Targeting Non‐B‐Form DNA in Living Cells

Under certain conditions, repetitive DNA motifs have the potential to adopt non‐B‐form DNA structures, such as hairpins, triplexes, Z‐DNA, quadruplexes, and i‐motifs. Some non‐B‐form DNAs have been proposed to cause mutations and, consequently, participate in several biologically important processes, including regulation, evolution, and human disease. Advancement in the knowledge of specific interactions between molecules and non‐B‐form DNAs at the molecular level in living cells is important for understanding their biological functions. In this review, we describe the latest studies on molecules that target non‐B‐form DNAs in vivo, with a focus on Z‐DNA, G‐quadruplexes, triplexes, i‐motifs, and hairpins.     

Synthesis of bis-indole carboxamides as G-quadruplex stabilizing and inducing ligands

The design and synthesis of a series of bis‐indole carboxamides with varying amine containing side chains as G‐quadruplex DNA stabilising small molecules are reported. Their interactions with quadruplexes have been evaluated by means of Förster resonance energy transfer (FRET) melting analysis, UV/Vis spectroscopy, circular dichroism spectroscopy and molecular modelling studies. FRET analysis indicates that these ligands exhibit significant selectivity for quadruplex over duplex DNA, and the position of the carboxamide side chains is of paramount importance in G‐quadruplex stabilisation. UV/Vis titration studies reveal that bis‐indole ligands bind tightly to quadruplexes and show a three‐ to fivefold preference for c‐kit2 over h‐telo quadruplex DNA. CD studies revealed that bis‐indole carboxamide with a central pyridine ring induces the formation of a single, antiparallel, conformation of the h‐telo quadruplex in the presence and absence of added salt. The chirality of h‐telo quadruplex was transferred to the achiral ligand (induced CD) and the formation of a preferred atropisomer was observed.     

Energetic Basis of AGCGA‐Rich DNA Folding into a Tetrahelical Structure

It was recently discovered that, besides well‐known G‐quadruplexes and i‐motifs, DNA may adopt another type of noncanonical structure called AGCGA‐quadruplexes. Here, the folding of the VK2 fragment from the regulatory region of the PLEKHG3 gene is studied and, for the first time, the energetic contributions that stabilize this unique fold are described. Similarly to the B‐DNA, it is stabilized by hydrophobic desolvation and, in contrast to G‐quadruplexes, also by specific binding of water molecules. Compared to B‐DNA, VK2 folding is enthalpically less favorable due to poorer base‐stacking interactions, resulting in substantial conformational flexibility. This entropically favorable conformational “breathing” stabilizes the AGCGA‐quadruplexes. In conclusion, AGCGA‐quadruplexes have a distinguishing thermodynamic fingerprint and the corresponding driving forces enabling their folding are consistent with the observed structural features.     

人类端粒DNA的分子识别及其作用机制探讨

核酸中富含短的G-碱基重复的序列可以形成一种复杂的高级结构,称为G-四链体（G-quadruplex）.在基因组中,借助生物信息学发现这类富G序列广泛分布在基因的启动子区,特别是那些参与到复制中去的基因,例如癌基因.同时发现这类序列在mRNA的5′非翻译区（5′UTR）也广泛存在.这类序列在染色体末段端粒部位的存在及功能已得到充分阐明.已知端粒富含G-碱基序列,其3′末端以单链状态存在,这使得在一些小分子的选择性作用下端粒序列很容易形成G-四链体结构,进而破坏端粒结构,影响端粒酶活性.已知端粒酶在超过85%的肿瘤中过量表达,因此,端粒酶已经成为抗癌药物设计的特殊靶点,是目前本领域的研究热点之一.已发现系列配体通过有效抑制端粒酶而表现高的抗肿瘤活性.本文主要综述了近年来端粒G-四链体分子识别及其药物靶向的最新进展,并对其作用机理做了进一步的分析和探讨.     

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.     

Structural basis of telomeric RNA quadruplex--acridine ligand recognition

Human telomeric DNA is now known to be transcribed into noncoding RNA sequences, termed TERRA. These sequences, which are believed to play roles in the regulation of telomere function, can form higher-order quadruplex structures and may themselves be the target of therapeutic intervention. The crystal structure of a TERRA quadruplex-acridine small-molecule complex at a resolution of 2.60 ?, is reported here and contrasts remarkably with the structure of the analogous DNA quadruplex complex. The bimolecular RNA complex has a parallel-stranded topology with propeller-like UUA loops. These loops are held in particular conformations by multiple hydrogen bonds involving the O2' hydroxyl groups of the ribonucleotide sugars and play an active role in binding the acridine molecules to the RNA quadruplex. By contrast, the analogous DNA quadruplex complex has simpler 1:1 acridine binding, with no loop involvement. There are significant loop conformational changes in the RNA quadruplex compared to the native TERRA quadruplex (Collie, G. W.; Haider, S. M.; Neidle, S.; Parkinson, G. N. Nucleic Acids Res. 2010, 38, 5569 - 5580), which have implications for the future design of small molecules targeting TERRA quadruplexes, and RNA quadruplexes more generally.     

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.     

Ligand Selectivity by Inserting GCGC-Tetrads into G-Quadruplex Structures

G-Quadruplexes (G4s) assembled from tandem G-rich repeat sequences exhibit significant biological functions and applications, which may well depend on their structural features, such as the planar arrangement of G-tetrads and flexibility of loop regions. It has been found that cytosine-intercalated G-repeat sequences also assemble to be quadruplex structures, involving the formation of nonplanar GCGC-tetrads. Herein, to investigate the effect of GCGC-tetrads on structural properties of G4s, some previously studied quadruplexes with or without GCGC-tetrads were selected, and were used to interact with various developed G4 ligands. Our data show that stacked G-tetrads in quadruplexes are important for the π–π stacking interactions, thus promoting the combination with end-stacking ligands, such as porphyrins or planar small molecules. This is confirmed by the observation that the quadruplex formed by d(GGGCT₄GGGC) with two internal G-tetrads and two external GCGC-tetrads can bind to planar ligands in the presence of specific G4-stabilizing cations, including K⁺ and Pb²⁺, and can realize the sensitive detection of Pb²⁺. However, the quadruplex composed of two external G-tetrads and two internal GCGC-tetrads formed by d(GCGGT₃GCGG) facilitates the binding of nonplanar ligands, such as triphenylmethane (TPM) dyes, owing to the structural flexibility induced by internal GCGC-tetrads. This work provides new insights into the interaction between DNA quadruplexes and specific ligands, which is beneficial to the development of quadruplex-based biosensors and the design of anticancer drugs.     

Structure and stability of higher-order human telomeric quadruplexes

G-quadruplex formation in the sequences 5'-(TTAGGG)(n) and 5'(TTAGGG)(n)TT (n = 4, 8, 12) was studied using circular dichroism, sedimentation velocity, differential scanning calorimetry, and molecular dynamics simulations. Sequences containing 8 and 12 repeats formed higher-order structures with two and three contiguous quadruplexes, respectively. Plausible structures for these sequences were determined by molecular dynamics simulations followed by experimental testing of predicted hydrodynamic properties by sedimentation velocity. These structures featured folding of the strand into contiguous quadruplexes with mixed hybrid conformations. Thermodynamic studies showed the strands folded spontaneous to contain the maximum number contiguous quadruplexes. For the sequence 5'(TTAGGG)(12)TT, more than 90% of the strands contained completely folded structures with three quadruplexes. Statistical mechanical-based deconvolution of thermograms for three quadruplex structures showed that each quadruplex melted independently with unique thermodynamic parmameters. Thermodynamic analysis revealed further that quadruplexes in higher-ordered structures were destabilized relative to their monomeric counterparts, with unfavorable coupling free energies. Quadruplex stability thus depends critically on the sequence and structural context.     

Tandem mass spectrometry of platinated quadruplex DNA

Quadruplexes are higher-order structures formed by G-rich DNA strands that are involved in various processes of cell cycle regulation, such as control of telomere length and participation in gene regulation. Because of these central biological functions, quadruplex DNA represents a promising target for cancer therapy, e.g. by applying organometallic drugs, such as cisplatin. High-resolution electrospray tandem mass spectrometry is evaluated as a technique for exploring structural features of unplatinated and platinated quadruplexes. Results of experiments on tetramolecular, bimolecular and monomolecular quadruplexes provide information about the extent of platination and the binding sites of the drug. The dissociation behavior of the different types of quadruplexes is compared. Tetramolecular quadruplexes were found to weave out a strand end in order to provide a platination site, and their fragmentation is characterized by the release of an unplatinated strand and the formation of a platinated triplex. Partial opening of the structure in combination with the loss of small fragments leads to truncated quadruplex ions. For the bimolecular quadruplexes studied, strand separation is the predominant dissociation pathway. Depending on the loop sequence, cross-linking of the loops by cisplatin is demonstrated. Distinct differences in the product ion spectra of unannealed and annealed monomolecular sequences provide proof of quadruplex formation and show that platination preferentially occurs at the terminal regions.     

Hierarchical Self‐Assembly of a Biomimetic Light‐Harvesting Antenna Based on DNA G‐Quadruplexes

A new modular approach to an artificial light‐harvesting antenna system is presented. The approach involves the hierarchical self‐assembly of porphyrin acceptor molecules to G‐quadruplexes tethered to coumarin donor moieties.     

Orienting Tetramolecular G‐Quadruplex Formation: The Quest for the Elusive RNA Antiparallel Quadruplex

DNA and RNA G‐quadruplexes (G4) are unusual nucleic acid structures involved in a number of key biological processes. RNA G‐quadruplexes are less studied although recent evidence demonstrates that they are biologically relevant. Compared to DNA quadruplexes, RNA G4 are generally more stable and less polymorphic. Duplexes and quadruplexes may be combined to obtain pure tetrameric species. Here, we investigated whether classical antiparallel duplexes can drive the formation of antiparallel tetramolecular quadruplexes. This concept was first successfully applied to DNA G4. In contrast, RNA G4 were found to be much more unwilling to adopt the forced antiparallel orientation, highlighting that the reason RNA adopts a different structure must not be sought in the loops but in the G‐stem structure itself. RNA antiparallel G4 formation is likely to be restricted to a very small set of peculiar sequences, in which other structural features overcome the formidable intrinsic barrier preventing its formation.     

Analytical potential of the quadruplex DNA-based FRET probes

DNA exhibits structural flexibility and may adopt also tetraplex structures known as guanine-quadruplexes or G-quadruplexes. These G-quadruplexes have recently received great attention because G-rich sequences are often found in genome and because of their potential links to mechanisms that relate to cancer, HIV, and other diseases. The unique structure of quadruplexes has also stimulated development of new analytical and bioanalytical assays based on fluorescence resonance energy transfer (FRET). Intramolecular folding of a flexible single-stranded DNA molecule into a compact G-quadruplex is a structural transition leading to closer proximity of its 5'- and 3'-ends. Thus, labeling both ends of a DNA strand with donor and acceptor fluorophores enables monitoring the quadruplex formation process by means of the FRET signal. This review shows how FRET technique contributes to G-quadruplex research and focuses mainly on analytical applications of FRET-labeled quadruplexes. Applications include studies of structural transitions of quadruplexes, FRET-based selection of ligands that bind to quadruplexes, design of molecular probes for protein recognition and development of sensors for detection of potassium ions in aqueous solution.     

Effect of loop orientation on quadruplex-TMPyP4 interaction

G-quadruplexes are believed to be potential targets for therapeutic intervention and this has resulted in designing of various quadruplex interacting ligands. Moreover, reports about existence of quadruplex forming sequences across the genome have propelled greater interest in understanding their interaction with small molecules. An intramolecular quadruplex sequence can adopt different conformations, owing to different orientation of loops in the structure. The differences in the loop orientation can affect their molecular recognition. Herein, we have studied the interaction of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H, 23H-porphine (TMPyP4), a well-known G quadruplex binding ligand with three DNA quadruplexes differing in loop orientations. Results obtained from UV, ITC, and SPR studies have coherently revealed that the TMPyP4 molecule shows preferential binding to parallel G-quadruplex ( c-myc and c-kit) over its antiparallel counterpart (human telomeric). The binding affinity for parallel quadruplex was (10(7)) 1 order of magnitude higher than that for antiparallel DNA quadruplex (10 ). The study shows two binding modes, stronger binding (10(7)) of TMPyP4 involving end stacking and a weaker external binding (10 ), while TMPyP4 shows only one binding mode with duplex with a binding affinity of the order of 10(6). Overall, the study emphasizes that differences in the loop orientation give rise to different conformations of quadruplex, which in turn govern its binding to small molecules, and thereby play a pivotal role in molecular recognition.     

Bioactive papaverine derivatives bind G-quadruplexes selectively

G-quadruplexes are a family of DNA secondary structures resulting from the folding of a guanine-rich sequence. Targeting quadruplexes by small molecules is an approach that is currently being studied with the aim of exploring their biological roles and developing new anti-cancer agents. There is evidence that the formation of G4 structures by telomeric DNA can be used to inhibit the enzyme activity of telomerase, and thereby to activate the pathway to senescence in tumour cells. It was previously shown that the papaverine oxidation products 6a,12a-diazadibenzo-[a,g]fluorenylium derivative (ligand I) and 2,3,9,10-tetramethoxy-12-oxo-12H-indolo[2,1-a]isoquinolinium chloride (ligand II) bind to G-quadruplex representing the human telomeric sequence. These ligands possess the ability to inhibit telomerase and polymerase action at the micromolar level. Here we report a DNA binding study on these two ligands and a new derivative 2-(2-carboxy-4,5-dimethoxyphenyl0-6,7-dimethoxyisoquiloliniuminner salt (ligand III) in order to evaluate their binding selectivity to samples of nucleic acids (ssDNA, dsDNA, triplexes, and quadruplexes). Simultaneous investigations on several DNA-ligand complexes carried out using an equilibrium dialysis approach revealed pronounced binding selectivity of ligand I and ligand II to tetraplex DNA structures over the doublestranded DNA forms.     

Tracking the Dynamic Folding and Unfolding of RNA G‐Quadruplexes in Live Cells

Because of the absence of methods for tracking RNA G‐quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G‐quadruplexes is so far limited. Herein, we report a new red‐emitting fluorescent probe, QUMA‐1 , for the selective, continuous, and real‐time visualization of RNA G‐quadruplexes in live cells. The applications of QUMA‐1 in several previously intractable applications, including live‐cell imaging of the dynamic folding, unfolding, and movement of RNA G‐quadruplexes and the visualization of the unwinding of RNA G‐quadruplexes by RNA helicase have been demonstrated. Notably, our real‐time results revealed the complexity of the dynamics of RNA G‐quadruplexes in live cells. We anticipate that the further application of QUMA‐1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G‐quadruplexes will uncover more information about the biological roles of RNA G‐quadruplexes.     

A Nitroxide‐Tagged Platinum(II) Complex Enables the Identification of a DNA G‐Quadruplex Binding Mode

We reported a novel strategy for investigating small molecule binding to G‐quadruplexes (GQs). A newly synthesized dinuclear platinum(II) complex (Pt₂L) containing a nitroxide radical was shown to selectively bind a GQ‐forming sequence derived from human telomere (hTel). Using the nitroxide moiety as a spin label, electron paramagnetic resonance (EPR) spectroscopy was carried out to investigate binding between Pt₂L and hTel GQ. Measurements indicated that two molecules of Pt₂L bind with one molecule of hTel GQ. The inter‐spin distance measured between the two bound Pt₂L, together with molecular docking analyses, revealed that Pt₂L predominately binds to the neighboring narrow and wide grooves of the G‐tetrads as hTel adopts the antiparallel conformation. The design and synthesis of nitroxide tagged GQ binders, and the use of spin‐labeling/EPR to investigate their interactions with GQs, will aid the development of small molecules for manipulating GQs involved in crucial biological processes.     

Diarylethynyl amides that recognize the parallel conformation of genomic promoter DNA G-quadruplexes

We report bis-phenylethynyl amide derivatives as a potent G-quadruplex binding small molecule scaffold. The amide derivatives were efficiently prepared in 3 steps by employing Sonogashira coupling, ester hydrolysis and a chemoselective amide coupling. Ligand-quadruplex recognition has been evaluated using a fluorescence resonance energy transfer (FRET) melting assay, surface plasmon resonance (SPR), circular dichroism (CD) and (1)H nuclear magnetic resonance (NMR) spectroscopy. While most of the G-quadruplex ligands reported so far comprise a planar, aromatic core designed to stack on the terminal tetrads of a G-quadruplex, these compounds are neither polycyclic, nor macrocyclic and have free rotation around the triple bond enabling conformational flexibility. Such molecules show very good binding affinity, excellent quadruplex:duplex selectivity and also promising discrimination between intramolecular promoter quadruplexes. Our results indicate that the recognition of the c-kit2 quadruplex by these ligands is achieved through groove binding, which favors the formation of a parallel conformation.     

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.