One Terminal Guanosine Flip of Intramolecular Parallel G-Quadruplex: Catalytic Enhancement of G-Quadruplex/Hemin DNAzymes

Numerous studies have shown compelling evidence that incorporation of an inversion of polarity site (IPS) in G-rich sequences can affect the topological and structural characteristics of G-quadruplexes (G4s). Herein, the influence of IPS on the formation of a previously studied intramolecular parallel G4 of d(G₃TG₃TG₃TG₃) (TTT) and its stacked higher-order structures is explored. Insertion of 3′–3′ or 5′–5′ IPS did not change the parallel folding pattern of TTT. However, both the species and position of the IPS in TTT have a significant impact on the G4 stability and end-stacking through the alteration of G4–G4 interfaces properties. The data demonstrate that one base flip in each terminal G-tetrad can stabilize parallel G4s and facilitate intermolecular packing of monomeric G4s. Such modifications can also enhance the fluorescence and enzymatic performances by promoting interactions between parallel G4s with N-methyl mesoporphyrin IX (NMM) and hemin, respectively.     

Reversible pH Switch of Two‐Quartet G‐Quadruplexes Formed by Human Telomere

A four‐repeat human telomere DNA sequence without the 3′‐end guanine, d[TAGGG(TTAGGG)₂TTAGG] (htel1‐ΔG23) has been found to adopt two distinct two G‐quartet antiparallel basket‐type G‐quadruplexes, TD and KDH⁺ in presence of KCl. NMR, CD, and UV spectroscopy have demonstrated that topology of KDH⁺ form is distinctive with unique protonated T18?A20⁺?G5 base triple and other capping structural elements that provide novel insight into structural polymorphism and heterogeneity of G‐quadruplexes in general. Specific stacking interactions amongst two G‐quartets flanking base triples and base pairs in TD and KDH⁺ forms are reflected in 10 K higher thermal stability of KDH⁺. Populations of TD and KDH⁺ forms are controlled by pH. The (de)protonation of A20 is the key for pH driven structural transformation of htel1‐ΔG23. Reversibility offers possibilities for its utilization as a conformational switch within different compartments of living cell enabling specific ligand and protein interactions.     

Guanine Substitutions Prevent Conformational Switch from Antiparallel to Parallel G-Quadruplex

Guanine quadruplexes, recently reported to form in vivo, represent a broad spectrum of non-canonical conformations of nucleic acids. The actual conformation might differ between water solutions and crowding or dehydrating solutions that better reflect the conditions in the cell. Here we show, using spectroscopic techniques, that most guanine substitutions prevent the conformational switch from antiparallel or hybrid forms to parallel ones when induced by dehydrating agents. The inhibitory effect does not depend on the position of the substitution, but, interestingly, on the type of substitution and, to some extent, on its destabilising potential. A parallel form might be induced in some cases by ligands such as N-methyl mesoporphyrin IX and even this ligand-induced switch is inhibited by guanine substitution. The ability or inability to have a conformation switch, based on actual conditions, might significantly influence potential conformation-dependent quadruplex interactions.     

Sugar Puckering Drives G-Quadruplex Refolding: Implications for V-Shaped Loops

A DNA G-quadruplex adopting a (3+1) hybrid structure was modified in two adjacent syn positions of the antiparallel strand with anti-favoring 2′-deoxy-2′-fluoro-riboguanosine (^FrG) analogues. The two substitutions promoted a structural rearrangement to a topology with the 5′-terminal G residue located in the central tetrad and the two modified residues linked by a V-shaped zero-nucleotide loop. Strikingly, whereas a sugar pucker in the preferred north domain is found for both modified nucleotides, the ^FrG analogue preceding the V-loop is forced to adopt the unfavored syn conformation in the new quadruplex fold. Apparently, a preferred C3′-endo sugar pucker within the V-loop architecture outweighs the propensity of the ^FrG analogue to adopt an anti glycosidic conformation. Refolding into a V-loop topology is likewise observed for a sequence modified at corresponding positions with two riboguanosine substitutions. In contrast, 2′-F-arabinoguanosine analogues with their favored south-east sugar conformation do not support formation of the V-loop topology. Examination of known G-quadruplexes with a V-shaped loop highlights the critical role of the sugar conformation for this distinct structural motif.     

G-Quadruplex Formation by DNA Sequences Deficient in Guanines: Two Tetrad Parallel Quadruplexes Do Not Fold Intramolecularly

Guanine quadruplexes (G4s) are noncanonical forms of nucleic acids that are frequently found in genomes. The stability of G4s depends, among other factors, on the number of G-tetrads. Three- or four-tetrad G4s and antiparallel two-tetrad G4s have been characterized experimentally; however, the existence of an intramolecular (i. e., not dimeric or multimeric) two-tetrad parallel-stranded DNA G4 has never been experimentally observed. Many sequences compatible with two-tetrad G4 can be found in important genomic regions, such as promoters, for which parallel G4s predominate. Using experimental and theoretical approaches, the propensity of the model sequence AATGGGTGGGTTTGGGTGGGTAA to form an intramolecular parallel-stranded G4 upon increasing the number of GGG-to-GG substitutions has been studied. Deletion of a single G leads to the formation of intramolecular G4s with a stacked G-triad, whose topology depends on the location of the deletion. Removal of another guanine from another G-tract leads to di- or multimeric G4s. Further deletions mostly prevent the formation of any stable G4. Thus, a solitary two-tetrad parallel DNA G4 is not thermodynamically stable and requires additional interactions through capping residues. However, transiently populated metastable two-tetrad species can associate to form stable dimers, the dynamic formation of which might play additional delicate roles in gene regulation. These findings provide essential information for bioinformatics studies searching for potential G4s in genomes.     

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.     

Quadruplex–Duplex Junction: A High-Affinity Binding Site for Indoloquinoline Ligands

A parallel quadruplex derived from the Myc promoter sequence was extended by a stem-loop duplex at either its 5′- or 3′-terminus to mimic a quadruplex–duplex (Q–D) junction as a potential genomic target. High-resolution structures of the hybrids demonstrate continuous stacking of the duplex on the quadruplex core without significant perturbations. An indoloquinoline ligand carrying an aminoalkyl side chain was shown to bind the Q–D hybrids with a very high affinity in the order K_a≈10⁷ m ⁻¹ irrespective of the duplex location at the quadruplex 3′- or 5′-end. NMR chemical shift perturbations identified the tetrad face of the Q–D junction as specific binding site for the ligand. However, calorimetric analyses revealed significant differences in the thermodynamic profiles upon binding to hybrids with either a duplex extension at the quadruplex 3′- or 5′-terminus. A large enthalpic gain and considerable hydrophobic effects are accompanied by the binding of one ligand to the 3′-Q–D junction, whereas non-hydrophobic entropic contributions favor binding with formation of a 2:1 ligand-quadruplex complex in case of the 5′-Q–D hybrid.     

Photoresponsive Formation of an Intermolecular Minimal G‐Quadruplex Motif

The ability of three different bifunctional azobenzene linkers to enable the photoreversible formation of a defined intermolecular two‐tetrad G‐quadruplex upon UV/Vis irradiation was investigated. Circular dichroism and NMR spectroscopic data showed the formation of G‐quadruplexes with K⁺ ions at room temperature in all three cases with the corresponding azobenzene linker in an E conformation. However, only the para–para‐substituted azobenzene derivative enables photoswitching between a nonpolymorphic, stacked, tetramolecular G‐quadruplex and an unstructured state after E–Z isomerization.     

Dyads of G-Quadruplex Ligands Triggering DNA Damage Response and Tumour Cell Growth Inhibition at Subnanomolar Concentration

Naphthalene diimide (NDI) dyads exhibiting a different substitution pattern and linker length have been synthesised and evaluated as G-quadruplex (G4) ligands, by investigating their cytotoxicity in selected cell lines. The dyads with the long C₇ linker exhibit extremely low IC₅₀ values, below 10 nm , on different cancer cell lines. Contrary, the dyads with the shorter C₄ linker were much less effective, with IC values increasing up to 1 μm . Among the three dyads with the longest linker, small differences in the IC₅₀ values emerge, suggesting that the linker length plays a more important role than the substitution pattern. We have further shown that the dyads are able to induce cellular DNA damage response, which is not limited to the telomeric regions and is likely the origin of their cytotoxicity. Both absorption titration and dynamic light scattering of the most cytotoxic dyads in the presence of hTel22 highlight their ability to induce effective G4 aggregation, acting as non-covalent cross-linking agents.     

Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding

Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand–G-quadruplex structure determination.     

Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry

Invited for this months cover is the group of Prof. Maxim V. Berezovski. The cover picture shows the two-dimensional separation of unfolded (green) and folded (red) forms of G-quadruplex (GQ) DNA. The first dimension is kinetic capillary electrophoresis (KCE) separation of unfolded and folded DNA with different K⁺ concentrations in solution; the second dimension is ion mobility mass spectrometry separation of DNA conformers in the gas phase. DNA folding into a compact GQ structure is mediated by K⁺ ions. For more details, see the Full Paper on p. 58 ff.     

Duplex‐Guided Refolding into Novel G‐Quadruplex (3+1) Hybrid Conformations

The oligomer d(GCGTG₃TCAG₃TG₃TG₃ACGC) with short complementary flanking sequences at the 5′‐ and 3′‐ends was shown to fold into three different DNA G‐quadruplex species. In contrast, a corresponding oligomer that lacks base complementarity between the two overhang sequences folds into a single parallel G‐quadruplex. The three coexisting quadruplex structures were unambiguously identified and structurally characterized through detailed spectral comparisons with well‐defined G‐quadruplexes formed upon the deliberate incorporation of syn‐favoring 8‐bromoguanosine analogues into specific positions of the G‐core. Two (3+1) hybrid structures coexist with the parallel fold and feature a novel lateral–propeller–propeller loop architecture that has not yet been confirmed experimentally. Both hybrid quadruplexes adopt the same topology and only differ in their pattern of anti→syn transitions and tetrad stackings.     

NMR Structure of a Triangulenium‐Based Long‐Lived Fluorescence Probe Bound to a G‐Quadruplex

An NMR structural study of the interaction between a small‐molecule optical probe (DAOTA‐M2) and a G‐quadruplex from the promoter region of the c‐myc oncogene revealed that they interact at 1:2 binding stoichiometry. NMR‐restrained structural calculations show that binding of DAOTA‐M2 occurs mainly through π–π stacking between the polyaromatic core of the ligand and guanine residues of the outer G‐quartets. Interestingly, the binding affinities of DAOTA‐M2 differ by a factor of two for the outer G‐quartets of the unimolecular parallel G‐quadruplex under study. Unrestrained MD calculations indicate that DAOTA‐M2 displays significant dynamic behavior when stacked on a G‐quartet plane. These studies provide molecular guidelines for the design of triangulenium derivatives that can be used as optical probes for G‐quadruplexes.     

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.     

Imide Condensation as a Strategy for the Synthesis of Core-Diversified G-Quadruplex Ligands with Anticancer and Antiparasitic Activity**

A facile imide coupling strategy for the one-step preparation of G-quadruplex ligands with varied core chemistries is described. The G-quadruplex stabilization of a library of nine compounds was examined using FRET melting experiments, and CD, UV-Vis, fluorescence and NMR titrations, identifying several compounds that were capable of stabilizing G-quadruplex DNA with interesting selectivity profiles. The best G4 ligand was identified as compound 3 , which was based on a perylene scaffold and exhibited 40-fold selectivity for a telomeric G-quadruplex over duplex DNA. Surprisingly, a tetra-substituted flexible core, compound 11 , also exhibited selective stabilization of G4 DNA over duplex DNA. The anticancer and antiparasitic activity of the library was also examined, with the lead compound 3 exhibiting nanomolar inhibition of Trypanosoma brucei with 78-fold selectivity over MRC5 cells. The cellular localization of this compound was also studied via fluorescence microscopy. We found that uptake was time dependant, with localization outside the nucleus and kinetoplast that could be due to strong fluorescence quenching in the presence of small amounts of DNA.     

原癌基因c-myc启动区G-四链体结构及靶向小分子配体*

随着DNA G-四链体结构的发现和现代分子生物学技术对其与癌症关系的揭示,DNA G-四链体逐渐成为抗肿瘤药物研究的新靶点。c-myc启动区 G-四链体由于在细胞生长、增殖、凋亡、衰老及肿瘤形成等过程中的重要作用,成为DNA G-四链体中最受关注的序列之一。本文旨在对c-myc启动区 G-四链体的结构及靶向c-myc G-四链体的小分子配体的研究进展进行综述。首先,介绍c-myc G-四链体的生物学意义;其次,对几种常用的c-myc G-四链体的结构进行解析;最后,对以c-myc为靶点的小分子配体的研究进展及其与G-四链体的作用模式进行综述,并对目前以c-myc G-四链体为靶点、已经走向临床实验的CX-3543的开发与作用机制进行介绍。     

Induced-fit recognition of DNA by small circular oligonucleotides

We have investigated the molecular interaction between cyclic and linear oligonucleotides. We have found that short cyclic oligonucleotides can induce hairpinlike structures in linear DNA fragments. By using NMR and CD spectroscopy we have studied the interaction of the cyclic oligonucleotide d with d, as well as with its two linear analogs d(GTCCCTCA) and d(CTCAGTCC). Here we report the NMR structural study of these complexes. Recognition between these oligonucleotides occurs through formation of four intermolecular Watson-Crick base pairs. The three-dimensional structure is stabilized by two tetrads, formed by facing the minor-groove side of the Watson-Crick base pairs. Overall, the structure is similar to those observed previously in other quadruplexes formed by minor-groove alignment of Watson-Crick base pairs. However, in this case the complexes are heterodimeric and are formed by two different tetrads (G:C:A:T and G:C:G:C). These complexes represent a new model of DNA recognition by small cyclic oligonucleotides, increasing the number of potential applications of these interesting molecules.     

A Light-up Logic Platform for Selective Recognition of Parallel G-Quadruplex Structures via Disaggregation-Induced Emission

The design of turn-on dyes with optical signals sensitive to the formation of supramolecular structures provides fascinating and underexplored opportunities for G-quadruplex (G4) DNA detection and characterization. Here, we show a new switching mechanism that relies on the recognition-driven disaggregation (on-signal) of an ultrabright coumarin-quinazoline conjugate. The synthesized probe selectively lights-up parallel G4 DNA structures via the disassembly of its supramolecular state, demonstrating outputs that are easily integrable into a label-free molecular logic system. Finally, our molecule preferentially stains the G4-rich nucleoli of cancer cells.     

Molecular Recognition of the Hybrid‐2 Human Telomeric G‐Quadruplex by Epiberberine: Insights into Conversion of Telomeric G‐Quadruplex Structures

Human telomeres can form DNA G‐quadruplex (G4), an attractive target for anticancer drugs. Human telomeric G4s bear inherent structure polymorphism, challenging for understanding specific recognition by ligands or proteins. Protoberberines are medicinal natural‐products known to stabilize telomeric G4s and inhibit telomerase. Here we report epiberberine (EPI) specifically recognizes the hybrid‐2 telomeric G4 predominant in physiologically relevant K⁺ solution and converts other telomeric G4 forms to hybrid‐2, the first such example reported. Our NMR structure in K⁺ solution shows EPI binding induces extensive rearrangement of the previously disordered 5′‐flanking and loop segments to form an unprecedented four‐layer binding pocket specific to the hybrid‐2 telomeric G4; EPI recruits the (?1) adenine to form a “quasi‐triad” intercalated between the external tetrad and a T:T:A triad, capped by a T:T base pair. Our study provides structural basis for small‐molecule drug design targeting the human telomeric G4.