Inverting the G‐Tetrad Polarity of a G‐Quadruplex by Using Xanthine and 8‐Oxoguanine

G‐quadruplexes are four‐stranded nucleic acid structures that are built from consecutively stacked guanine tetrad (G‐tetrad) assemblies. The simultaneous incorporation of two guanine base lesions, xanthine (X) and 8‐oxoguanine (O), within a single G‐tetrad of a G‐quadruplex was recently shown to lead to the formation of a stable G?G?X?O tetrad. Herein, a judicious introduction of X and O into a human telomeric G‐quadruplex‐forming sequence is shown to reverse the hydrogen‐bond polarity of the modified G‐tetrad while preserving the original folding topology. The control exerted over G‐tetrad polarity by joint X?O modification will be valuable for the design and programming of G‐quadruplex structures and their properties.     

Ion‐Responsive Hemin–G‐Quadruplexes for Switchable DNAzyme and Enzyme Functions

Programmed nucleic acid sequences undergo K⁺ ion‐induced self‐assembly into G‐quadruplexes and separation of the supramolecular structures by the elimination of K⁺ ions by crown ether or cryptand ion‐receptors. This process allows the switchable formation and dissociation of the respective G‐quadruplexes. The different G‐quadruplex structures bind hemin, and the resulting hemin–G‐quadruplex structures reveal horseradish peroxidase DNAzyme catalytic activities. The following K⁺ ion/receptor switchable systems are described: 1) The K⁺‐induced self‐assembly of the Mg²⁺‐dependent DNAzyme subunits into a catalytic nanostructure using the assembly of G‐quadruplexes as bridging unit. 2) The K⁺‐induced stabilization of the anti‐thrombin G‐quadruplex nanostructure that inhibits the hydrolytic functions of thrombin. 3) The K⁺‐induced opening of DNA tweezers through the stabilization of G‐quadruplexes on the “tweezers’ arms" and the release of a strand bridging the tweezers into a closed structure. In all of the systems reversible, switchable, functions are demonstrated. For all systems two different signals are used to follow the switchable functions (fluorescence and the catalytic functions of the derived hemin–G‐quadruplex DNAzyme).     

Discrimination of G‐Quadruplexes from Duplex and Single‐Stranded DNAs with Fluorescence and Energy‐Transfer Fluorescence Spectra of Crystal Violet

G‐rich nucleic acid sequences with the potential to form G‐quadruplex structures are common in biologically important regions. Most of these sequences are present with their complementary strands, so the development of a sensitive biosensor to distinguish G‐quadruplex and duplex structures and to determine the competitive ability of quadruplex to duplex structures has received a great deal of attention. In this work, the interactions between two triphenylmethane dyes (malachite green (MG) and crystal violet (CV)) and G‐quadruplex, duplex, or single‐stranded DNAs were studied by fluorescence spectroscopy and energy‐transfer fluorescence spectroscopy. Good discrimination between quadruplexes and duplex or single‐stranded DNAs can be achieved by using the fluorescence spectrum of CV or the energy‐transfer fluorescence spectra of CV and MG. In addition, by using energy‐transfer fluorescence titrations of CV with G‐quadruplexes, the binding‐stoichiometry ratios of CV to G‐quadruplexes can be determined. By using the fluorescence titrations of G‐quadruplex–CV complexes with C‐rich complementary strands, the fraction of G‐rich oligonucleotide that engages in G‐quadruplex structures in the presence of the complementary sequence can be measured. This study may provide a simple method for discrimination between quadruplexes and duplex or single‐stranded DNAs and for measuring G‐quadruplex percentages in the presence of the complementary C‐rich sequences.     

Molecular‐Dynamics Studies of Single‐Stranded Hexitol,Altritol, Mannitol,and Ribose Nucleic Acids (HNA,MNA, ANA,and RNA,Resp.) and of the Stability of HNA⋅RNA,ANA⋅RNA,and MNA⋅RNA Duplexes

The influence of the orientation of a 3′‐OH group on the conformation and stability of hexitol oligonucleotides in complexes with RNA and as single strands in aqueous solution was investigated by molecular‐dynamics (MD) simulations with AMBER 4.1. The particle mesh Ewald (PME) method was used for the treatment of long‐range electrostatic interactions. An equatorial orientation of the 3′‐OH group in the single‐stranded D ‐mannitol nucleic acid (MNA) m(GCGTAGCG) and in the complex with the RNA r(CGCAUCGC) has an unfavorable influence on the helical stability. Frequent H‐bonds between the 3′‐OH group and the O−C(6′) of the phosphate backbone of the following nucleotide explain the distorted conformation of the MNA⋅RNA complex as well as that of the single MNA strand. This is consistent with experimental results that show lowered hybridization potentials for MNA⋅RNA complexes. An axial orientation of the 3′‐OH group in the D ‐altritol nucleic acid (ANA) a(GCGTAGCG) leads to a stable complex with the complementary RNA r(CGCAUCGC), as well as to a more highly preorganized single‐stranded ANA chain. The averaged conformation of the ANA⋅RNA complex is similar to that of A‐RNA, with only minor changes in groove width, helical curvature, and H‐bonding pattern. The relative stabilities of ANA⋅RNA vs. HNA⋅RNA (HNA=D ‐hexitol nucleic acid without 3′‐OH group) can be explained by differences in restricted movements, H‐bonds, and solvation effects.     

The Readout of Base‐Pair Information in Adenine–Thymine α‐D‐Arabinonucleosides

Structurally modified nucleosides are central players in the field of nucleic acid chemistry. Adenine–thymine (AT) pyrimido[4,5‐d]pyrimidine furanosyl and pyranosyl arabinonucleosides have been synthesized for the first time. Single‐crystal X‐ray diffraction analysis reveals novel base pairs that, in synergy with the sugar residues, direct the emergence of distinct networks containing channels and cavities. The microscopic noncovalent connections can be translated into macroscopic levels in which robust organogels are formed by the furanoside but not the pyranoside. The influences of the sugars are also displayed by the different shaped superstructures of the free nucleosides in solution. The readout of the information in the base moiety is therefore tailored by the sugar configuration, and the interplays exert subtle effects on the structures, from solid to gel and to the solution state. The potential for forming these appealing base pairs and higher structures enables these intriguing nucleosides to serve as unique building blocks in various areas or to construct innovative nucleic acid structures.     

A Photoreactive G‐Quadruplex Ligand Triggered by Green Light

A photoreactive molecular dye targeting the G‐quadruplex nucleic acid (G4) of the human telomeric sequence Tel22, and several mutated analogues, was activated by green light (λ=532 nm). Highly selective covalent modification of G4 versus single‐stranded and double‐stranded DNA was achieved with efficiency up to 64 %. The phenoxyl radical was generated and detected by laser‐flash photolysis as a reactive intermediate that targeted loop thymine residues. These insights may suggest a non‐invasive tool for selective nucleic acid tagging and “pull‐down” cellular applications.     

Nonenzymatic template-directed reactions on altritol oligomers, preorganized analogues of oligonucleotides

Altritol nucleic acids (ANAs) are RNA analogues with a phosphorylated D-altritol backbone. The nucleobase is attached at the 2-(S)-position of the carbohydrate moiety. We report that ANA oligomers are superior to the corresponding DNA, RNA, and HNA (hexitol nucleic acid) in supporting efficient nonenzymatic template-directed synthesis of complementary RNAs from nucleoside-5'-phosphoro-2-methyl imidazolides. Activated ANA and HNA monomers do not oligomerize efficiently on DNA, RNA, HNA, or ANA templates.     

Four-stranded DNA structures can be stabilized by two different types of minor groove G:C:G:C tetrads

Four-stranded nucleic acid structures are central to many processes in biology and in supramolecular chemistry. It has been shown recently that four-stranded DNA structures are not only limited to the classical guanine quadruplex but also can be formed by tetrads resulting from the association of Watson-Crick base pairs. Such an association may occur through the minor or the major groove side of the base pairs. Structures stabilized by minor groove tetrads present distinctive features, clearly different from the canonical guanine quadruplex, making these quadruplexes a unique structural motif. Within our efforts to study the sequence requirements for the formation of this unusual DNA motif, we have determined the solution structure of the cyclic oligonucleotide dpCCGTCCGT by two-dimensional NMR spectroscopy and restrained molecular dynamics. This molecule self-associates, forming a symmetric dimer stabilized by two G:C:G:C tetrads with intermolecular G-C base pairs. Interestingly, although the overall three-dimensional structure is similar to that found in other cyclic and linear oligonucleotides of related sequences, the tetrads that stabilize the structure of dpCCGTCCGT are different to other minor groove G:C:G:C tetrads found earlier. Whereas in previous cases the G-C base pairs aligned directly, in this new tetrad the relative position of the two base pairs is slipped along the axis defined by the base pairs. This is the first time that a quadruplex structure entirely stabilized by slipped minor groove G:C:G:C tetrads is observed in solution or in the solid state. However, an analogous arrangement of G-C base pairs occurs between the terminal residues of contiguous duplexes in some DNA crystals. This structural polymorphism between minor groove GC tetrads may be important in stabilization of higher order DNA structures.     

Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G‐quadruplex aptamer and thrombin

The dynamic binding status between the thrombin and its G‐quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence‐coupled capillary electrophoresis method. A 29‐nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G‐quadruplex from unstructured 29‐nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6‐carboxyfluorescein labeled G‐quadruplex aptamer was measured. Third, the stability of G‐quadruplex aptamer–thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29‐nucleic acid thrombin binding aptamer could compete with G‐quadruplex aptamer and thus disassociated the G‐quadruplex structure into an unstructured aptamer. These data suggest that our in‐house established fluorescence‐coupled capillary electrophoresis assay could be applied to binding studies of the G‐quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available.     

Evaluation of alkaloids binding to 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 the binding interaction of six alkaloids with parallel intermolecular G‐quadruplex [d(TGGGGT)]₄, and five alkaloids including berberine, jatrorrhizine, palmatine, tetrandrine, and fangchinoline showed complexation with the target DNA. Relative binding affinities were estimated on the basis of mass spectrometric data. The slight differences in chemical structures of berberine, jatrorrhizine, and palmatine had little influence on their binding affinities to [d(TGGGGT)]₄. Tetrandrine and fangchinoline selectively bound to [d(TGGGGT)]₄ versus duplex DNA. Collision‐induced dissociation (CID) experiments showed that the complexes with berberine, jatrorrhizine, and palmatine dissociated via strand separation and ligand retaining in the strand while the complexes with tetrandrine and fangchinoline were dissociated via ligand elimination. A comparison of dissociation patterns in CID experiments of complexes with the alkaloids to those with the traditional G‐quadruplex DNA binders suggested an end‐stacking binding mode for tetrandrine and fangchinoline and an intercalation binding mode for berberine, jatrorrhizine, and palmatine to the target DNA. The current work not only provides deep insight into alkaloid/[d(TGGGGT)]₄ complexes and useful guidelines for design of efficient anticancer agents but also demonstrates the utility of ESI‐MS as a powerful tool for evaluating interaction between ligand and quadruplex DNA. Copyright © 2012 John Wiley & Sons, Ltd.     

Selective and Cooperative Ligand Binding to Antiparallel Human Telomeric DNA G‐Quadruplexes

The quest for ligands that specifically bind to particular G‐quadruplex nucleic acid structures is particularly important to conceive molecules with specific effects on gene expression or telomere maintenance, or conceive structure‐specific molecular probes. Using electrospray mass spectrometry in native conditions, we reveal a highly cooperative and selective 2:1 binding of Cu^II‐tolylterpyridine complexes to human telomeric G‐quadruplexes. Circular dichroism and comparisons of affinities for different sequences reveal a marked preference for antiparallel structures with diagonal loops and/or wide‐medium–narrow‐medium groove‐width order. The cooperativity is attributed to conformational changes in the polymorphic telomeric G‐quadruplex sequences, which convert preferably into an antiparallel three‐quartet topology upon binding of two ligands.     

Solution Structure of a Hexitol Nucleic Acid Duplex with Four Consecutive T⋅T Base Pairs

The structure of the hexitol nucleic acid (HNA) h(GCGCTTTTGCGC) was determined by NMR spectroscopy. This unnatural nucleic acid was developed as a mimic for A‐RNA. In solution, the studied sequence is forming a symmetric double‐stranded structure with four central consecutive T⋅T wobble pairs flanked by G⋅C Watson‐Crick base pairs. The stem regions adopt an A‐type helical structure. Discrete changes in backbone angles are altering the course of the helix axis in the internal loop region. Two H‐bonds are formed in each wobble pair, and base stacking is preserved in the duplex, explaining the stability of the duplex. This structure elucidation provides information about the influence of a (T)₄ fragment on local helix geometries as well as on the nature of the T⋅T mismatch base pairing in a TTTT tract.     

Light‐Induced Reversible Reconfiguration of DNA‐Based Constitutional Dynamic Networks: Application to Switchable Catalysis

The light‐induced reversible and cyclic reconfiguration of constitutional dynamic networks, consisting of supramolecular nucleic acid structures as constituents and a photoisomerizable trans/cis‐azobenzene‐functionalized nucleic acid as the trigger is demonstrated. In addition, the cyclic photochemical reconfiguration of the constitutional dynamic networks guides the switchable on/off operation of an emerging hemin/G‐quadruplex DNAzyme.     

Structural and thermodynamic studies of the interaction of distamycin A with the parallel quadruplex structure [d(TGGGGT)]4

The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by 1H NMR spectroscopy and isothermal titration calorimetry (ITC). To unambiguously assert that distamycin A interacts with the grooves of the quadruplex [d(TGGGGT)]4, we have analyzed the NMR titration profile of a modified quadruplex, namely [d(TGGMeGGT)]4, and we have applied the recently developed differential frequency-saturation transfer difference (DF-STD) method, for assessing the ligand-DNA binding mode. The three-dimensional structure of the 4:1 distamycin A/[d(TGGGGT)]4 complex has been determined by an in-depth NMR study followed by dynamics and mechanics calculations. All results unequivocally indicate that distamycin molecules interact with [d(TGGGGT)]4 in a 4:1 binding mode, with two antiparallel distamycin dimers that bind simultaneously two opposite grooves of the quadruplex. The affinity between distamycin A and [d(TGGGGT)]4 enhances ( approximately 10-fold) when the ratio of distamycin A to the quadruplex is increased. In this paper we report the first three-dimensional structure of a groove-binder molecule complexed to a DNA quadruplex structure.     

A Flexible Terpyridine Derivative Interacts Specifically with G‐Quadruplexes

G‐quadruplexes formed by nucleic acids are implicated in pathologies ranging from cancers to neurodegenerative diseases. We evaluated interactions of 29 bi‐ and terpyridine derivatives with G‐quadruplexes and duplexes. FRET‐melting, circular dichroism, and ¹H NMR spectroscopy showed that one terpyridine derivative interacted very selectively with G‐quadruplexes. This G‐quadruplex ligand inhibited helicase activity and should influence G‐quadruplex‐related biological processes.     

Isoguanine and 5‐Methyl‐Isocytosine Bases,In Vitro and In Vivo

The synthesis, base‐pairing properties and in vitro and in vivo characteristics of 5‐methyl‐isocytosine (isoC^Me) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)–DNA(d) mosaic backbone, are described. The required h‐isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by T_m measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d‐isoG base mispairing follows the order T>G>C while the h‐isoG base mispairing follows the order G>C>T. The h‐ and d‐isoC^Me bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoC^Me misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base‐pair interpretation for the deoxyribose and hexitol isoC^Me/isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality.     

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.     

Ruthenium Complex “Light Switches” that are Selective for Different G‐Quadruplex Structures

Recognition and regulation of G‐quadruplex nucleic acid structures is an important goal for the development of chemical tools and medicinal agents. The addition of a bromo‐substituent to the dipyridylphenazine (dppz) ligands in the photophysical “light switch”, [Ru(bpy)₂dppz]²⁺, and the photochemical “light switch”, [Ru(bpy)₂dmdppz]²⁺, creates compounds with increased selectivity for an intermolecular parallel G‐quadruplex and the mixed‐hybrid G‐quadruplex, respectively. When [Ru(bpy)₂dppz‐Br]²⁺ and [Ru(bpy)₂dmdppz‐Br]²⁺ are incubated with the G‐quadruplexes, they have a stabilizing effect on the DNA structures. Activation of [Ru(bpy)₂dmdppz‐Br]²⁺ with light results in covalent adduct formation with the DNA. These complexes demonstrate that subtle chemical modifications of Ru^II complexes can alter G‐quadruplex selectivity, and could be useful for the rational design of in vivo G‐quadruplex probes.     

Photo‐Cross‐Linking Probes for Trapping G‐Quadruplex DNA

We have developed a straightforward synthetic pathway to a set of six photoactivatable G‐quadruplex ligands with a validated G4‐binding motif (the bisquinolinium pyridodicarboxamide PDC‐360A) tethered through various spacers to two different photo‐cross‐linking groups: benzophenone and an aryl azide. The high quadruplex‐versus‐duplex selectivity of the PDC core was retained in the new derivatives and resulted in selective alkylation of two well‐known G‐quadruplexes (human telomeric G4 and oncogene promoter c‐myc G4) under conditions of harsh competition. The presence of two structurally different photoactivatable functions allowed the selective alkylation of G‐quadruplex structures at specific nucleobases and irreversible G4 binding. The topology and sequence of the quadruplex matrix appear to influence strongly the alkylation profile, which differs for the telomeric and c‐myc quadruplexes. The new compounds are photoactive in cells and thus provide new tools for studying G4 biology.