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.     

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.     

Cationic Pentaheteroaryls as Selective G‐Quadruplex Ligands by Solvent‐Free Microwave‐Assisted Synthesis

We report herein a solvent‐free and microwaved‐assisted synthesis of several water soluble acyclic pentaheteroaryls containing 1,2,4‐oxadiazole moieties ( 1 – 7 ). Their binding interactions with DNA quadruplex structures were thoroughly investigated by FRET melting, fluorescent intercalator displacement assay (G4‐FID) and CD spectroscopy. Among the G‐quadruplexes considered, attention was focused on telomeric repeats together with the proto‐oncogenic c‐kit sequences and the c‐myc oncogene promoter. Compound 1 , and to a lesser extent 2 and 5 , preferentially stabilise an antiparallel structure of the telomeric DNA motif, and exhibit an opposite binding behaviour to structurally related polyoxazole ( TOxaPy ), and do not bind duplex DNA. The efficiency and selectivity of the binding process was remarkably controlled by the structure of the solubilising moieties.     

Towards the Development of Structure‐Selective G‐Quadruplex‐Binding Indolo[3,2‐b]quinolines

The interaction of phenyl‐substituted indolo[3,2‐b]quinolines with DNA G‐quadruplexes of different topology were studied by using a combination of spectroscopic and calorimetric methodologies. N5‐Methylated indoloquinoline derivatives (^MePIQ) with an aminoalkyl side chain exhibit high affinities for the parallel‐stranded MYC quadruplex and a (3+1)‐hybrid structure combined with an excellent discrimination against the antiparallel thrombin‐binding aptamer (TBA) and the human telomeric (HT) quadruplexes. Dissociation constants for the binding of the ligand to the MYC quadruplex are in the submicromolar range, being below the corresponding dissociation constants for the antiparallel‐stranded quadruplexes by about one order of magnitude. Competition experiments with double‐helical DNA reveal the impact of indoloquinoline structural features on the selectivity for the parallel quadruplex relative to duplex DNA. Based on a calorimetric analysis binding to MYC is shown to be equally driven by favorable enthalpic and entropic contributions with no significant impact on the type of cation present.     

Copper‐Induced Topology Switching and Thrombin Inhibition with Telomeric DNA G‐Quadruplexes

The topological diversity of DNA G‐quadruplexes may play a crucial role in its biological function. Reversible control over a specific folding topology was achieved by the synthesis of a chiral, glycol‐based pyridine ligand and its fourfold incorporation into human telomeric DNA by solid‐phase synthesis. Square‐planar coordination to a Cu^II ion led to the formation of a highly stabilizing intramolecular metal–base tetrad, substituting one G‐tetrad in the parent unimolecular G‐quadruplex. For the Tetrahymena telomeric repeat, Cu^II‐triggered switching from a hybrid‐dominated conformer mixture to an antiparallel topology was observed. Cu^II‐dependent control over a protein–G‐quadruplex interaction was shown for the thrombin–tba pair (tba=thrombin‐binding aptamer).     

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.     

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.     

Neutral and Negatively Charged Phosphate Modifications Altering Thermal Stability,Kinetics of Formation and Monovalent Ion Dependence of DNA G‐Quadruplexes

The effect of phosphate group modifications on formation and properties of G‐quadruplexes (G4s) has not been investigated in detail. Here, we evaluated the structural, thermodynamic and kinetic properties of the parallel G‐quadruplexes formed by oligodeoxynucleotides d(G₄T), d(TG₄T) and d(TG₅T), in which all phosphates were replaced with N‐methanesulfonyl (mesyl) phosphoramidate or phosphoryl guanidine groups resulting in either negatively charged or neutral DNA sequences, respectively. We established that all modified sequences were able to form G‐quadruplexes of parallel topology; however, the presence of modifications led to a decrease in thermal stability relative to unmodified G4s. In contrast to negatively charged G4s, assembly of neutral G4 DNA species was faster in the presence of sodium ions than potassium ions, and was independent of the salt concentration used. Formation of mixed G4s composed of both native and neutral G‐rich strands has been detected using native gel electrophoresis, size‐exclusion chromatography and ESI‐MS. In summary, our results indicate that the phosphate modifications studied are compatible with G‐quadruplex formation, which could be used for the design of biologically active compounds.     

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).     

Ion‐Selective Formation of a Guanine Quadruplex on DNA Origami Structures

DNA origami nanostructures are a versatile tool that can be used to arrange functionalities with high local control to study molecular processes at a single‐molecule level. Here, we demonstrate that DNA origami substrates can be used to suppress the formation of specific guanine (G) quadruplex structures from telomeric DNA. The folding of telomeres into G‐quadruplex structures in the presence of monovalent cations (e.g. Na⁺ and K⁺) is currently used for the detection of K⁺ ions, however, with insufficient selectivity towards Na⁺. By means of FRET between two suitable dyes attached to the 3′‐ and 5′‐ends of telomeric DNA we demonstrate that the formation of G‐quadruplexes on DNA origami templates in the presence of sodium ions is suppressed due to steric hindrance. Hence, telomeric DNA attached to DNA origami structures represents a highly sensitive and selective detection tool for potassium ions even in the presence of high concentrations of sodium ions.     

Solution NMR Structure of a Ligand/Hybrid‐2‐G‐Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding

Telomeric G‐quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G‐quadruplex that adopts the biologically relevant hybrid‐2 conformation in a ligand‐bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G‐quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G‐quadruplex. The ligand is sandwiched between one terminal G‐tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G‐quadruplex structure as observed for other G‐quadruplexes in different conformations, invalidating simple docking approaches to ligand–G‐quadruplex structure determination.     

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.     

An Unprecedented Knot‐like G‐Quadruplex Peripheral Motif

A knot‐like G‐quadruplex peripheral structure is formed by a 7‐nt DNA sequence DL7 (TGTTGGT), in which six out of its seven nucleobases participate in compact base‐pairing interactions. Here, the solution NMR structure of a 24‐nt DNA oligonucleotide containing the DL7 sequence shows the interaction between a two‐layer anti‐parallel G‐quadruplex core and the peripheral knot‐like structure, including the construction of two sharp turns in the DNA backbone. The formation of this novel structural element highlights the intricate properties of single‐stranded DNA folding in presence of G‐quadruplex‐forming motifs. We demonstrated the compatibility of the DL7 knot‐like structure with various G‐quadruplexes, which could have implications in drug design and DNA engineering.     

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.     

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.     

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.     

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.     

Selective and Cooperative Ligand Binding to Antiparallel Human Telomeric DNA G‐Quadruplexes

The quest for ligands that specifically bind to particular G‐quadruplex nucleic acid structures is particularly important to conceive molecules with specific effects on gene expression or telomere maintenance, or conceive structure‐specific molecular probes. Using electrospray mass spectrometry in native conditions, we reveal a highly cooperative and selective 2:1 binding of Cu^II‐tolylterpyridine complexes to human telomeric G‐quadruplexes. Circular dichroism and comparisons of affinities for different sequences reveal a marked preference for antiparallel structures with diagonal loops and/or wide‐medium–narrow‐medium groove‐width order. The cooperativity is attributed to conformational changes in the polymorphic telomeric G‐quadruplex sequences, which convert preferably into an antiparallel three‐quartet topology upon binding of two ligands.