An Aggregating Amphiphilic Squaraine: A Light‐up Probe That Discriminates Parallel G‐Quadruplexes

G‐quadruplexes (G4s) are peculiar DNA or RNA tertiary structures that are involved in the regulation of many biological events within mammalian cells, bacteria, and viruses. Although their role as versatile therapeutic targets has been emphasized for 35 years, G4 selectivity over ubiquitous double‐stranded DNA/RNA, as well as G4 differentiation by small molecules, still remains challenging. Here, a new amphiphilic dicyanovinyl‐substituted squaraine, SQgl , is reported to act as an NIR fluorescent light‐up probe discriminating an extensive panel of parallel G4s while it is non‐fluorescent in the aggregated state. The squaraine can form an unconventional sandwich π‐complex binding two quadruplexes, which leads to a strongly fluorescent (Φ _F=0.61) supramolecular architecture. SQgl is highly selective against non‐quadruplex and non‐parallel G4 sequences without altering their topology, as desired for applications in selective in vivo high‐resolution imaging and theranostics.     

Identification of Compounds that Selectively Stabilize Specific G‐Quadruplex Structures by Using a Thioflavin T‐Displacement Assay as a Tool

Small molecules are used in the G‐quadruplex (G4) research field in vivo and in vitro, and there are increasing demands for ligands that selectively stabilize different G4 structures. Thioflavin T (ThT) emits an enhanced fluorescence signal when binding to G4 structures. Herein, we show that ThT can be competitively displaced by the binding of small molecules to G4 structures and develop a ThT‐displacement high‐throughput screening assay to find novel and selective G4‐binding compounds. We screened approximately 28 000 compounds by using three different G4 structures and identified eight novel G4 binders. Analysis of the structural conformation and stability of the G4 structures in presence of these compounds demonstrated that the four compounds enhance the thermal stabilization of the structures without affecting their structural conformation. In addition, all four compounds also increased the G4‐structure block of DNA synthesis by Taq DNA polymerase. Also, two of these compounds showed selectivity between certain Schizosaccharomyces pombe G4 structures, thus suggesting that these compounds or their analogues can be used as selective tools for G4 DNA studies.     

The Development of G‐Quadruplex‐Based Assays for the Detection of Small Molecules and Toxic Substances

G‐Quadruplexes can be induced to form guanine‐rich DNA sequences by certain small molecules or metal ions. In concert with an appropriate signal transducer, such as a fluorescent dye or a phosphorescent metal complex, the ligand‐recognition event can be transduced into a luminescent response. This focus review aims to highlight recent examples of aptamer‐based and metal‐mediated G‐quadruplex assays for the detection of small molecules and toxic substances in the last three years. We discuss the mechanisms and features of the different assays and present an outlook and a perspective for the future of this field.     

Crystallization of DNA‐Capped Gold Nanoparticles in High‐Concentration,Divalent Salt Environments

The multiparametric nature of nanoparticle self‐assembly makes it challenging to circumvent the instabilities that lead to aggregation and achieve crystallization under extreme conditions. By using non‐base‐pairing DNA as a model ligand instead of the typical base‐pairing design for programmability, long‐range 2D DNA–gold nanoparticle crystals can be obtained at extremely high salt concentrations and in a divalent salt environment. The interparticle spacings in these 2D nanoparticle crystals can be engineered and further tuned based on an empirical model incorporating the parameters of ligand length and ionic strength.     

Crystallization of DNA‐Capped Gold Nanoparticles in High‐Concentration,Divalent Salt Environments

The multiparametric nature of nanoparticle self‐assembly makes it challenging to circumvent the instabilities that lead to aggregation and achieve crystallization under extreme conditions. By using non‐base‐pairing DNA as a model ligand instead of the typical base‐pairing design for programmability, long‐range 2D DNA–gold nanoparticle crystals can be obtained at extremely high salt concentrations and in a divalent salt environment. The interparticle spacings in these 2D nanoparticle crystals can be engineered and further tuned based on an empirical model incorporating the parameters of ligand length and ionic strength.     

Delineation of G‐Quadruplex Alkylation Sites Mediated by 3,6‐Bis(1‐methyl‐4‐vinylpyridinium iodide)carbazole‐Aniline Mustard Conjugates

A new G‐quadruplex (G‐4)‐directing alkylating agent BMVC‐C3M was designed and synthesized to integrate 3,6‐bis(1‐methyl‐4‐vinylpyridinium iodide)carbazole (BMVC) with aniline mustard. Various telomeric G‐4 structures (hybrid‐2 type and antiparallel) and an oncogene promoter, c‐MYC (parallel), were constructed to react with BMVC‐C3M, yielding 35 % alkylation yield toward G‐4 DNA over other DNA categories (<6 %) and high specificity under competition conditions. Analysis of the intact alkylation adducts by electrospray ionization mass spectroscopy (ESI‐MS) revealed the stepwise DNA alkylation mechanism of aniline mustard for the first time. Furthermore, the monoalkylation sites and intrastrand cross‐linking sites were determined and found to be dependent on G‐4 topology based on the results of footprinting analysis in combination with mass spectroscopic techniques and in silico modeling. The results indicated that BMVC‐C3M preferentially alkylated at A15 (H26), G12 (H24), and G2 (c‐MYC), respectively, as monoalkylated adducts and formed A15–C3M–A21 (H26), G12–C3M–G4 (H24), and G2–C3M–G4/G17 (c‐MYC), respectively, as cross‐linked dialkylated adducts. Collectively, the stability and site‐selective cross‐linking capacity of BMVC‐C3M provides a credible tool for the structural and functional characterization of G‐4 DNAs in biological systems.     

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.     

Synthesis,Characterization, Crystal Structure,and Biological Activities of Transition Metal Complexes with 1‐Phenyl‐3‐methyl‐5‐hydroxypyrazole‐4‐methylene‐8′‐quinolineimine

The Schiff base ligand, 1‐phenyl‐3‐methyl‐5‐hydroxypyrazole‐4‐methylene‐8′‐quinolineimine, and its Cu^II, Zn^II, and Ni^II complexes were synthesized and characterized. The crystal structure of the Zn^II complex was determined by single‐crystal X‐ray diffraction, indicating that the metal ions and Schiff base ligand can form mononuclear six‐coordination complexes with 1:1 metal‐to‐ligand stoichiometry at the metal ions as centers. The binding mechanism and affinity of the ligand and its metal complexes to calf thymus DNA (CT DNA) were investigated by UV/Vis spectroscopy, fluorescence titration spectroscopy, EB displacement experiments, and viscosity measurements, indicating that the free ligand and its metal complexes can bind to DNA via an intercalation mode with the binding constants at the order of magnitude of 105–106 M ^–1, and the metal complexes can bind to DNA more strongly than the free ligand alone. In addition, antioxidant activities of the ligand and its metal complexes were investigated through scavenging effects for hydroxyl radical in vitro, indicating that the compounds show stronger antioxidant activities than some standard antioxidants, such as mannitol. The ligand and its metal complexes were subjected to cytotoxic tests, and experimental results indicated that the metal complexes show significant cytotoxic activity against lung cancer A 549 cells.     

Macrocyclic Polyoxazoles as G‐Quadruplex Ligands

This review deals with recent progress in the synthesis and evaluation of our telomestatin‐inspired macrocyclic polyoxazoles as G‐quadruplex (G4) ligands. The hexaoxazole derivatives (6OTDs) interact with and stabilize G4‐forming oligonucleotides, depending upon the character of the side chain functional groups. Cationic functional groups are particularly effective due to their secondary interaction with phosphate in the DNA backbone. On the other hand, heptaoxazole derivatives (7OTDs) showed potent G4‐binding and stabilization activity regardless of the functional groups on the side chain. A caged G4 ligand, Y2Nv2‐6OTD ( 7 ), and a fluorescent G4 ligand, L1BOD‐7OTD ( 13 ), have been synthesized.     

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.     

Metal‐complex formation and DNA interaction of 5, 10,15,20‐tetrakis(1‐methyl‐4‐pyridiyl)‐porphine: Study of the mechanistic aspects

The macrocyclic porphyrin 5,10,15,20‐tetrakis(1‐methyl‐4‐pyridiyl)‐porphine is studied in its ability to coordinate Cu(II) even at very low pH values and to interact, as a copper complex, with calf‐thymus (CT‐DNA). The kinetics and equilibria for metal‐ligand complexes formation are spectrophotometrically studied, particularly focussing on the mechanistic information provided by the kinetic approach. The rate constants of complex formation is much lower than that of water exchange at Cu(II); this behavior is ascribed to an equilibrium between two porphyrin populations, only one of them being reactive. Concerning the interaction of the copper–porphyrin complex (D) with CT‐DNA, it has been found that the complex binds to G–C base pairs by intercalation while forms external complex with the A–T base pairs. The kinetic results agree with a reaction mechanism that takes into account the slow shuffling from an AT‐bound form (DAT) to a GC‐bound form (DGC) of the copper complex (D), finally leading to a more stable DGC* intercalated form. Kinetic and equilibrium parameters for the copper complex binding to the nucleic acid are obtained, and the binding mechanism is discussed. A mechanism is proposed where D reacts simultaneously with (G–C) and (A–T) base pairs. The resulting bound forms interconvert according to a “shuffling” process, which involves formation of an intermediate (DGC) form. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 42: 79–89, 2010     

A Quadruplex‐Based,Label‐Free,and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

We demonstrate a unique quadruplex‐based fluorescence assay for sensitive, facile, real‐time, and label‐free detection of RNase H activity and inhibition by using a G‐quadruplex formation strategy. In our approach, a RNA–DNA substrate was prepared, with the DNA strand designed as a quadruplex‐forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G‐rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G‐quadruplex binder, N‐methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high‐throughput screening of enzyme inhibitors and demonstrates that the structure of the G‐quadruplex can be used as a functional tool in specific fields in the future.     

Binding of a Telomestatin Derivative Changes the Mechanical Anisotropy of a Human Telomeric G‐Quadruplex

Mechanical anisotropy is an essential property for biomolecules to assume structural and functional roles in mechanobiology. However, there is insufficient information on the mechanical anisotropy of ligand–biomolecule complexes. Herein, we investigated the mechanical property of individual human telomeric G‐quadruplexes bound to telomestatin, using optical tweezers. Stacking of the ligand to the G‐tetrad planes changes the conformation of the G‐quadruplex, which resembles a balloon squeezed in certain directions. Such a squeezed balloon effect strengthens the G‐tetrad planes, but dislocates and weakens the loops in the G‐quadruplex upon ligand binding. These dynamic interactions indicate that the binding between the ligand and G‐quadruplex follows the induced‐fit model. We anticipate that the altered mechanical anisotropy of the ligand–G‐quadruplex complex can add additional level of regulations on the motor enzymes that process DNA or RNA molecules.     

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.     

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).     

Anticancer Activity of Hydrogen‐Bond‐Stabilized Half‐Sandwich RuII Complexes with Heterocycles

Neutral half‐sandwich organometallic ruthenium(II) complexes of the type [(η⁶‐cymene)RuCl₂(L)] ( H1 – H10 ), where L represents a heterocyclic ligand, have been synthesized and characterized spectroscopically. The structures of five complexes were also established by single‐crystal X‐ray diffraction confirming a piano‐stool geometry with η⁶ coordination of the arene ligand. Hydrogen bonding between the N? H group of the heterocycle and a chlorine atom attached to Ru stabilizes the metal–ligand interaction. Complexes coordinated to a mercaptobenzothiazole framework ( H1 ) or mercaptobenzoxazole ( H6 ) showed high cytotoxicity against several cancer cells but not against normal cells. In vitro studies have shown that the inhibition of cancer cell growth involves primarily G1‐phase arrest as well as the generation of reactive oxygen species (ROS). The complexes are found to bind DNA in a non‐intercalative fashion and cause unwinding of plasmid DNA in a cell‐free medium. Surprisingly, the cytotoxic complexes H1 and H6 differ in their interaction with DNA, as observed by biophysical studies, they either cause a biphasic melting of the DNA or the inhibition of topoisomerase IIα activity, respectively. Substitution of the aromatic ring of the heterocycle or adding a second hydrogen‐bond donor on the heterocycle reduces the cytotoxicity.     

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.     

Introduction of Ketamine as a G‐Quadruplex‐Binding Ligand Using Platinum Nanoparticle Modified Carbon Paste Electrode

We designed an electrochemical platform by modifying a carbon paste electrode (CPE) with platinum nanoparticles to study the interaction between ketamine and the G‐quadruplex structure of human telomeric DNA (G₄HTD). The drug ketamine (Kt) was used as the model ligand and its ability for stabilizing the G‐quadruplex structure was examined. The modified CPE (NPtCPE) was characterized by differential pulse voltammetry (DPV) and atomic force microscopy (AFM). The interaction of Kt with G₄HTD was studied by DPV and the DPV current decreased with increasing Kt concentration. The results from UV‐vis and circular dichroism (CD) spectroscopy showed a prominent intercalation mode between G₄HTD and Kt.     

Monomer–Dimer Equilibrium for the 5′–5′ Stacking of Propeller‐Type Parallel‐Stranded G‐Quadruplexes: NMR Structural Study

Guanine‐rich sequence motifs, which contain tracts of three consecutive guanines connected by single non‐guanine nucleotides, are abundant in the human genome and can form a robust G‐quadruplex structure with high stability. Herein, by using NMR spectroscopy, we investigate the equilibrium between monomeric and 5′–5′ stacked dimeric propeller‐type G‐quadruplexes that are formed by DNA sequences containing GGGT motifs. We show that the monomer–dimer equilibrium depends on a number of parameters, including the DNA concentration, DNA flanking sequences, the concentration and type of cations, and the temperature. We report on the high‐definition structure of a simple monomeric G‐quadruplex containing three single‐residue loops, which could serve as a reference for propeller‐type G‐quadruplex structures in solution.     

Improved purification of immunoglobulin G from plasma by mixed‐mode chromatography

Efficient loading of immunoglobulin G in mixed‐mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed‐mode ligand, 4‐(1H‐imidazol‐1‐yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15–64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification.