Enhanced stability of G-quadruplexes from conformationally constrained aep-PNA backbone

Nucleic (DNA) acids having contiguous stretch of G sequence form quadruplex structure, which is very critical to control cell division. Recently the existence of G-quadruplex in RNA is also reported in presence of monovalent metal ion. PNA is a promising DNA analogue which binds strongly to DNA to form PNA:DNA duplex or PNA(2):DNA triplex. PNA also forms quadruplexes such G-quadruplex and i-motif in G and C-rich sequences respectively. aep-PNA containing a prolyl ring is one of several PNA analogues that provide rigidity and chirality in backbone and has binding affinity to natural DNA which is higher than that of PNA. Here we examine the ability of aep-PNA-G to form a quadruplex by UV, CD and mass spectroscopic techniques.     

Effect of loop orientation on quadruplex-TMPyP4 interaction

G-quadruplexes are believed to be potential targets for therapeutic intervention and this has resulted in designing of various quadruplex interacting ligands. Moreover, reports about existence of quadruplex forming sequences across the genome have propelled greater interest in understanding their interaction with small molecules. An intramolecular quadruplex sequence can adopt different conformations, owing to different orientation of loops in the structure. The differences in the loop orientation can affect their molecular recognition. Herein, we have studied the interaction of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H, 23H-porphine (TMPyP4), a well-known G quadruplex binding ligand with three DNA quadruplexes differing in loop orientations. Results obtained from UV, ITC, and SPR studies have coherently revealed that the TMPyP4 molecule shows preferential binding to parallel G-quadruplex ( c-myc and c-kit) over its antiparallel counterpart (human telomeric). The binding affinity for parallel quadruplex was (10(7)) 1 order of magnitude higher than that for antiparallel DNA quadruplex (10 ). The study shows two binding modes, stronger binding (10(7)) of TMPyP4 involving end stacking and a weaker external binding (10 ), while TMPyP4 shows only one binding mode with duplex with a binding affinity of the order of 10(6). Overall, the study emphasizes that differences in the loop orientation give rise to different conformations of quadruplex, which in turn govern its binding to small molecules, and thereby play a pivotal role in molecular recognition.     

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.     

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.     

Topology variation and loop structural homology in crystal and simulated structures of a bimolecular DNA quadruplex

The topology of DNA quadruplexes depends on the nature and number of the nucleotides linking G-quartet motifs. To assess the effects of a three-nucleotide TTT linker, the crystal structure of the DNA sequence d(G(4)T(3)G(4)) has been determined at 1.5 A resolution, together with that of the brominated analogue d(G(4)(Br)UTTG(4)) at 2.4 A resolution. Both sequences form bimolecular intermolecular G-quadruplexes with lateral loops. d(G(4)(Br)UTTG(4)) crystallized in the monoclinic space group P2(1) with three quadruplex molecules in the asymmetric unit, two associating together as a head-to-head stacked dimer, and the third as a single head-to-tail dimer. The head-to-head dimers have two lateral loops on the same G-quadruplex face and form an eight-G-quartet stack, with a linear array of seven K(+) ions between the quartets. d(G(4)T(3)G(4)) crystallized in the orthorhombic space group C222 and has a structure very similar to the head-to-tail dimer in the P2(1) unit cell. The sequence studied here is able to form several different folds; however, all four quadruplexes in the two structures have lateral loops, in contrast to the diagonal loops reported for the analogous quadruplex with T(4) loops. A total of seven independent T(3) loops were observed in the two structures. These can be classified into two discrete conformational classes, suggesting that these represent preferred loop conformations that are independent of crystal-packing forces.     

Diarylethynyl amides that recognize the parallel conformation of genomic promoter DNA G-quadruplexes

We report bis-phenylethynyl amide derivatives as a potent G-quadruplex binding small molecule scaffold. The amide derivatives were efficiently prepared in 3 steps by employing Sonogashira coupling, ester hydrolysis and a chemoselective amide coupling. Ligand-quadruplex recognition has been evaluated using a fluorescence resonance energy transfer (FRET) melting assay, surface plasmon resonance (SPR), circular dichroism (CD) and (1)H nuclear magnetic resonance (NMR) spectroscopy. While most of the G-quadruplex ligands reported so far comprise a planar, aromatic core designed to stack on the terminal tetrads of a G-quadruplex, these compounds are neither polycyclic, nor macrocyclic and have free rotation around the triple bond enabling conformational flexibility. Such molecules show very good binding affinity, excellent quadruplex:duplex selectivity and also promising discrimination between intramolecular promoter quadruplexes. Our results indicate that the recognition of the c-kit2 quadruplex by these ligands is achieved through groove binding, which favors the formation of a parallel conformation.     

FRET templated by G-quadruplex DNA: a specific ternary interaction using an original pair of donor/acceptor partners

G-quadruplex represents a suitable scaffold for FRET (fluorescence resonance energy transfer) since its two external quartets offer two well-defined binding sites for concomitant trapping of donor/acceptor partners. Combining selective G-quadruplex binders (macrocyclic bis(quinacridine) BOQ(1) or monomeric quinacridine MMQ(1), donor) with a highly fluorescent DNA probe (thiazole orange, acceptor), we designed a structure-specific FRET-system based on an unprecedented noncovalent ternary complex. This system could be potentially usable as a signature for quadruplex-DNA conformation in solution, but also might offer a unique means for observing cation and ligand binding influence on quadruplex topology.     

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.     

Specific recognition of human telomeric G-quadruplex DNA with small molecules and the conformational analysis by ESI mass spectrometry and circular dichroism spectropolarimetry

Electrospray ionization mass spectrometry (ESI-MS) was utilized to investigate the binding affinity and stoichiometry of small molecules with human telomeric G-quadruplex DNA. The binding-affinity order obtained for the (AGGGTT)(4) quadruplex was: Tel01>ImImImbetaDp>PyPyPygammaImImImbetaDp. The specific binding of Tel01 and PyPyPygammaImImImbetaDp in one system consisting of human telomeric G-quadruplex and duplex DNA was observed directly for the first time. This revealed that PyPyPygammaImImImbetaDp has a binding specificity for the duplex DNA, whereas Tel01 selectively recognizes the G-quadruplex DNA. Moreover, both ESI-MS and circular dichroism (CD) spectra indicated that Tel01 favored the formation and stabilization of the antiparallel G-quadruplex, and a structural transition of the (AGGGTT)(4) sequence from a coexistence of parallel and antiparallel G-quadruplexes to a parallel G-quadruplex induced by annealing.     

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.     

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.     

Structure and stability of higher-order human telomeric quadruplexes

G-quadruplex formation in the sequences 5'-(TTAGGG)(n) and 5'(TTAGGG)(n)TT (n = 4, 8, 12) was studied using circular dichroism, sedimentation velocity, differential scanning calorimetry, and molecular dynamics simulations. Sequences containing 8 and 12 repeats formed higher-order structures with two and three contiguous quadruplexes, respectively. Plausible structures for these sequences were determined by molecular dynamics simulations followed by experimental testing of predicted hydrodynamic properties by sedimentation velocity. These structures featured folding of the strand into contiguous quadruplexes with mixed hybrid conformations. Thermodynamic studies showed the strands folded spontaneous to contain the maximum number contiguous quadruplexes. For the sequence 5'(TTAGGG)(12)TT, more than 90% of the strands contained completely folded structures with three quadruplexes. Statistical mechanical-based deconvolution of thermograms for three quadruplex structures showed that each quadruplex melted independently with unique thermodynamic parmameters. Thermodynamic analysis revealed further that quadruplexes in higher-ordered structures were destabilized relative to their monomeric counterparts, with unfavorable coupling free energies. Quadruplex stability thus depends critically on the sequence and structural context.     

Investigating a quadruplex-ligand interaction by unfolding kinetics

We have investigated the interaction of the intramolecular human telomeric DNA G-quadruplex with a hemicyanine-peptide ligand, by studying the rate of quadruplex opening with a complementary DNA oligonucleotide. By employing a minimal kinetic model, the relationship between the observed rate of quadruplex opening and the ligand concentration has enabled estimation of the dissociation constant. A van't Hoff analysis revealed the enthalpy and entropy changes of binding to be -77 +/- 22 kJ mol(-1) and -163 +/- 75 J mol(-1) K(-1), respectively. Arrhenius analyses of the rate constants of opening free and bound quadruplex gave activation energies of 118 +/- 2 and 98 +/- 10 kJ mol(-1), respectively. These results indicate that the presence of the ligand has only a small effect on the activation energy, suggesting that the unbinding of the ligand occurs after the transition state for quadruplex unfolding.     

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.     

Energetics of the human Tel-22 quadruplex-telomestatin interaction: a molecular dynamics study

The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadruplex as an attractive target for cancer therapeutic intervention. In this context, telomestatin, a G-quadruplex-specific ligand known to bind and stabilize G-quadruplex, is of great interest. Knowledge of the three-dimensional structure of telomeric quadruplex and its complex with telomestatin in solution is a prerequisite for structure-based rational drug design. Here, we report the relative stabilities of human telomeric quadruplex (AG3[T2AG3]3) structures under K+ ion conditions and their binding interaction with telomestatin, as determined by molecular dynamics simulations followed by energy calculations. The energetics study shows that, in the presence of K+ ions, mixed hybrid-type Tel-22 quadruplex conformations are more stable than other conformations. The binding free energy for quadruplex-telomestatin interactions suggests that 1:2 binding is favored over 1:1 binding. To further substantiate our results, we also calculated the change in solvent-accessible surface area (DeltaSASA) and heat capacity (DeltaCp) associated with 1:1 and 1:2 binding modes. The extensive investigation performed for quadruplex-telomestatin interaction will assist in understanding the parameters influencing the quadruplex-ligand interaction and will serve as a platform for rational drug design.     

Discrimination of G‐Quadruplexes from Duplex and Single‐Stranded DNAs with Fluorescence and Energy‐Transfer Fluorescence Spectra of Crystal Violet

G‐rich nucleic acid sequences with the potential to form G‐quadruplex structures are common in biologically important regions. Most of these sequences are present with their complementary strands, so the development of a sensitive biosensor to distinguish G‐quadruplex and duplex structures and to determine the competitive ability of quadruplex to duplex structures has received a great deal of attention. In this work, the interactions between two triphenylmethane dyes (malachite green (MG) and crystal violet (CV)) and G‐quadruplex, duplex, or single‐stranded DNAs were studied by fluorescence spectroscopy and energy‐transfer fluorescence spectroscopy. Good discrimination between quadruplexes and duplex or single‐stranded DNAs can be achieved by using the fluorescence spectrum of CV or the energy‐transfer fluorescence spectra of CV and MG. In addition, by using energy‐transfer fluorescence titrations of CV with G‐quadruplexes, the binding‐stoichiometry ratios of CV to G‐quadruplexes can be determined. By using the fluorescence titrations of G‐quadruplex–CV complexes with C‐rich complementary strands, the fraction of G‐rich oligonucleotide that engages in G‐quadruplex structures in the presence of the complementary sequence can be measured. This study may provide a simple method for discrimination between quadruplexes and duplex or single‐stranded DNAs and for measuring G‐quadruplex percentages in the presence of the complementary C‐rich sequences.     

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.     

Platinum phenanthroimidazole complexes as G-quadruplex DNA selective binders

Complexes that bind and stabilize G-quadruplex DNA structures are of significant interest due to their potential to inhibit telomerase and halt tumor cell proliferation. We here report the synthesis of the first Pt(II) G-quadruplex selective molecules, containing pi-extended phenanthroimidazole ligands. Binding studies of these complexes with duplex and quadruplex d(T(4)G(4)T(4))(4) DNA were performed. Intercalation to duplex DNA was established through UV/Vis titration, CD spectroscopy, and thermal denaturation studies. Significantly stronger binding affinity of these phenanthroimidazole Pt(II) complexes to G-quadruplex DNA was observed by UV/Vis spectroscopy and competitive equilibrium dialysis studies. Observed binding constants to quadruplex DNA were nearly two orders of magnitude greater than for duplex DNA. Circular dichroism studies show that an increase in pi-surface leads to a significant increase in the thermal stability of the Pt(II)/quadruplex DNA complex. The match in the pi-surface of these phenanthroimidazole Pt(II) complexes with quadruplex DNA was further substantiated by molecular modeling studies. Numerous favorable pi-stacking interactions with the large aromatic surface of the intermolecular G-quadruplex, and unforeseen hydrogen bonds between the ancillary ethylenediamine ligands and the quadruplex phosphate backbone are predicted. Thus, both biological and computational studies suggest that coupling the square-planar geometry of Pt(II) with pi-extended ligands results in a simple and modular method to create effective G-quadruplex selective binders, which can be readily optimized for use in telomerase-based antitumor therapy.     

Site-specific probing of oxidative reactivity and telomerase function using 7,8-dihydro-8-oxoguanine in telomeric DNA

Telomeres at the ends of human chromosomes contain the repeating sequence 5'-d[(TTAGGG)(n)]-3'. Oxidative damage of guanine in DNAs that contain telomeric and nontelomeric sequence generates 7,8-dihydro-8-oxoguanine (8OG) preferentially in the telomeric segment, because GGG sequences are more reactive in duplex DNA. We have developed a general strategy for probing site-specific oxidation reactivity in diverse biological structures through substitution of minimally modified building blocks that are more reactive than the parent residue, but preserve the parent structure. In this study, 8OG was substituted for guanine at G(8), G(9), G(14), or G(15) in the human telomeric oligonucleotide 5'-d[AGGGTTAG(8)G(9)GTT AG(14)G(15)GTTAGGGTGT]-3'. Replacement of G by 8OG in telomeric DNA can affect the formation of intramolecular G quadruplexes, depending on the position of substitution. When 8OG was incorporated in the 5'-position of a GGG triplet, G quadruplex formation was observed; however, substitution of 8OG in the middle of a GGG triplet produced multiple structures. A clear correspondence between structure and reactivity was observed when oligonucleotides containing 8OG in the 5'-position of a GGG triplet were prepared in the quadruplex or duplex forms and interrogated by mediated electrocatalytic oxidation with Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine). The rate constant for one-electron oxidation of a single 8OG in the 5'-position of a GGG triplet was (6.2 +/- 1.7) x 10(4) M(-1) s(-1) in the G quadruplex form. The rate constant was 2-fold lower for the same telomeric sequence in the duplex form ((3.0 +/- 1.3) x 10(4) M(-1) s(-1)). The position of 8OG in the GGG triplet affects telomerase activity and synthesis of telomeric repeat products. Telomerase activity was decreased significantly when 8OG was substituted in the 5'-position of the GGG triplet, but not when 8OG was substituted in the middle of the triplet. Thus, biological oxidation of G to 8OG in telomeres has the potential to modulate telomerase activity. Further, small molecules that inhibit telomerase by stabilizing telomeric G quadruplexes may not be as effective under oxidative stress.     

Selective G-quadruplex DNA stabilizing agents based on bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (～0.2-0.4 μM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the T(m) of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (ΔT(m) ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures.