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.     

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 G‐Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

A hemin‐binding DNA G‐quadruplex (also known as a hemin aptamer or DNAzyme) has been previously reported to be able to enhance the peroxidase activity of hemin. In this work, we described a DNAzyme structure that had an effector‐recognizing part appearing as a single stranded DNA linkage flanked by two split G‐quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G‐quadruplex halves and thus efficiently suppressed the enzymatic activity of the G‐quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2′‐azinobis(3‐ethylbenzthiazoline)‐6‐sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re‐engineered G‐quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G‐quadruplex is an especially useful module to design low‐cost and label‐free sensors toward various biologically or environmentally interesting targets.     

选择性检测谷胱甘肽的荧光探针

本文设计合成了一种基于BODIPY衍生物选择性检测谷胱甘肽的比率式荧光探针1。荧光探针1中BODIPY的3位连有苯乙炔基团,5位连有咪唑盐离去基团,利用其与谷胱甘肽和半胱氨酸反应机理的不同实现了对谷胱甘肽的选择性检测。紫外可见吸收光谱和荧光光谱实验结果表明探针分子1与谷胱甘肽反应后的光谱发生明显红移,可以实现对谷胱甘肽的比率式检测。探针分子1对谷胱甘肽有极高的选择性,不受其它氨基酸尤其是半胱氨酸的干扰。荧光滴定实验表明探针分子1可实现对谷胱甘肽的定量检测,检测限为3.3×10-8 mol/L。探针分子成功地应用于活体细胞中检测谷胱甘肽。     

A Chimeric DNA/RNA Antiparallel Quadruplex with Improved Stability

Nucleic acid quadruplexes are proposed to play a role in the regulation of gene expression, are often present in aptamers selected for specific binding functions and have potential applications in medicine and biotechnology. Therefore, understanding their structure and thermodynamic properties and designing highly stable quadruplexes is desirable for a variety of applications. Here, we evaluate DNA→RNA substitutions in the context of a monomolecular, antiparallel quadruplex, the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG) in the presence of either K⁺ or Sr²⁺. TBA predominantly folds into a chair-type configuration containing two G-tetrads, with G residues in both syn and anti conformation. All chimeras with DNA→RNA substitutions (G→g) at G residues requiring the syn conformation demonstrated strong destabilization. In contrast, G→g substitutions at Gs with anti conformation increased stability without affecting the monomolecular chair-type topology. None of the DNA→RNA substitutions in loop positions affected the quadruplex topology; however, these substitutions varied widely in their stabilizing or destabilizing effects in an unpredictable manner. This analysis allowed us to design a chimeric DNA/RNA TBA construct that demonstrated substantially improved stability relative to the all-DNA construct. These results have implications for a variety of quadruplex-based applications including for the design of dynamic nanomachines.     

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.     

Stabilization of G‐Quadruplex DNA with Platinum(II) Schiff Base Complexes: Luminescent Probe and Down‐Regulation of c‐myc Oncogene Expression

The interactions of a series of platinum(II) Schiff base complexes with c‐myc G‐quadruplex DNA were studied. Complex [PtL ^1a ] ( 1 a ; H₂L ^1a =N,N′‐bis(salicylidene)‐4,5‐methoxy‐1,2‐phenylenediamine) can moderately inhibit c‐myc gene promoter activity in a cell‐free system through stabilizing the G‐quadruplex structure and can inhibit c‐myc oncogene expression in cultured cells. The interaction between 1 a and G‐quadruplex DNA has been examined by ¹H NMR spectroscopy. By using computer‐aided structure‐based drug design for hit‐to‐lead optimization, an in silico G‐quadruplex DNA model has been constructed for docking‐based virtual screening to develop new platinum(II) Schiff base complexes with improved inhibitory activities. Complex [PtL ³ ] ( 3 ; H₂L ³ = N,N′‐bis{4‐[1‐(2‐propylpiperidine)oxy]salicylidene}‐4,5‐methoxy‐1,2‐phenylenediamine) has been identified with a top score in the virtual screening. This complex was subsequently prepared and experimentally tested in vitro for its ability to stabilize or induce the formation of the c‐myc G‐quadruplex. The inhibitory activity of 3 (IC₅₀=4.4 μM ) is tenfold more than that of 1 a . The interaction between 1 a or 3 with c‐myc G‐quadruplex DNA has been examined by absorption titration, emission titration, molecular modeling, and NMR titration experiments, thus revealing that both 1 a and 3 bind c‐myc G‐quadruplex DNA through an external end‐stacking mode at the 3’ terminal face of the G‐quadruplex. Such binding of G‐quadruplex DNA with 3 is accompanied by up to an eightfold increase in the intensity of photoluminescence at λ_max=652 nm. Complex 3 also effectively down‐regulated the expression of c‐myc in human hepatocarcinoma cells.     

基于新型不对称BODIPY的GSH荧光探针

本文通过在BODIPY母体中引入丙二腈,设计合成了一个新型不对称BODIPY荧光染料CN-B-Cl。由于丙二腈强吸电作用,荧光染料CN-B-Cl具有优异的化学活性,能够与含巯基的化合物在buffer体系中迅速发生芳香亲核取代反应;与GSH反应生成硫取代BODIPY,而与Cys/Hcy反应生成氮取代BODIPY。根据不同取代基BODIPY化合物发光性能的不同,该荧光探针可选择性区分GSH与Cys/Hcy。     

Engineering Bisquinolinium/Thiazole Orange Conjugates for Fluorescent Sensing of G‐Quadruplex DNA

Lighting up : A G‐quadruplex‐specific fluorescent probe was designed combining the specificity of the pyridodicarboxamide motif for guanine quadruplexes and the fluorescence properties of thiazole orange. While the assembly of the two partners through a flexible linker leads to a nonselective probe, merging them in a single, rigid scaffold leads to a dye that elicits the properties required for G‐quadruplex sensing.

     

一种可再生高选择性的硫化氢荧光探针

为了方便地检测环境样品中的硫化氢,利用香豆素酰肼肟配体构建了一个基于其铜配合物的可再生高选择性的硫化氢荧光探针(1-Cu²⁺)。 顺磁性Cu²⁺的荧光猝灭作用使探针的荧光很弱。 Na₂S溶液的加入可显著增强其荧光,其它常见阴离子(F^-,Cl^-,Br^-,I^-,CO32-,HPO42-,H₂PO4-,NO2-,NO3-,SO42-,CH₃COO^-,N3-,S₂O32-,CN^-)对配合物探针的荧光影响很小,共存时也不会干扰探针对硫化氢的增强响应。 Cu²⁺的加入能够再生探针(1-Cu²⁺),通过依次加入Cu²⁺和S^2- ,可重复地检测S^2-。 该探针响应时间快(~5 s),在0.5~5.0 μmol/L的范围内对H₂S响应呈线性,检测限低至37 nmol/L。     

A Highly Selective Turn-On Fluorescent Probe for the Detection of Zinc

A novel turn-on fluorescence probe L has been designed that exhibits high selectivity and sensitivity with a detection limit of 9.53 × 10⁻⁸ mol/L for the quantification of Zn²⁺. ¹H-NMR spectroscopy and single crystal X-ray diffraction analysis revealed the unsymmetrical nature of the structure of the Schiff base probe L. An emission titration experiment in the presence of different molar fractions of Zn²⁺ was used to perform a Job’s plot analysis. The results showed that the stoichiometric ratio of the complex formed by L and Zn²⁺ was 1:1. Moreover, the molecular structure of the mononuclear Cu complex reveals one ligand L coordinates with one Cu atom in the asymmetric unit. On adding CuCl₂ to the ZnCl₂/L system, a Cu-Zn complex was formed and a strong quenching behavior was observed, which inferred that the Cu²⁺ displaced Zn²⁺ to coordinate with the imine nitrogen atoms and hydroxyl oxygen atoms of probe L.     

Design and Synthesis of 2,2′‐Diindolylmethanes to Selectively Target Certain G‐Quadruplex DNA Structures

G‐quadruplex (G4) structures carry vital biological functions, and compounds that selectively target certain G4 structures have both therapeutic potential and value as research tools. Along this line, 2,2′‐diindolylmethanes have been designed and synthesized in this work based on the condensation of 3,6‐ or 3,7‐disubstituted indoles with aldehydes. The developed class of compounds efficiently stabilizes G4 structures without inducing conformational changes in such structures. Furthermore, the 2,2′‐diindolylmethanes target certain G4 structures more efficiently than others and this G4 selectivity can be altered by chemical modifications of the compounds.     

A Study of Interaction between Flavonoids and the Parallel Quadruplex Structure [d(TGGGGT)]4 by Electrospray Ionization Mass Spectrometry

In this study, electrospray ionization mass spectrometry (ESI‐MS) was used to investigate interaction of 21 flavonoids (10 aglycones and 11 glycosides) with the parallel quadruplex structure [d(TGGGGT)]₄. Relative binding affinities of flavonoids toward [d(TGGGGT)]₄ were estimated based on the fraction of bound DNA. It was found that [d(TGGGGT)]₄ showed a binding preference to the flavonoid glycosides over flavonoid aglycones. It was deduced that glycosylation played a key role for the [d(TGGGGT)]₄‐binding properties of flavonoid glycosides. Upon collision‐induced dissociation, complexes of flavonoid/[d(TGGGGT)]₄ underwent the loss of flavonoids, suggesting an end‐stacking binding mode. The current work demonstrates that ESI‐MS is a powerful tool in the study of interaction between drugs and nucleic acids.     

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.     

A Highly Selective and Sensitive Ratiometric Fluorescent Probe for Hypochlorite and Its Application

A novel water-soluble hybrid fluorescein-coumarin dye(FLNC) fluorescence ratiometric probe to recognize OCl^- was designed and synthesized. The most prominent feature of the probe is that the molecular structure contains two different fluorophores, and the fluorescein moiety was connected to coumarin moiety by diacylhydrazine linker. When OCl^- was added, the two fluorophores in the FLNC probe could have a rapid respond simultaneously, with high selectivity and sensitivity towards OCl^- among other reactive oxygen species/reactive nitrogen species(ROS/RNS) and anions, and the detection limit was measured to be 0.30 μmol/L. In addition, the color changes of the solution could be observed by the naked eye and have a short response time(completed within 1 min), which suggestes that the probe FLNC has the potential to be used for real-time detection.     

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.