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.     

Theoretical Study of Origin of Triple Fluorescences of a Squaraine Dye, Bis[4-(N,N-dimethylamino)phenyl]-squaraine

A new scheme of photo‐fluorescent emission origin, described as S₀ (relaxed state)→S_n (Frank‐Condon state)→ S_m (relaxed state)→S₀ (Frank‐Condon state), is presented to explain the multiple fluorescent emissions of squaraine dyes observed experimentally according to the configuration interaction singles calculations of relaxed excited states of a model compound, bis[4‐(N,N‐dimethylamino)phenyl]squaraine (SQ). It is exhibited that all triple fluorescent emissions of SQ have their significant origin in vertical electron transitions of different relaxed excited states. In addition, some important absorption peaks appearing in higher energy region are most likely to be responsible for the higher energy band observed in solid states of many squaraine dyes.     

Recent developments in novel blue/green/red/NIR small fluorescent probes for in cellulo tracking of RNA/DNA G-quadruplexes

The detection and stabilization of G-quadruplexes (G4s) in living systems is of enormous applicability in the fields of chemical biology and therapeutic materials. Whereas DNA serves as a genetic material, RNA functions in the regulation and expression of genetic materials. Even there is various report on fluorescent probes invitro G4s recognitions, in this review we highlighted briefly, in-cellulo identification of G4s along with conventional methods principles. Although there are varieties of G4-forming sequences in the genome, targeting a specific type (topology) in living cells is highly challenging because of the high instability of G4s in cellular/subcellular systems. In contrast, several reports describe the in vitro identification of G4s, along with in-cell demonstrations, using efficient fluorescent probes, through either intrinsic or extrinsic approaches. In the intrinsic mode, the sensing results from the use of highly selective synthetic fluorescent oligonucleotides or proteins (a labeling approach). In the extrinsic mode, quencher-free small molecular probes are used to recognize specific G4s under physiological conditions. Because of their robustness, simplicity, and ease of handling, this review describes recent trends in the use of blue/green, green, red, and near-infrared (NIR) fluorescent probes for the recognition of G4s in live cells-and, particularly, those approaches employing quencher-free probes. Also highlighted are a few labeled probes, and their in cellulo localizations, which were accomplished upon the formation of non-canonical G4s under specified conditions and supplemented by exogenous G4-forming components, without harnessing cellular physiological conditions.     

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.     

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.     

Selective,pH-Dependent Colorimetric and Fluorimetric Detection of Quadruplex DNA with 4-Dimethylamino(phenyl)-Substituted Berberine Derivatives

The 9- and 12-dimethylaminophenyl-substituted berberine derivatives 3 a and 3 b were readily synthesized by Suzuki-Miyaura reactions and shown to be useful fluorescent probes for the optical detection of quadruplex DNA (G4-DNA). Their association with the nucleic acids was investigated by spectrometric titrations, CD and LD spectroscopy, and with DNA-melting analysis. Both ligands bind to duplex DNA by intercalation and to G4-DNA by terminal π stacking. At neutral conditions, they bind with higher affinity (K_b=10⁵−10⁶ M⁻¹) to representative quadruplex forming oligonucleotides 22AG , c-myc , c-kit , and a2 , than to duplex calf thymus (ct) DNA (K_b=5-7×10⁴ M⁻¹). At pH 5, however, the affinity of 3 a towards G4-DNA 22AG is higher (K_b=1.2×10⁶ M⁻¹), whereas the binding constant towards ct DNA is lower (K_b=3.9×10³ M⁻¹) than under neutral conditions. Notably, the association of the ligand with DNA results in characteristic changes of the absorption and emission properties under specific conditions, which may be used for optical DNA detection. Other than the parent berberine, the ligands do not show a noticeable increase of their very low intrinsic emission intensity upon association with DNA at neutral conditions. In contrast, a fluorescence light-up effect was observed upon association to duplex (Φ_fl=0.01) and quadruplex DNA (Φ_fl=0.04) at pH 5. This fluorimetric response to G4-DNA association in combination with the distinct, red-shifted absorption under these conditions provides a simple and conclusive optical detection of G4-DNA at lower pH.     

Squaraine Rotaxane as a Reversible Optical Chloride Sensor

A mechanically interlocked squaraine rotaxane is comprised of a deep‐red fluorescent squaraine dye inside a tetralactam macrocycle. NMR studies show that Cl^? binding to the rotaxane induces macrocycle translocation away from the central squaraine station, a process that is completely reversed when the Cl^? is removed from the solution. Steady‐state fluorescence and excited‐state lifetime measurements show that this reversible machine‐like motion modulates several technically useful optical properties, including a three‐fold increase in deep‐red fluorescence emission that is observable to the naked eye. The excited states were characterized quantitatively by time‐correlated single photon counting, femtosecond transient absorption spectroscopy, and nanosecond laser flash photolysis. Cl^? binding to the rotaxane increases the squaraine excited singlet state lifetime from 1.5 to 3.1 ns, and decreases the excited triplet state lifetime from >200 to 44 μs. Apparently, the surrounding macrocycle quenches the excited singlet state of the encapsulated squaraine dye and stabilizes the excited triplet state. Prototype dipsticks were prepared by adsorbing the lipophilic rotaxane onto the ends of narrow, C18‐coated, reverse‐phase silica gel plates. The fluorescence intensity of a dipstick increased eighteen‐fold upon dipping in an aqueous solution of tetrabutylammonium chloride (300 mM ) and was subsequently reversed by washing with pure water. It is possible to develop the dipsticks for colorimetric determination of Cl^? levels by the naked eye. After dipping into aqueous tetrabutylammonium chloride, a dipstick’s color slowly fades at a rate that depends on the amount of Cl^? in the aqueous solution. The fading process is due primarily to hydrolytic bleaching of the squaraine chromophore within the rotaxane. That is, association of Cl^? to immobilized rotaxane induces macrocycle translocation and exposure of the electrophilic C₄O₂ core of the squaraine station, which is in turn attacked by the ambient moisture to produce a bleached product.     

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.     

Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 1 a ), its derivative with a quaternary aminoalkyl chain ( 1 b ), and the symmetric 2,6‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 2 a ), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA‐binding and DNA‐probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4‐disubstituted) dyes 1 a and 1 b bind to quadruplex DNA structures with a near‐micromolar affinity and a fair selectivity with respect to double‐stranded (ds) DNA [K_a(G4)/K_a(ds)=2.5–8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80–100‐fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double‐stranded DNA. This optical selectivity allows these dyes to be used as quadruplex‐DNA‐selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2 a displays a strong binding preference for double‐stranded DNA [K_a(ds)/K_a(G4)=40–100), presumably due to binding in the minor groove. In addition, 2 a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20–50‐fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA‐recognition properties and fluorimetric response of organic dyes.     

A Bis(methylpiperazinylstyryl)phenanthroline as a Fluorescent Ligand for G‐Quadruplexes

G‐quadruplex (G4)‐forming sequences are prevalent in the genome and are considered to play important roles in gene regulation, and hence have been viewed as potential therapeutic targets in oncology. However, the structures and functions of most G4s in the genome are poorly understood. Therefore, the development of fluorescent probes and ligands for G4s is important for G4 research and drug discovery. Herein, we report a new G4 ligand, 2,9‐bis[4‐(4‐methylpiperazin‐1‐yl)styryl]‐1,10‐phenanthroline (BMSP), which was synthesized by a simple process. BMSP exhibits almost no fluorescence in aqueous buffer. The interaction of BMSP with G4s greatly enhances its fluorescence with a large Stokes’ shift of 160 nm. Antiparallel human telomeric G4s exhibit the strongest binding affinity (K_d≈0.13 μm ) to BMSP and induce a fluorescence enhancement of up to 150‐fold. BMSP binds to G4s through π–π stacking on the terminal G‐quartets. BMSP can enter live cells, and it strongly inhibits the growth of cancer cells rather than causing cell death. Our results suggest that BMSP has the potential to serve both as a fluorescent probe for some G4s and as a chemotherapeutic agent for cancer treatment.     

Cover Picture: Expanding the Toolbox of Target Directed Bio-Orthogonal Synthesis: In Situ Direct Macrocyclization by DNA Templates (Angew. Chem. Int. Ed. 7/2023)

Herein, we demonstrate for the first time that noncanonical DNA can direct macrocyclization-like challenging reactions to synthesize gene modulators. The planar G-quartets present in DNA G-quadruplexes (G4s) provide a size complementary reaction platform for the bio-orthogonal macrocyclization of bifunctional azide and alkyne fragments over oligo- and polymerization. G4s immobilized on gold-coated magnetic nanoparticles have been used as target templates to enable easy identification of a selective peptidomimetic macrocycle. Structurally similar macrocycles have been synthesized to understand their functional role in the modulation of gene function. The innate fluorescence of the in situ formed macrocycle has been utilized to monitor its cellular localization using a G4 antibody and its in cell formation from the corresponding azide and alkyne fragments. The successful execution of in situ macrocyclization in vitro and in cells would open up a new dimension for target-directed therapeutic applications.     

Synthesis and Electronic Properties of 1,2‐Hemisquarimines and Their Encapsulation in a Cucurbit[7]uril Host

The synthesis of a new class of robust squaraine dyes, colloquially named 1,2‐hemisquarimines (1,2‐HSQiMs), through the microwave‐assisted condensation of aniline derivatives with the 1,2‐squaraine core is reported. In CH₃CN, 1,2‐HSQiMs show a broad absorption band with a high extinction coefficient and a maximum at around λ=530 nm, as well as an emission band centered at about λ=574 nm, that are pH dependent. Protonation of the imine nitrogen causes a redshift of both absorption and emission maxima, with a concomitant increase in the lifetime of the emitting excited state. Encapsulation of the chromophore into a cucurbit[7]uril host revealed fluorescence enhancement and increased photostability in water. The redox characteristics of 1,2‐HSQiMs indicate that charge injection into TiO₂ is possible; this opens up promising perspectives for their use as photosensitizers for solar energy conversion.     

The Thiophene “Sigma‐Hole” as a Concept for Preorganized,Specific Recognition of G⋅C Base Pairs in the DNA Minor Groove

In spite of its importance in cell function, targeting DNA is under‐represented in the design of small molecules. A barrier to progress in this area is the lack of a variety of modules that recognize G ? C base pairs (bp) in DNA sequences. To overcome this barrier, an entirely new design concept for modules that can bind to mixed G ? C and A ? T sequences of DNA is reported herein. Because of their successes in biological applications, minor‐groove‐binding heterocyclic cations were selected as the platform for design. Binding to A ? T sequences requires hydrogen‐bond donors whereas recognition of the G‐NH₂ requires an acceptor. The concept that we report herein uses pre‐organized N‐methylbenzimidazole (N‐MeBI) thiophene modules for selective binding with mixed bp DNA sequences. The interaction between the thiophene sigma hole (positive electrostatic potential) and the electron‐donor nitrogen of N‐MeBI preorganizes the conformation for accepting an hydrogen bond from G‐NH₂. The compound–DNA interactions were evaluated with a powerful array of biophysical methods and the results show that N‐MeBI‐thiophene monomer compounds can strongly and selectively recognize single G ? C bp sequences. Replacing the thiophene with other moieties significantly reduces binding affinity and specificity, as predicted by the design concept. These results show that the use of molecular features, such as sigma‐holes, can lead to new approaches for small molecules in biomolecular interactions.     

Stabilization of Human Telomeric G‐Quadruplex and Inhibition of Telomerase Activity by Propeller‐Shaped Trinuclear PtII Complexes

Two novel propeller‐shaped, trigeminal‐ligand‐containing, flexible trinuclear Pt^II complexes, {[Pt(dien)]₃(ptp)}(NO₃)₆ ( 1 ) and {[Pt(dpa)]₃(ptp)}(NO₃)₆ ( 2 ) (dien: diethylenetriamine; dpa: bis‐(2‐pyridylmethyl)amine; ptp: 6′‐(pyridin‐3‐yl)‐3,2′:4′,3′′‐terpyridine), have been designed and synthesized, and their interactions with G‐quadruplex (G4) sequences are characterized. A combination of biophysical and biochemical assays reveals that both Pt^II complexes exhibit higher affinity for human telomeric (hTel) and c‐myc promoter G4 sequences than duplex DNA. Complex 1 binds and stabilizes hTel G4 sequence more effectively than complex 2 . Both complexes are found to induce and stabilize either antiparallel or parallel conformation of G4 structures. Molecular docking studies indicate that complex 1 binds into the large groove of the antiparallel hTel G4 structure (PDB ID: 143D) and complex 2 stacks onto the exposed G‐quartet of the parallel hTel G4 structure (PDB ID: 1KF1). Telomeric repeat amplification protocol assays demonstrate that both complexes are good telomerase inhibitors, with IC₅₀ values of (16.0±0.4) μM and (4.20±0.25) μM for 1 and 2 , respectively. Collectively, the results suggest that these propeller‐shaped flexible trinuclear Pt^II complexes are effective and selective G4 binders and good telomerase inhibitors. This work provides valuable information for the interaction between multinuclear metal complexes with G4 DNA.     

DNA-Intercalative Platinum Anticancer Complexes Photoactivated by Visible Light

Photoactivatable agents offer the prospect of highly selective cancer therapy with low side effects and novel mechanisms of action that can combat current drug resistance. 1,8-Naphthalimides with their extended π system can behave as light-harvesting groups, fluorescent probes and DNA intercalators. We conjugated N-(carboxymethyl)-1,8-naphthalimide (gly-R-Nap) with an R substituent on the naphthyl group to photoactive diazido Pt^IV complexes to form t,t,t-[Pt(py)₂(N₃)₂(OH)(gly-R-Nap)], R=H ( 1 ), 3-NO₂ ( 2 ) or 4-NMe₂ ( 3 ). They show enhanced photo-oxidation, cellular accumulation and promising photo-cytotoxicity in human A2780 ovarian, A549 lung and PC3 prostate cancer cells with visible light activation, and low dark cytotoxicity. Complexes 1 and 2 exhibit pre-intercalation into DNA, resulting in enhanced photo-induced DNA crosslinking. Complex 3 has a red-shifted absorption band at 450 nm, allowing photoactivation and photo-cytotoxicity with green light.     

The crystal structure of the CeNA:RNA hybrid ce(GCGTAGCG):r(CGCUACGC)

Cyclohexenyl nucleic acids (CeNA) are characterised by the carbon–carbon double bond replacing the O4′‐oxygen atom of the natural D ‐2′‐deoxyribose sugar ring in DNA. CeNAs exhibit a high conformational flexibility, are stable against nuclease activity and their hybridisation is RNA selective. Additionally, CeNA has been shown to induce an enhanced biological activity when incorporated in siRNA. This makes CeNA a good candidate for siRNA and synthetic aptamer applications. The crystal structure of the synthetic CeNA:RNA hybrid ce(GCGTAGCG):r(CGCUACGC) has been solved with a resolution of 2.50 Å. The CeNA:RNA duplex adopts an anti‐parallel, right‐handed double helix with standard Watson–Crick base pairing. Analyses of the helical parameters revealed the octamer to form an A‐like double helix. The cyclohexenyl rings mainly adopt the ³H₂ conformation, which resembles the C3′‐endo conformation of RNA ribose ring. This C3′‐endo ring puckering was found in most of the RNA residues and is typical for A‐family helices. The crystal structure is stabilised by the presence of hexahydrated magnesium ions. The fact that the CeNA:RNA hybrid adopts an A‐type double helical conformation confirms the high potential of CeNAs for the construction of efficient siRNAs which can be used for therapeutical applications.     

Structure‐Based Design of Platinum(II) Complexes as c‐myc Oncogene Down‐Regulators and Luminescent Probes for G‐Quadruplex DNA

A series of platinum(II) complexes with tridentate ligands was synthesized and their interactions with G‐quadruplex DNA within the c‐myc gene promoter were evaluated. Complex 1 , which has a flat planar 2,6‐bis(benzimidazol‐2‐yl)pyridine (bzimpy) scaffold, was found to stabilize the c‐myc G‐quadruplex structure in a cell‐free system. An in silico G‐quadruplex DNA model has been constructed for structure‐based virtual screening to develop new Pt^II‐based complexes with superior inhibitory activities. By using complex 1 as the initial structure for hit‐to‐lead optimization, bzimpy and related 2,6‐bis(pyrazol‐3‐yl)pyridine (dPzPy) scaffolds containing amine side‐chains emerge as the top candidates. Six of the top‐scoring complexes were synthesized and their interactions with c‐myc G‐quadruplex DNA have been investigated. The results revealed that all of the complexes have the ability to stabilize the c‐myc G‐quadruplex. Complex 3 a ([Pt^II L2R ] ⁺ ; L2 =2,6‐bis[1‐(3‐piperidinepropyl)‐1H‐enzo[d]imidazol‐2‐yl]pyridine, R =Cl) displayed the strongest inhibition in a cell‐free system (IC₅₀=2.2 μM ) and was 3.3‐fold more potent than that of 1 . Complexes 3 a and 4 a ([Pt^II L3R ]⁺; L3 =2,6‐bis[1‐(3‐morpholinopropyl)‐1H‐pyrazol‐3‐yl]pyridine, R =Cl) were found to effectively inhibit c‐myc gene expression in human hepatocarcinoma cells with IC₅₀ values of ≈17 μM , whereas initial hit 1 displayed no significant effect on gene expression at concentrations up to 50 μM . Complexes 3 a and 4 a have a strong preference for G‐quadruplex DNA over duplex DNA, as revealed by competition dialysis experiments and absorption titration; 3 a and 4 a bind G‐quadruplex DNA with binding constants (K) of approximately 10⁶–10⁷ dm³ mol^?1, which are at least an order of magnitude higher than the K values for duplex DNA. NMR spectroscopic titration experiments and molecular modeling showed that 4 a binds c‐myc G‐quadruplex DNA through an external end‐stacking mode at the 3′‐terminal face of the G‐quadruplex. Intriguingly, binding of c‐myc G‐quadruplex DNA by 3 b is accompanied by an increase of up to 38‐fold in photoluminescence intensity at λ_max=622 nm.     

A new selective ‘turn-on’ small fluorescent cationic probe for recognition of RNA in cells

Fluorescent imaging probes have revolutionised cell biology by monitoring cellular objects. However, the lack of fluorescent probes with high selectivity for RNA has been a drawback. Thus, selective RNA binding for fluorescent sensors is essential. Here, we report the selective fluorescence enhancement upon addition of RNA. By exploiting a selective recognition of small tetra-cationic probe 1 for RNA, we also explain the possible binding mode for RNA. As a membrane-permeant fluorescence probe, 1 provides selective imaging of RNA not only in human neuroblastoma tumour SH-SY5Y cell line used for Parkinson's disease but also in the unicellular green alga cells. Further exploitation could open new opportunities in neurotoxin and cancer biology.     

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.