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.     

Interaction of Metal Complexes with G‐Quadruplex DNA

Guanine‐rich sequences of DNA can assemble into tetrastranded structures known as G‐quadruplexes. It has been suggested that these secondary DNA structures could be involved in the regulation of several key biological processes. In the human genome, guanine‐rich sequences with the potential to form G‐quadruplexes exist in the telomere as well as in promoter regions of certain oncogenes. The identification of these sequences as novel targets for the development of anticancer drugs has sparked great interest in the design of molecules that can interact with quadruplex DNA. While most reported quadruplex DNA binders are based on purely organic templates, numerous metal complexes have more recently been shown to interact effectively with this DNA secondary structure. This Review provides an overview of the important roles that metal complexes can play as quadruplex DNA binding molecules, highlighting the unique properties metals can confer to these molecules.     

An Octahedral Cobalt(III) Complex with Axial NH3 Ligands that Templates and Selectively Stabilises G-quadruplex DNA

Guanine-rich sequences of DNA are known to readily fold into tetra-stranded helical structures known as G-quadruplexes (G4). Due to their biological relevance, G4s are potential anticancer drug targets and therefore there is significant interest in molecules with high affinity for these structures. Most G4 binders are polyaromatic planar compounds which π–π stack on the G4′s guanine tetrad. However, many of these compounds are not very selective since they can also intercalate into duplex DNA. Herein we report a new class of binder based on an octahedral cobalt(III) complex that binds to G4 via a different mode involving hydrogen bonding, electrostatic interactions and π–π stacking. We show that this new compound binds selectivity to G4 over duplex DNA (particularly to the G-rich sequence of the c-myc promoter). This new octahedral complex also has the ability to template the formation of G4 DNA from the unfolded sequence. Finally, we show that upon binding to G4, the complex prevents helicase Pif1-p from unfolding the c-myc G4 structure.     

Impact of planarity of unfused aromatic molecules on G-quadruplex binding: learning from isaindigotone derivatives

G-quadruplex structures are a new class of attractive targets for DNA-interactive anticancer agents. The primary building block of this structure is the G-quartet, which is composed of four coplanar guanines and serves as the major binding site for small molecules. NMR studies and molecular dynamics simulations have suggested that the planarity of G-quartet surface has been highly dynamic in solution. To better investigate how the planarity of unfused aromatic ligand impacts on its quadruplex binding properties, a variety of planarity controllable isaindigotone derivatives were designed and synthesized. The interaction of G-quadruplex DNA with these designed ligands was systematically explored using a series of biophysical studies. The FRET-melting, SPR, and CD spectroscopy results showed that reducing the planarity of their unfused aromatic core resulted in their decreased binding affinity and stabilization ability for G-quadruplex. NMR studies also suggested that these compounds could stack on the G-quartet surface. Such results are in parallel with subsequent molecular modeling studies. A detailed binding energy analysis indicated that van der Waals energy (ΔE(vdw)) and entropy (TΔS) are responsible for their decreased quadruplex binding and stabilization effect. All these results provided insight information about how quadruplex recognition could be controlled by adjusting the planarity of ligands, which shed light on further development of unfused aromatic molecules as optimal G-quadruplex binding ligands.     

Macrocyclization of bis-indole quinolines for selective stabilization of G-quadruplex DNA structures

The recognition of G-quadruplex (G4) DNA structures as important regulatory elements in biological mechanisms, and the connection between G4s and the evolvement of different diseases, has sparked interest in developing small organic molecules targeting G4s. However, such compounds often lack drug-like properties and selectivity. Here, we describe the design and synthesis of a novel class of macrocyclic bis-indole quinolines based on their non-macrocyclic lead compounds. The effects of the macrocyclization on the ability to interact with G4 DNA structures were investigated using biophysical assays and molecular dynamic simulations. Overall, this revealed compounds with potent abilities to interact with and stabilize G4 structures and a clear selectivity for both G4 DNA over dsDNA and for parallel/hybrid G4 topologies, which could be attributed to the macrocyclic structure. Moreover, we obtained knowledge about the structure–activity relationship of importance for the macrocyclic design and how structural modifications could be made to construct improved macrocyclic compounds. Thus, the macrocyclization of G4 ligands can serve as a basis for the optimization of research tools to study G4 biology and potential therapeutics targeting G4-related diseases.

Macrocyclization improves the selectivity, affinity, and ability to stabilize G4 DNA structures.     

Affinity Chromatography-Based Assays for the Screening of Potential Ligands Selective for G-Quadruplex Structures

DNA G-quadruplexes (G4s) are key structures for the development of targeted anticancer therapies. In this context, ligands selectively interacting with G4s can represent valuable anticancer drugs. Aiming at speeding up the identification of G4-targeting synthetic or natural compounds, we developed an affinity chromatography-based assay, named G-quadruplex on Oligo Affinity Support (G4-OAS), by synthesizing G4-forming sequences on commercially available polystyrene OAS. Then, due to unspecific binding of several hydrophobic ligands on nude OAS, we moved to Controlled Pore Glass (CPG). We thus conceived an ad hoc functionalized, universal support on which both the on-support elongation and deprotection of the G4-forming oligonucleotides can be performed, along with the successive affinity chromatography-based assay, renamed as G-quadruplex on Controlled Pore Glass (G4-CPG) assay. Here we describe these assays and their applications to the screening of several libraries of chemically different putative G4 ligands. Finally, ongoing studies and outlook of our G4-CPG assay are reported.     

Selective Binding and Redox-Activity on Parallel G-Quadruplexes by Pegylated Naphthalene Diimide-Copper Complexes

G-quadruplexes (G4s) are higher-order supramolecular structures, biologically important in the regulation of many key processes. Among all, the recent discoveries relating to RNA-G4s, including their potential involvement as antiviral targets against COVID-19, have triggered the ever-increasing need to develop selective molecules able to interact with parallel G4s. Naphthalene diimides (NDIs) are widely exploited as G4 ligands, being able to induce and strongly stabilize these structures. Sometimes, a reversible NDI-G4 interaction is also associated with an irreversible one, due to the cleavage and/or modification of G4s by functional-NDIs. This is the case of NDI-Cu-DETA, a copper(II) complex able to cleave G4s in the closest proximity to the target binding site. Herein, we present two original Cu(II)-NDI complexes, inspired by NDI-Cu-DETA, differently functionalized with 2-(2-aminoethoxy)ethanol side-chains, to selectively drive redox-catalyzed activity towards parallel G4s. The selective interaction toward parallel G4 topology, controlled by the presence of 2-(2-aminoethoxy)ethanol side chains, was already firmly demonstrated by us using core-extended NDIs. In the present study, the presence of protonable moieties and the copper(II) cavity, increases the binding affinity and specificity of these two NDIs for a telomeric RNA-G4. Once defined the copper coordination relationship and binding constants by competition titrations, ability in G4 stabilization, and ROS-induced cleavage were analyzed. The propensity in the stabilization of parallel topology was highlighted for both of the new compounds HP2Cu and PE2Cu. The results obtained are particularly promising, paving the way for the development of new selective functional ligands for binding and destructuring parallel G4s.     

Neomycin-capped aromatic platforms: quadruplex DNA recognition and telomerase inhibition

A series of aminoglycoside-capped macrocyclic structures has been prepared using intramolecular bis-tethering of neomycin on three aromatic platforms (phenanthroline, acridine, quinacridine). Based on NMR and calculations studies, it was found that the cyclic compounds adopt a highly flexible structure without conformational restriction of the aminoglycoside moiety. FRET-melting stabilization measurements showed that the series displays moderate to high affinity for the G4-conformation of human telomeric repeats, this effect being correlated with the size of the aromatic moiety. In addition, a FRET competition assay evidenced the poor binding ability of all macrocycles for duplex DNA and a clear binding preference for loop-containing intramolecular G4 structures compared to tetramolecular parallel G4 DNA. Finally, TRAP experiments demonstrated that the best G4-binder (quinacridine ) is also a potent and selective telomerase inhibitor with an IC(50) in the submicromolar range (200 nM).     

Cationic helicenes as selective G4 DNA binders and optical probes for cellular imaging

The important role that G-quadruplex DNA (G4 DNA) structures play in regulating biological processes is becoming widely recognised. These structures have also been proposed to be attractive drug targets. Therefore, there has been significant interest in developing small molecules that can selectively bind to G4 DNA over other topologies. In this paper we investigate the interaction between DNA and helical compounds (helicenes) based on a central carbocation trisubstituted with aromatic rings. We show that the non-planar structure of these helicenes results in a significantly reduced affinity for dsDNA when compared to their planar analogues, whilst maintaining a high affinity for G4 DNA. Additionally, the right- and left-handed enantiomers of one of these helicenes recognise the chiral DNA environments of G4 and dsDNA differently. We show that upon DNA binding the helicenes display a fluorescence switch-on effect, which we have successfully used for cellular imaging in live and fixed U2OS cells, staining mitochondria and the nucleus, respectively.

G-quadruplex DNA (G4 DNA) structures are selectively recognised by helical optical probes.     

Selective Chemical Modification to the Higher-Order Structures of Nucleic Acids

DNA and RNA can adopt a variety of stable higher-order structural motifs, including G-quadruplex (G4 s), mismatches, and bulges. Many of these secondary structures are closely related to the regulation of gene expression. Therefore, the higher-order structure of nucleic acids is one of the candidate therapeutic targets, and the development of binding molecules targeting the higher-order structure of nucleic acids has been pursued vigorously. Furthermore, as one of the methodologies for detecting the higher-order structures of these nucleic acids, developing techniques for the selective chemical modification of the higher-order structures of nucleic acids is also underway. In this personal account, we focus on the following higher-order structures of nucleic acids, double-stranded DNA containing the abasic site, T−T/U−U mismatch structure, and G-quadruplex structure, and describe the development of molecules that bind to and chemically modify these structures.     

A Redox‐Activated G‐Quadruplex DNA Binder Based on a Platinum(IV)–Salphen Complex

There has been increasing interest in the development of small molecules that can selectively bind to G‐quadruplex DNA structures. The latter have been associated with a number of key biological processes and therefore are proposed to be potential targets for drug development. Herein, we report the first example of a reduction‐activated G‐quadruplex DNA binder. We show that a new octahedral platinum(IV)–salphen complex does not interact with DNA in aqueous media at pH 7.4; however, upon addition of bioreductants such as ascorbic acid or glutathione, the compound is readily reduced to the corresponding square planar platinum(II) complex. In contrast to the parent platinum(IV) complex, the in situ generated platinum(II) complex has good affinity for G‐quadruplex DNA.     

Synthesis and evaluation of analogues of 10H-indolo[3,2-b]quinoline as G-quadruplex stabilising ligands and potential inhibitors of the enzyme telomerase

We report here the synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of several rationally-designed quindoline analogues, substituted at the 2- and 7- positions. The ability of these compounds to interact with and stabilise an intramolecular G-quadruplex DNA against increases in temperature was evaluated by a fluorescence-based (FRET) melting assay. The resulting T(m) values were found to correlate with their potency for telomerase inhibition, as measured in an in vitro telomerase TRAP assay. The interactions of a number of compounds with a quadruplex DNA molecular structure were simulated by molecular modelling methods. It is concluded that this class of compound represents a new chemical type suitable for further development as telomerase inhibitors.     

Assessing The Key Photophysical Properties of Triangulenium Dyes for DNA Binding by Alteration of the Fluorescent Core

Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe ( DAOTA-M2 ) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.     

Novel water soluble bis(μ‐chloro) bridged Cu(II) binuclear and mononuclear complexes: Synthesis,characterization and biological evaluation

A water soluble chloro bridged binuclear copper(II) complex (3) and mononuclear complex (4) have been synthesized from chloro substituted 2‐oxo‐1,2‐dihydroquinolin‐3‐yl‐methylene‐2 hydroxybenzohydrazide 1 and 2 and CuCl₂·2H₂O. The structures of the complexes have been determined by single crystal X‐ray diffraction. The binding interactions of the ligands and complexes with CT‐DNA and protein have been evaluated by absorption and emission spectroscopic method. CT‐DNA and ethidium bromide (EB) competitive studies revealed that the compounds could interact with CT‐DNA through intercalation binding mode. Interactions of the compounds with BSA were also studied by UV−visible, fluorescence and synchronous fluorescence spectroscopic methods which showed that the compounds had a strong binding affinity with BSA through static quenching process. The cytotoxic effect of the compounds examined on cancer cell lines, such as A549 (lung cancer) and MCF7 (breast cancer) cell lines showed that all four compounds exhibited substantial cytotoxic activity.     

Dynamic imaging of single DNA-protein interactions using atomic force microscopy

Atomic force microscopy (AFM) imaging of static DNA-protein complexes, in air and in liquid, can be used to directly obtain quantitative and qualitative information on the structure of different complexes. For example, DNA length, the location of preferential binding sites for proteins and bending of DNA as a result of the complexation can all be measured. Recording consecutive AFM images of DNA and protein molecules under conditions that they are still able to move and interact, or dynamic AFM imaging, however, can reveal information on the dynamic aspects of the interactions between these molecules. Here, an overview is given of the technical challenges that need to be considered for successful dynamic AFM imaging studies of individual DNA-protein interactions. Necessary technical improvements to the AFM set-up and the development of new sample preparation methods are described in this paper.     

Biological contour,molecular docking and antiproliferative studies of DNA targeted histidine based transition metal(II) complexes: Invention and its depiction

A novel series of histidine derived transition metal complexes were synthesized and characterized by multispectral techniques such as UV‐Vis., FT IR, EPR, NMR, ESI‐mass analysis and other physico‐chemical methods like elemental analysis, molar conductivity, magnetic susceptibility. The synthesized compounds were attempted for their biological prospective. The biological studies involved are DNA interaction (binding and damage), antimicrobial, antioxidant, antiproliferative and molecular docking. DNA interaction studies were carried out with the help of UV‐Vis absorption titration, viscosity measurement and cyclic voltammetric techniques which revealed that the synthesized compounds could interact with CT‐DNA through intercalative binding mode. A gel electrophoresis assay demonstrated the ability of complexes to cleave the supercoiled pUC18 DNA. The antioxidant property shows that the metal complexes have preferable ability to scavenge hydroxyl radical than the ligand. Moreover, the antimicrobial assay indicates that these complexes are good antimicrobial agents against various pathogens. Furthermore, the in vitro antiproliferative activities of the complexes were examined on HeLa, Hep G2 and NIH 3 T3 cell lines using an MTT assay. The morphological changes were investigated using Hoechst 33258 staining apoptosis assay. In addition, molecular docking studies were executed to considerate the nature of binding of the synthesized complexes with protein and DNA.     

Specific Binding of a d‐RNA G‐Quadruplex Structure with an l‐RNA Aptamer

G‐quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4‐binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l ‐RNA aptamer that targets a d ‐RNA G‐quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l ‐RNA aptamer, Ap3‐7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA‐rG4–RHAU53 binding. Our approach and findings open a new door in further developing G4‐specific tools for diverse applications.     

Enhanced sampling molecular dynamics simulations correctly predict the diverse activities of a series of stiff-stilbene G-quadruplex DNA ligands

Ligands with the capability to bind G-quadruplexes (G4s) specifically, and to control G4 structure and behaviour, offer great potential in the development of novel therapies, technologies and functional materials. Most known ligands bind to a pre-formed topology, but G4s are highly dynamic and a small number of ligands have been discovered that influence these folding equilibria. Such ligands may be useful as probes to understand the dynamic nature of G4 in vivo, or to exploit the polymorphism of G4 in the development of molecular devices. To date, these fascinating molecules have been discovered serendipitously. There is a need for tools to predict such effects to drive ligand design and development, and for molecular-level understanding of ligand binding mechanisms and associated topological perturbation of G4 structures. Here we study the G4 binding mechanisms of a family of stiff-stilbene G4 ligands to human telomeric DNA using molecular dynamics (MD) and enhanced sampling (metadynamics) MD simulations. The simulations predict a variety of binding mechanisms and effects on G4 structure for the different ligands in the series. In parallel, we characterize the binding of the ligands to the G4 target experimentally using NMR and CD spectroscopy. The results show good agreement between the simulated and experimentally observed binding modes, binding affinities and ligand-induced perturbation of the G4 structure. The simulations correctly predict ligands that perturb G4 topology. Metadynamics simulations are shown to be a powerful tool to aid development of molecules to influence G4 structure, both in interpreting experiments and to help in the design of these chemotypes.

Enhanced sampling molecular dynamics simulations and solution-phase experiments come together to demonstrate the diverse effects of G4-interactive small molecules.     

Using Macrocyclic G-Quadruplex Ligands to Decipher the Interactions Between Small Molecules and G-Quadruplex DNA

This study aims to deepen the knowledge of the current state of rational G4-ligand design through the design and synthesis of a novel set of compounds based on indoles, quinolines, and benzofurans and their comparisons with well-known G4-ligands. This resulted in novel synthetic methods and G4-ligands that bind and stabilize G4 DNA with high selectivity. Furthermore, the study corroborates previous studies on the design of G4-ligands and adds deeper explanations to why a) macrocycles offer advantages in terms of G4-binding and -selectivity, b) molecular pre-organization is of key importance in the development of strong novel binders, c) an electron-deficient aromatic core is essential to engage in strong arene-arene interactions with the G4-surface, and d) aliphatic amines can strengthen interactions indirectly through changing the arene electrostatic nature of the compound. Finally, fundamental physicochemical properties of selected G4-binders are evaluated, underscoring the complexity of aligning the properties required for efficient G4 binding and stabilization with feasible pharmacokinetic properties.     

Templated Formation of Discrete RNA and DNA:RNA Hybrid G‐Quadruplexes and Their Interactions with Targeting Ligands

G‐rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper‐catalyzed azide‐alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G‐quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context.