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Quadruplex-duplex (Q–D) junctions constitute unique structural motifs in genomic sequences. Through comprehensive calorimetric as well as high-resolution NMR structural studies, Q–D junctions with a hairpin-type snapback loop coaxially stacked onto an outer G-tetrad were identified to be most effective binding sites for various polycyclic quadruplex ligands. The Q–D interface is readily recognized by intercalation of the ligand aromatic core structure between G-tetrad and the neighboring base pair. Based on the thermodynamic and structural data, guidelines for the design of ligands with enhanced selectivity towards a Q–D interface emerge. Whereas intercalation at Q–D junctions mostly outcompete stacking at the quadruplex free outer tetrad or intercalation between duplex base pairs to varying degrees, ligand side chains considerably contribute to the selectivity for a Q–D target over other binding sites. In contrast to common perceptions, an appended side chain that additionally interacts within the duplex minor groove may confer only poor selectivity. Rather, the Q–D selectivity is suggested to benefit from an extension of the side chain towards the exposed part of the G-tetrad at the junction. The presented results will support the design of selective high-affinity binding ligands for targeting Q–D interfaces in medicinal but also technological applications.

Canonical G‐quadruplexes can adopt a variety of different topologies depending on the arrangement of propeller, lateral, or diagonal loops connecting the four G‐columns. A novel intramolecular G‐quadruplex structure is derived through inversion of the last G‐tract of a three‐layered parallel fold, associated with the transition of a single propeller into a lateral loop. The resulting (3+1) hybrid fold features three syn⋅anti⋅anti⋅anti G‐tetrads with a 3’‐terminal all‐syn G‐column. Although the ability of forming a duplex stem‐loop between G‐tracts seems beneficial for a propeller‐to‐lateral loop rearrangement, unmodified G‐rich sequences resist folding into the new (3+1) topology. However, refolding can be driven by the incorporation of syn‐favoring guanosine analogues into positions of the fourth G‐stretch. The presented hybrid‐type G‐quadruplex structure as determined by NMR spectroscopy may provide for an additional scaffold in quadruplex‐based technologies., Starting with a parallel‐stranded DNA G‐quadruplex, refolding into a novel (3+1) hybrid topology with a propeller‐propeller‐lateral loop architecture and an all‐syn 3’‐terminal G‐tract is reported. Although supported by additional stacking of a lateral duplex hairpin onto an outer G‐tetrad, the structural rearrangement must be enforced through the specific incorporation of syn‐favoring 8‐bromoguanosine analogues into the terminal G‐run.

The KCNN4 gene encoding a potassium channel protein whose expression has been correlated with tumor progression was found to comprise a guanine-rich minisatellite region with the ability to form a ...

White spot disease (WSD) is one of the most devastating viral infections of crustaceans caused by the white spot syndrome virus (WSSV). A conserved sequence WSSV131 in the DNA genome of WSSV was found to fold into a polymorphic G‐quadruplex structure. Supported by two mutant sequences with single G→T substitutions in the third G4 tract of WSSV131, circular dichroism and NMR spectroscopic analyses demonstrate folding of the wild‐type sequence into a three‐tetrad parallel topology comprising three propeller loops with a major 1 : 3 : 1 and a minor 1 : 2 : 2 loop length arrangement. A thermodynamic analysis of quadruplex formation by differential scanning calorimetry (DSC) indicates a thermodynamically more stable 1 : 3 : 1 loop isomer. DSC also revealed the formation of additional highly stable multimeric species with populations depending on potassium ion concentration., The right spot for white spot: A G‐quadruplex‐forming sequence identified within the DNA genome of white spot syndrome virus folds into a major and a thermodynamically less stable minor loop isomer. The parallel quadruplex scaffold might constitute a potential target for viral drug development against white spot disease.

A G-rich sequence was designed to allow folding into either a stable parallel or hybrid-type topology. With the parent sequence featuring coexisting species, various related sequences with single and double mutations and with a shortened central propeller loop affected the topological equilibrium. Two simple modifications, likewise introduced separately to all sequences, were employed to lock folds into one of the topologies without noticeable structural alterations. The unique combination of sequence mutations, high-resolution NMR structural information, and the thermodynamic stability for both topological competitors identified critical loop residue interactions. In contrast to first loop residues, which are mostly disordered and exposed to solvent in both propeller and lateral loops bridging a narrow groove, the last loop residue in a lateral three-nucleotide loop is engaged in stabilizing stacking interactions. The propensity of single-nucleotide loops to favor all-parallel topologies by enforcing a propeller-like conformation of an additional longer loop is shown to result from their preference in linking two outer tetrads of the same tetrad polarity. Taken together, the present studies contribute to a better structural and thermodynamic understanding of delicate loop interactions in genomic and artificially designed quadruplexes, e.g. when employed as therapeutics or in other biotechnological applications.

Guanine(G)-rich DNA or RNA sequences can assemble or intramolecularly fold into G-quadruplexes formed through the stacking of planar G·G·G·G tetrads in the presence of monovalent cations. These secondary nucleic acid structures have convincingly been shown to also exist within a cellular environment exerting important regulatory functions in physiological processes. For identifying nucleic acid segments prone to quadruplex formation, a putative quadruplex sequence motif encompassing closely spaced tracts of three or more guanosines is frequently employed for bioinformatic search algorithms. Depending on the number and type of intervening residues as well as on solution conditions, such sequences may fold into various canonical G4 topologies with continuous G-columns. On the other hand, a growing number of sequences capable of quadruplex formation feature G-deficient guanine tracts, escaping the conservative consensus motif. By folding into non-canonical quadruplex structures, they adopt unique topologies depending on their specific sequence context. These include G-columns with only two guanines, bulges, snapback loops, D- and V-shaped loops as well as interlocked structures. This review focuses on G-quadruplex species carrying such distinct structural motifs. It evaluates characteristic features of their non-conventional scaffold and highlights principles of stabilizing interactions that also allow for their folding into stable G-quadruplex structures., G-rich nucleic acid sequences encompassing G-tracts of varying lengths can fold into different non-canonical G-quadruplexes with distinct structural features.

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., An indoloquinoline‐based G4 ligand binds with very high affinity at the quadruplex–duplex junction of a parallel G‐quadruplex featuring a stem‐loop duplex extension. Thermodynamic profiles and modes of binding differ for hybrids with duplex extensions at either the 5′‐ or 3′‐face of the G‐quadruplex.

A hybrid-type G-quadruplex is modified with LNA (locked nucleic acid) and 2′-F-riboguanosine in various combinations at the two syn positions of its third antiparallel G-tract. LNA substitution in the central tetrad causes a complete rearrangement to either a V-loop or antiparallel structure, depending on further modifications at the 5′-neighboring site. In the two distinct structural contexts, LNA-induced stabilization is most effective compared to modifications with other G surrogates, highlighting a potential use of LNA residues for designing not only parallel but various more complex G4 structures. For instance, the conventional V-loop is a structural element strongly favored by an LNA modification at the V-loop 3′-end in contrast with an alternative V-loop, clearly distinguishable by altered conformational properties and base-backbone interactions as shown in a detailed analysis of V-loop structures.

Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.

Various genomic DNA sequences including a MYC promoter sequence are amenable to the formation of a G-quadruplex featuring a snap-back loop with the incorporation of a 3'-terminal guanine into the quadruplex core. To evaluate relative stabilities and ligand binding in more detail, optical, microcalorimetric, and NMR structural studies were performed on both a minimal mutant sequence Pu22T that exclusively folds into a snap-back loop quadruplex and a parallel MYC quadruplex proposed to be the most relevant fold of the MYC promoter in a cellular environment. Similar thermal stabilities for Pu22T and MYC suggest the coexistence of both quadruplexes when derived from a sequence able to fold into both topologies. Isothermal titration calorimetry indicates a mostly identical enthalpy-driven strong binding of an indoloquinoline ligand but with a reduced number of high-affinity binding sites in Pu22T in line with a novel modified FRET competitive melting assay. Corroborated by fluorescence titrations using 2-aminopurine as a fluorescent probe, NMR chemical shift footprints show binding of the ligand at the Pu22T 5'-outer tetrad with the formation of a binding pocket. On the other hand, steric restrictions due to the snap-back loop severely restrict ligand stacking on the 3'-outer tetrad of Pu22T.

White spot syndrome virus (WSSV) is considered one of the most infectious and lethal viruses that affect shrimp. Bioinformatic studies revealed several G-quadruplex forming sequences at the open reading frame region. Moreover, the sequences are widely conserved through all deposited WSSV sequences. Introductory structural studies on two sequences, namely WSSV131 and WSSV172, are proposed to form a quadruplex. While WSSV172 forms a mixture of quadruplex topologies, WSSV131 is suggested to form a parallel topology, as indicated by the NMR spectra and circular dichroism (CD) ellipticity pattern. CD spectra also suggested that the major parallel species of the WSSV131 sequence are found to be stable above 60 °C. Ultimately, these results may open a new strategy for WSSV treatment by targeting the quadruplex confirmation with a quadruplex binding ligand.

Mixed 2'-F-riboguanosine and 2'-F-arabinoguanosine disubstitutions of a hybrid-type G-quadruplex are found to induce a refolding into two alternative structures with different types of V-loops upon positional exchange of the two G analogs. While conformational preferences of the incorporated G surrogates fail to fully account for the observed rearrangements, additional hydrogen bonds with a fluorine acceptor are suggested to be critical determinants of the two distinct V-loop conformers imposing different tetrad polarities.

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., Refolding of a (3+1) hybrid G‐quadruplex into a quadruplex with an interrupted G‐tract and a V‐shaped loop is enforced by two 2′‐deoxy‐2′‐fluoro‐riboguanosine substitutions. The analogues flanking the V‐loop in the rearranged structure adopt syn and anti glycosidic torsion angles and a north‐type sugar pucker, the latter being identified as a critical parameter for V‐loop formation.

Nucleic acids can adopt various secondary structures including double-, triple-, and tetra-stranded helices that differ by the specific hydrogen bond mediated pairing pattern between their nucleobase constituents. Whereas double-helical DNA relies on Watson–Crick base pairing to play a prominent role in storing genetic information, G-quadruplexes are tetra-stranded structures that are formed by the association of guanine bases from G-rich DNA and RNA sequences. During the last few decades, G-quadruplexes have attracted considerable interest after the realization that they form and exert regulatory functions in vivo. In addition, quadruplex architectures have also been recognized as versatile and powerful tools in a growing number of technological applications. To appreciate the astonishing structural diversity of these tetra-stranded structures and to give some insight into basic interactions that govern their folding, this article gives an overview of quadruplex structures and rules associated with the formation of different topologies. A brief discussion will also focus on nonconventional quadruplexes as well as on general principles when targeting quadruplexes with ligands. Graphic abstract

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G‐quadruplexes have attracted growing interest in recent years due to their occurrence in vivo and their possible biological functions. In addition to being promising targets for drug design, these four‐stranded nucleic acid structures have also been recognized as versatile tools for various technological applications. Whereas a large number of studies have yielded insight into their remarkable structural diversity, our current knowledge on G‐quadruplex stabilities as a function of sequence and environmental factors only gradually emerges with an expanding collection of thermodynamic data. This minireview provides an overview of general rules that may be used to better evaluate quadruplex thermodynamic stabilities but also discusses present challenges in predicting most stable folds for a given sequence and environment., Biological functions of G‐quadruplexes and their properties as exploited in various technological applications depend on their major fold adopted under specific conditions. The present minireview guides through structural and environmental factors that have been identified to play a major role in the formation and stability of G‐quadruplexes.

Multiple G‐tracts within the promoter region of the c‐myc oncogene may fold into various G‐quadruplexes with the recruitment of different tracts and guanosine residues for the G‐core assembly. Thermodynamic profiles for the folding of wild‐type and representative truncated as well as mutated sequences were extracted by comprehensive DSC experiments. The unique G‐quadruplex involving consecutive G‐tracts II–V with formation of two one‐nucleotide and one central two‐nucleotide propeller loop, previously proposed to be the biologically most relevant species, was found to be the most stable fold in terms of its Gibbs free energy of formation at ambient temperatures. Its stability derives from its short propeller loops but also from the favorable type of loop residues. Whereas quadruplex folds with long propeller loops are significantly disfavored, a snap‐back loop structure formed by incorporating a 3’‐terminal guanosine into the empty position of a tetrad seems highly competitive based on its thermodynamic stability. However, its destabilization by extending the 3’‐terminus questions the significance of such a species under in vivo conditions., Multiple guanosine tracts within the promoter region of the c‐myc oncogene may fold into various G‐quadruplex structures to act as gene regulatory elements. A thermodynamic profiling of quadruplex folding identifies most stable topologies at ambient temperatures.

The ability to control the folding topology of DNA G-quadruplexes allows for rational design of quadruplex-based scaffolds for potential use in various therapeutic and technological applications. By exploiting the distinct conformational properties of some base- and sugar-modified guanosine surrogates, conformational transitions can be induced through their judicious incorporation at specific sites in the quadruplex core. Changes may involve tetrad polarity inversions with conservation of the global fold or complete refolding to new topologies. Reliable predictions relating to low-energy conformers formed upon specific chemical perturbations of the system and the rational design of modified sequences suffer from our still limited understanding of the subtle interplay of various favorable and unfavorable interactions within a particular quadruplex scaffold. However, aided by an increasing number of systematic substitution experiments and high-resolution structures of modified quadruplex variants, critical interactions, in addition to glycosidic bond angle propensities, are starting to emerge as important contributors to modification-driven quadruplex refolding.

Binding of indoloquinolines with different aliphatic side chains to a parallel G-quadruplex derived from the MYC promoter sequence was characterized by optical and calorimetric measurements. ITC experiments performed at different temperatures enabled the determination of molar heat capacity changes upon quadruplex binding and a partitioning of the total binding free enthalpy into contributing terms with hydrophobic effects being major driving forces for all derivatives. Whereas affinities increase for indoloquinolines equipped with a long and positively charged side arm, the highest contribution of specific intermolecular interactions anticipated to impart enhanced specificity is found for a ligand with an uncharged ether aliphatic tail. Obtained thermodynamic signatures may considerably aid in the rational selection of ligand side chains for G-quadruplex binders with enhanced affinity or selectivity.

A DNA G-quadruplex adopting a (3+1)-hybrid structure was substituted at its 5'-tetrad by riboguanosine (rG) analogs. Incorporation of anti-favoring rG at appropriate syn-positions of the 5'-outer tetrad induced conformational rearrangements to yield a quadruplex featuring a 5'-tetrad with reversed polarity. A high-resolution structure of a disubstituted quadruplex variant as well as direct NMR experimental evidence reveals a non-conventional C-H⋅⋅⋅O hydrogen bond in a medium groove between the 2'-OH of an rG residue adopting a C2'-endo sugar pucker and H8 of a 3'-neighboring anti-G residue. In contrast, a C3'-endo sugar conformation for another guanine ribonucleotide prevents formation of a corresponding hydrogen bond but relocates its 2'-OH substituent from the quadruplex narrow groove into a medium groove. Both the formation of favorable CHO hydrogen bridges and unfavorable interactions of the 2'-hydroxyl group in a narrow groove will promote RNA folding into a parallel topology featuring all-anti core residues and four grooves of medium size.

A (3+1)-hybrid-type G-quadruplex was substituted within its central tetrad by a single 2'-fluoro-modified guanosine. Driven by the anti-favoring nucleoside analogue, a novel quadruplex fold with inversion of a single G-tract and conversion of a propeller loop into a lateral loop emerges. In addition, scalar couplings across hydrogen bonds demonstrate the formation of intra- and inter-residual F⋅⋅⋅H8-C8 pseudo-hydrogen bonds within the modified quadruplexes. Alternative folding can be rationalized by the impact of fluorine on intermediate species on the basis of a kinetic partitioning mechanism. Apparently, chemical or other environmental perturbations are able to redirect folding of a quadruplex, possibly modulating its regulatory role in physiological processes.

A G-quadruplex adopting a (3 + 1)-hybrid structure was substituted at its 5′-tetrad by 2′-deoxy-2′-fluoro-arabinoguanosine (FaraG) analogs. Incorporation of anti-favoring FaraG at syn-positions of the 5′-outer tetrad induced a reversal of the tetrad polarity without noticeably compromising the quadruplex stability. This conformational change is shown to be promoted by nonconventional C–H···F hydrogen bonds acting within the anti-FaraG residues.

Binding of a positively charged indoloquinoline derivative to a G-quadruplex formed by the G-rich promoter element of the c-MYC oncogene was subjected to a rigorous isothermal calorimetric analysis. Binding of the indoloquinoline is primarily enthalpy-driven but is also promoted by a favorable entropy term. Both binding enthalpy ΔH° and binding entropy ΔS° exhibit a noticeable temperature dependence with almost complete enthalpy–entropy compensation as a result of a negative change in heat capacity ΔCp°. Salt-dependent polyelectrolyte effects only moderately contribute to the overall free energy of association. More details on the binding process are revealed in an attempt to dissect the total free energy into individual contributory terms. Accordingly, specific intermolecular interactions between the indoloquinoline ligand and G-quadruplex substantially contribute in addition to hydrophobic effects in promoting the association. Comparing thermodynamic profiles for various quadruplex ligands indicates dif...

A benzothiazole derivative was identified as potent ligand for DNA G-quadruplex structures. Fluorescence titrations revealed selective binding to quadruplexes of different topologies including parallel, antiparallel, and (3+1) hybrid structures. The parallel c-MYC sequence was found to constitute the preferred target with dissociation constants in the micromolar range. Binding of the benzothiazole-based ligand to c-MYC was structurally and thermodynamically characterized in detail by employing a comprehensive set of spectroscopic and calorimetric techniques. Job plot analyses and mass spectral data indicate noncooperative ligand binding to form complexes with 1:1 and 2:1 stoichiometries. Whereas stacking interactions are suggested by optical methods, NMR chemical shift perturbations also indicate significant rearrangements of both 5'- and 3'-flanking sequences upon ligand binding. Additional isothermal calorimetry studies yield a thermodynamic profile of the ligand-quadruplex association and reveal enthalpic contributions to be the major driving force for binding. Structural and thermodynamic information obtained in the present work provides the basis for the rational development of benzothiazole derivatives as promising quadruplex binding agents.

A human telomere sequence that folds into an intramolecular (3 + 1)-hybrid G-quadruplex was modified by the incorporation of 2′-fluoro-2′-deoxyriboguanosines (FG) into syn positions of its outer tetrad. A circular dichroism and NMR spectral analysis reveals a nearly quantitative switch of the G-tetrad polarity with concerted syn↔anti transitions of all four G residues. These observations follow findings on a FG-substituted (3 + 1)-hybrid quadruplex with a different fold, suggesting a more general propensity of hybrid-type quadruplexes to undergo a tetrad polarity reversal. Two out of the three FG analogs in both modified quadruplexes adopt an S-type sugar pucker, challenging a sole contribution of N-type sugars in enforcing an anti glycosidic torsion angle associated with the tetrad flip. NMR restrained three-dimensional structures of the two substituted quadruplexes reveal a largely conserved overall fold but significant rearrangements of the overhang and loop nucleotides capping the flipped tetrad. Suga...

Methylene blue (MB) - nucleic acid interactions are of considerable interest due to the photosensitizing activity of the dye with potential applications in medicine and biotechnology. Covalent attachment of the MB to an oligonucleotide through a flexible heptamethylene linker enabled a positioning of the dye moiety to specific sites through triplex formation with a target duplex. NMR studies demonstrated interactions of MB with the nucleic acids. In sequences with the MB moiety facing the triplex-duplex junction with an alternating CG duplex overhang next to a T·A·T triple-helical tract, proton resonances experienced severe linebroadening upon MB binding and point to kinetically labile complexes with exchange among different binding modes. For sequences with the MB moiety facing a terminal T·A·T base triad of the triplex tract, structural heterogeneity decreased when compared to a triplex without MB attached to the third strand. Also, the thermal stability of the latter construct increased significantly in the presence of MB, indicating external end stacking as predominant binding mode. Without any obvious disruptions of sequential imino-imino NOE contacts within the triplex and duplex tracts, a most favorable intercalation between T·A·T base triples or CG base pairs is not supported by the present data under our experimental conditions.

The thermodynamic characterization of G4-ligand interactions has shown to be a powerful adjunct to structural information in the rational design and optimization of potent G-quadruplex ligands for use in therapeutics, diagnostics, or other technological applications. Isothermal titration calorimetry (ITC) can resolve energetic contributions to complex formation and constitutes the only available experimental method to directly measure binding enthalpies. A general protocol for using ITC in studies on quadruplex-ligand interactions with details on the experimental setup, data analysis, and potential pitfalls is presented. The methodologies used are illustrated on results obtained from the targeting of a parallel DNA G-quadruplex with a G4-binding indoloquinoline derivative.

The oligomer d(GCGTG3 TCAG3 TG3 TG3 ACGC) 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.

DNA-G-Quadruplexe wurden systematisch an anti-dG-Positionen mit einzelnen Riboguanosin-Bausteinen (rG) modifiziert. Zirkulardichroismus- und NMR-Experimente bestatigen den Erhalt der nativen Quadruplex-Topologie fur den Grosteil der DNA-RNA-Hybridstrukturen. Veranderungen in der C8-chemischen Verschiebung von Guanosinen an der 3′-Seite einer rG-Substitution im Quadruplex-Kern legen C8-H⋅⋅⋅O-Wasserstoffbrucken mit dem O2′ des C2′-endo-Ribonukleotids nahe. Eine geometrische Analyse bekannter, hochaufgeloster Strukturen deutet darauf hin, dass entsprechende Wechselwirkungen ein allgemeineres Merkmal in RNA-Quadruplexen sind und moglicherweise zur beobachteten Praferenz fur parallele Topologien beitragen.

The thermodynamic characterization of G4-ligand interactions has shown to be a powerful adjunct to structural information in the rational design and optimization of potent G-quadruplex ligands for use in therapeutics, diagnostics, or other technological applications. Isothermal titration calorimetry (ITC) can resolve energetic contributions to complex formation and constitutes the only available experimental method to directly measure binding enthalpies. A general protocol for using ITC in studies on quadruplex-ligand interactions with details on the experimental setup, data analysis, and potential pitfalls is presented. The methodologies used are illustrated on results obtained from the targeting of a parallel DNA G-quadruplex with a G4-binding indoloquinoline derivative.

A G-quadruplex forming sequence from the MYC promoter region was modified with syn-favoring 8-bromo-2'-deoxyguanosine residues. Depending on the number and position of modifications in the intramolecular parallel G-quadruplex, substitutions with the bromoguanosine analogue at the 5'-tetrad induce conformational rearrangements with concerted all-anti to all-syn transitions for all residues of the modified G-quartet. No unfavorable steric interactions of the C8-substituents in the medium grooves are apparent in the high-resolution structure as determined for a tetrasubstituted MYC quadruplex that exclusively forms the all-syn isomer. In contrast, considerable steric clashes with 5'-phosphate oxygen atoms for those analogues that follow a less flexible 1-nucleotide loop in the native all-anti conformation seem to constitute the major driving force for the tetrad inversion and allow for the rational design of appropriately substituted sequences. Correlations found between the population of species subjected to a tetrad flip and melting temperatures indicate that more effective conformational transitions are compromised by lower thermal stabilities of the modified parallel quadruplexes.

Naphthalene- and coumarin-substituted dithiolene ligands were synthesized, and their coordination behavior towards different metal ions, including biologically relevant ions like Cu+, Cu2+, and Fe3+, was tested. Both ligands bind nickel selectively and show no evidence of binding any other metal investigated. Particularly, the compound bearing the fluorophore coumarin can serve as a colorimetric (“naked eye”) and on-off fluorometric marker for the detection of Ni2+ ions with a characteristic near-infrared (NIR) signature even in the presence of various transition-metal ions, which are normally difficult to differentiate from nickel. Competitive fluorescence experiments showed that the concomitant presence of Ag+, Al3+, Ca2+, Cd2+, Co2+, Cu+, Cu2+, Fe3+, Hg2+, Mg2+, Mn2+, Pb2+, Sn2+, Zn2+, and MoO42– could not inhibit or diminish the Ni2+-specific fluorescence quenching, emphasizing the inherent Ni2+ ion selectivity of the system. The coordination behavior of the marker and Ni2+ were studied in detail by fluorescence titration experiments in THF/MeOH solution. X-ray structures were determined to complement characterization of the precursors and the naphthalene–dithiolene–nickel complex.

The biocatalytic synthesis of chiral amines has become a valuable addition to the chemists' tool-box. However, the efficient asymmetric synthesis of functionalised amines bearing more than one ster ...

A unimolecular G-quadruplex with a hybrid-type topology and propeller, diagonal, and lateral loops was examined for its ability to undergo structural changes upon specific modifications. Substituting 2′-deoxy-2′-fluoro analogues with a propensity to adopt an anti glycosidic conformation for two or three guanine deoxyribonucleosides in syn positions of the 5′-terminal G-tetrad significantly alters the CD spectral signature of the quadruplex. An NMR analysis reveals a polarity switch of the whole tetrad with glycosidic conformational changes detected for all four guanine nucleosides in the modified sequence. As no additional rearrangement of the overall fold occurs, a novel type of G-quadruplex is formed with guanosines in the four columnar G-tracts lined up in either an all-syn or an all-anti glycosidic conformation.

Binding of an indoloquinoline derivative with an aminoalkyl side chain to a truncated sequence from the MYC promoter region was studied through isothermal titration calorimetry (ITC). The targeted MYC3 sequence lacks 3'-flanking nucleotides and forms a monomeric parallel quadruplex (G4) with a blunt-ended 3'-outer tetrad under the solution conditions employed. Analysis of ITC isotherms reveals multiple binding equilibria with the initial formation of a 1:2 ligand/quadruplex complex. Evaluation of electrophoretic mobilities as well as NMR spectral data confirm ligand-induced dimerization of MYC3 quadruplexes with the ligand sandwiched between the two 3'-outer tetrads. Additional ligand molecules in excess bind to the 5'-outer tetrads of the sandwich complex. Such a ligand-promoted G4 dimerization may be exploited for the controlled assembly or disassembly of G4 aggregates to expand on present quadruplex-based technologies.

Even though there is an urgent need for new antifungals with improved clinical properties, the substrate promiscuity of tailoring enzymes has been poorly studied as a source of new structural diversity for polyene macrolides. We explore the acceptance of different polyene macrolides by the glutamine amidotransferase PscA and the catalytic effect of different homologous P450 cytochromes on a common scaffold, the pimaricin precursor 4,5-desepoxypimaricin. By combining these two parallel strategies, we present three new pimaricin derivatives and a new lucensomycin variant. Our results show that P450 cytochromes devoted to the modification of the polyol region of polyene macrolides are not as substrate-specific as previously thought and highlight PscA as a versatile small-ring polyene-modifying enzyme that allows the preparation of new carboxamide derivatives. We also provide useful information for the future production of previously unconceived epoxidized polyene macrolide antifungals.

Bifunctional dibenzobarrelene-based PC(sp3)P pincer iridium complex 1 is known as an efficient catalyst in acceptorless dehydrogenation of alcohols and hydrogenation/hydroformylation of alkenes. In order to shed light on the mechanism of the hydrogen formation/activation, we performed variable-temperature IR and NMR (1H, 31P) analysis of intra- and intermolecular interactions involving a hydride ligand and hydroxymethyl cooperating group in 1 and its analogues. The results of the spectroscopic measurements in different media (dichloromethane, toluene, DMSO, and mixed solvents) were compared with the quantum chemical (DFT/M06 and B3PW91) calculations. The obtained data imply flexibility of the dibenzobarrelene-based scaffold, unprecedented for conventional pincer ligands. Both the CH2OH-substituted complex 1 and its COOEt analogue 3 prefer facial configuration of the PCP ligand with a P–Ir–P angle of ca. 100°. Such geometries are stabilized by Ir···O interaction with the dangling functional group and diffe...

Indoloquinoline alkaloids isolated from natural sources are known for their broad spectrum of biological activities. The cytotoxic action of some derivatives is thought to arise from their interaction with genomic DNA driven by favorable stacking interactions of the intercalated indoloquinoline skeleton with the DNA base pairs. In addition to double-helical DNA, indoloquinoline derivatives have also been found to bind DNA triplexes as well as G-quadruplexes with high affinity, opening interesting perspectives towards the development of indoloquinoline-based DNA binding ligands with numerous potential applications in therapeutics, diagnostics and microbiology. The present paper presents an overview of studies dealing primarily with the binding of indoloquinoline analogs to double-helical, triple-helical or quadruplex targets. Available thermodynamic and structural data that also include few experimentally determined three-dimensional structures of DNA-drug complexes highlight key elements for an improved binding affinity but also important structural aspects for the design of structure-selective analogs, prerequisite for many potential applications.

A TSiT dinucleotide linked through a nonionic diisopropylsilyl backbone has been synthesized and studied for its self-association through base–base recognition together with a free thymidine nucleoside. To characterize hydrogen-bonded associates in more detail, NMR measurements were performed in a freonic solvent at temperatures as low as 113 K in the slow hydrogen bond exchange regime. For the thymidine, TT base pairs with both the 2- and 4-carbonyl engaged in hydrogen bonds to the imino proton were observed. Whereas hydrogen bonds to the O4 acceptor are stronger as evidenced by a more deshielded proton in the hydrogen bridge when compared to hydrogen bonds to the O2 acceptor, the latter is nevertheless slightly favored over O4 in the H-bond formation of TT base pairs. The diisopropylsilyl linkage of the TSiT dinucleotide has no significant impact on the geometry and strength of formed NH–O2 and NH–O4 hydrogen bonds indicating, that the silyl backbone does not compromise the alignment of bases and does not pose any restrictions to the cyclic hydrogen bond formation between thymidines of the two strands. However, NMR results point to an exclusive formation of TSiT duplexes with an antiparallel strand orientation.

Uridine (U) imino proton chemical shifts, measured in the slow hydrogen-bond exchange regime at low temperatures in a freonic solution, show that electron-withdrawing 5-bromo and 5-fluoro substituents on the uracil base strengthen NHN hydrogen bonds in (X)U·A base pairs formed by the free nucleosides. Whereas the halogens do not alter the preferential formation of Watson-Crick geometries, self-associates of the halouracils point to a more favorable engagement of the 2-carbonyl as proton acceptor in the cyclic hydrogen bonds, suggesting increased formation of reverse geometries and wobble base pairs with guanine when compared to the thymine base. Employing (15)N-labeled 5-bromouridine, no noticeable population of minor enol tautomers is found in the freonic mixture at 113 K.

A MYC sequence forming an intramolecular G-quadruplex with a parallel topology was modified by the incorporation of 8-bromoguanosine (BrG) analogues in one of its outer G-tetrads. The propensity of the BrG analogues to adopt a syn glycosidic torsion angle results in an exceptional monomolecular quadruplex conformation featuring a complete flip of one tetrad while keeping a parallel orientation of all G-tracts as shown by circular dichroism and nuclear magnetic resonance spectroscopic studies. When substituting three of the four G-tetrad residues with BrG analogues, two coexisting quadruplex conformational isomers with an all-syn and all-anti outer G-quartet are approximately equally populated in solution. A dynamic interconversion of the two quadruplexes with an exchange rate (kex) of 0.2 s–1 is demonstrated through the observation of exchange crosspeaks in rotating frame Overhauser effect spectroscopy and nuclear Overhauser effect spectroscopy experiments at 50 °C. The kinetic properties suggest disrupti...

DNA G-quadruplexes were systematically modified by single riboguanosine (rG) substitutions at anti-dG positions. Circular dichroism and NMR experiments confirmed the conservation of the native quadruplex topology for most of the DNA–RNA hybrid structures. Changes in the C8 NMR chemical shift of guanosines following rG substitution at their 3′-side within the quadruplex core strongly suggest the presence of C8−H⋅⋅⋅O hydrogen-bonding interactions with the O2′ position of the C2′-endo ribonucleotide. A geometric analysis of reported high-resolution structures indicates that such interactions are a more general feature in RNA quadruplexes and may contribute to the observed preference for parallel topologies.

Base pairs formed by the inosine nucleoside (I) play an important role in many physiological processes as well as in various DNA technologies. Relative stabilities and favored base pair geometries of free inosine wobble base pairs in aprotic solvents have been determined through (1)H NMR measurements at room temperature and at very low temperatures in a freonic solvent. As indicated by its significantly deshielded imino proton, the Watson-Crick-type I·C base pair forms a remarkably strong NHN hydrogen bond. For the thermodynamically less stable I·A wobble base pair, two configurations of similar population coexist at 133 K in the slow hydrogen bond exchange regime, namely a Watson-Crick(I)-Watson-Crick(A) geometry and a Watson-Crick(I)-Hoogsteen(A) geometry. I·U base pairs are stabilized by two rather weak hydrogen bonds and are significantly disfavored over inosine self-associates in a low-temperature Freon solution.

Phenyl-substituted indoloquinolines were studied for their biological activity and their DNA binding affinity. Water-soluble aminoalkyl derivatives were prepared and have shown significant in vitro anticancer activity. Unlike previous reports on the potential role of duplex DNA as target for various indoloquinoline based drugs, duplex UV melting experiments and fluorescence titrations suggest only weak and moderately strong binding of the phenyl-substituted indoloquinolines at 120 mM and 20 mM Na+ concentrations, respectively. Binding is suggested by ethidium displacement and circular dichroism experiments to be associated with drug intercalation between base pairs.

Several triplex-forming 9-mer oligonucleotide (TFO) conjugates with a methyl- or methoxy-substituted 5-phenyl-6H-indolo[3,2-b]quinoline (PIQ) attached at the 5'-terminus or 3'-terminus or at an internal C5 thymine position were synthesized and tested for their ability to form and stabilize a triple helix with a double-helical DNA target employing UV melting experiments, fluorescence titrations, and isothermal titration calorimetry (ITC). A considerable thermal stabilization by up to 14 degrees C at pH 6.0 was observed for the 5'- and 3'-conjugates with little influence on the type of substituent but also for a conjugate with the ligand tethered by a short linker to the interior of the 9-mer TFO. A detailed thermodynamic characterization of the unmodified TFO and its 5'-conjugate with a methyl-substituted ligand by ITC experiments yielded a DeltaDeltaG degrees of -1.8 kcal mol(-1) at pH 6.0 for the TFO-attached PIQ-triplex interaction and also revealed a favorable entropic contribution as the major determinant for the free energy of PIQ binding in the conjugate. The pH dependence of triplex thermal stability highlights the importance of ring protonation of the triplex-bound ligand for its effective interaction and triplex stabilization near physiological conditions.

DNA binding of two hybrid ligands composed of an alkylating pyrrolo[2,1- c ][1,4]benzodiazepine (PBD) moiety tethered to either a naphthalimide or a phenyl benzimidazole chromophore was studied by DNA melting experiments, UV and fluorescence titrations, CD spectroscopy and isothermal titration calorimetry (ITC). Binding of both hybrids results in a remarkable thermal stabilization with an increase of DNA melting temperatures by up to 40 °C for duplexes that allow for a covalent attachment of the PBD moiety to guanine bases in their minor groove. CD spectroscopic measurements suggest that the naphthalimide moiety of the drug interacts through intercalation. In contrast, the PBD-benzimidazole hybrid binds in the DNA minor groove with a preference for (A,T) 4 G sequences. Whereas the binding of both ligands is enthalpy-driven and associated with a negative entropy, the benzimidazole hybrid exhibits a less favourable binding enthalpy that is counterbalanced by a more favourable entropic term when compared to the naphthalimide hybrid.