‐quadruplexes are four‐stranded nucleic acid structures that are built from consecutively stacked guanine tetrad (G‐tetrad) assemblies. The simultaneous incorporation of two guanine base lesions, xanthine (X) and 8‐oxoguanine (O), within a single G‐tetrad of a G‐quadruplex was recently shown to lead to the formation of a stable G?G?X?O tetrad. Herein, a judicious introduction of X and O into a human telomeric G‐quadruplex‐forming sequence is shown to reverse the hydrogen‐bond polarity of the modified G‐tetrad while preserving the original folding topology. The control exerted over G‐tetrad polarity by joint X?O modification will be valuable for the design and programming of G‐quadruplex structures and their properties.     

Synthesis of bis-indole carboxamides as G-quadruplex stabilizing and inducing ligands

The design and synthesis of a series of bis‐indole carboxamides with varying amine containing side chains as G‐quadruplex DNA stabilising small molecules are reported. Their interactions with quadruplexes have been evaluated by means of Förster resonance energy transfer (FRET) melting analysis, UV/Vis spectroscopy, circular dichroism spectroscopy and molecular modelling studies. FRET analysis indicates that these ligands exhibit significant selectivity for quadruplex over duplex DNA, and the position of the carboxamide side chains is of paramount importance in G‐quadruplex stabilisation. UV/Vis titration studies reveal that bis‐indole ligands bind tightly to quadruplexes and show a three‐ to fivefold preference for c‐kit2 over h‐telo quadruplex DNA. CD studies revealed that bis‐indole carboxamide with a central pyridine ring induces the formation of a single, antiparallel, conformation of the h‐telo quadruplex in the presence and absence of added salt. The chirality of h‐telo quadruplex was transferred to the achiral ligand (induced CD) and the formation of a preferred atropisomer was observed.     

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.     

Molecular Recognition and Visual Detection of G‐Quadruplexes by a Dicarbocyanine Dye

The interactions of a dicarbocyanine dye 3,3′‐diethylthiadicarbocyanine, DiSC₂(5) , with DNA G‐quadruplexes were studied by means of a combination of various spectroscopic techniques. Aggregation of excess dye as a result of its positive charge is promoted by the presence of the polyanionic quadruplex structure. Specific high‐affinity binding to the parallel quadruplex of the MYC promoter sequence involves stacking of DiSC₂(5) on the external G‐tetrads; the 5′‐terminal tetrad is the favored binding site. Significant energy transfer between DNA and the dye in the UV spectral region is observed upon DiSC₂(5) binding. The transfer efficiency strongly depends on the DNA secondary structure as well as on the G‐quadruplex topology. These photophysical features enable the selective detection of DNA quadruplexes through sensitized DiSC₂(5) fluorescence in the visible region.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.     

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.     

Photo‐Cross‐Linking Probes for Trapping G‐Quadruplex DNA

We have developed a straightforward synthetic pathway to a set of six photoactivatable G‐quadruplex ligands with a validated G4‐binding motif (the bisquinolinium pyridodicarboxamide PDC‐360A) tethered through various spacers to two different photo‐cross‐linking groups: benzophenone and an aryl azide. The high quadruplex‐versus‐duplex selectivity of the PDC core was retained in the new derivatives and resulted in selective alkylation of two well‐known G‐quadruplexes (human telomeric G4 and oncogene promoter c‐myc G4) under conditions of harsh competition. The presence of two structurally different photoactivatable functions allowed the selective alkylation of G‐quadruplex structures at specific nucleobases and irreversible G4 binding. The topology and sequence of the quadruplex matrix appear to influence strongly the alkylation profile, which differs for the telomeric and c‐myc quadruplexes. The new compounds are photoactive in cells and thus provide new tools for studying G4 biology.     

Duplex‐Guided Refolding into Novel G‐Quadruplex (3+1) Hybrid Conformations

The oligomer d(GCGTG₃TCAG₃TG₃TG₃ACGC) with short complementary flanking sequences at the 5′‐ and 3′‐ends was shown to fold into three different DNA G‐quadruplex species. In contrast, a corresponding oligomer that lacks base complementarity between the two overhang sequences folds into a single parallel G‐quadruplex. The three coexisting quadruplex structures were unambiguously identified and structurally characterized through detailed spectral comparisons with well‐defined G‐quadruplexes formed upon the deliberate incorporation of syn‐favoring 8‐bromoguanosine analogues into specific positions of the G‐core. Two (3+1) hybrid structures coexist with the parallel fold and feature a novel lateral–propeller–propeller loop architecture that has not yet been confirmed experimentally. Both hybrid quadruplexes adopt the same topology and only differ in their pattern of anti→syn transitions and tetrad stackings.