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.     

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.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.     

Towards the Development of Structure‐Selective G‐Quadruplex‐Binding Indolo[3,2‐b]quinolines

The interaction of phenyl‐substituted indolo[3,2‐b]quinolines with DNA G‐quadruplexes of different topology were studied by using a combination of spectroscopic and calorimetric methodologies. N5‐Methylated indoloquinoline derivatives (^MePIQ) with an aminoalkyl side chain exhibit high affinities for the parallel‐stranded MYC quadruplex and a (3+1)‐hybrid structure combined with an excellent discrimination against the antiparallel thrombin‐binding aptamer (TBA) and the human telomeric (HT) quadruplexes. Dissociation constants for the binding of the ligand to the MYC quadruplex are in the submicromolar range, being below the corresponding dissociation constants for the antiparallel‐stranded quadruplexes by about one order of magnitude. Competition experiments with double‐helical DNA reveal the impact of indoloquinoline structural features on the selectivity for the parallel quadruplex relative to duplex DNA. Based on a calorimetric analysis binding to MYC is shown to be equally driven by favorable enthalpic and entropic contributions with no significant impact on the type of cation present.     

Engineering the quadruplex fold: nucleoside conformation determines both folding topology and molecularity in guanine quadruplexes

Nucleic acid quadruplexes, based on the guanine quartet, can arise from one or several strands, depending on the sequence. Those consisting of a single strand are usually folded in one of two principal topologies: antiparallel, in which all or half of the guanine stretches are antiparallel to each other, or parallel, in which all guanine stretches are parallel to each other. In the latter, all guanine nucleosides possess the anti conformation about the glycosidic bond, while in the former, half possess the anti conformation, and half possess the syn conformation. While antiparallel is the more common fold, examples of biologically important, parallel quadruplexes are becoming increasingly common. Thus, it is of interest to understand the forces that determine the quadruplex fold. Here, we examine the influence of individual nucleoside conformation on the overall folding topology by selective substitution of rG for dG. We can reverse the antiparallel fold of the thrombin binding aptamer (TBA) by this approach. Additionally, this substitution converts a unimolecular quadruplex into a bimolecular one. Similar reverse substitutions in the all-RNA analogue of TBA result in a parallel to antiparallel change in topology and alter the strand configuration from bimolecular to unimolecular. On the basis of the specific substitutions made, we conclude that the strong preference of guanine ribonucleosides for the anti conformation is the driving force for the change in topology. These results demonstrate how conformational properties of guanine nucleosides govern not only the quadruplex folding topology but also impact quadruplex molecularity and provide a means to control these properties.     

Quantitative Detection of G‐Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination

G‐quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G‐quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE , which showed distinguishable fluorescence lifetime responses between G‐quadruplexes and other DNA topologies, and fluorescence quantum yield (Φ_f) enhancement upon G‐quadruplex binding. We determined two NBTE ‐G‐quadruplex complex structures with high Φ_f values by NMR spectroscopy. The structures indicated NBTE interacted with G‐quadruplexes using three arms through π–π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φ_f values and lifetime response of NBTE upon G‐quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G‐quadruplex DNA in live cells with NBTE and found G‐quadruplex DNA content in cancer cells is 4‐fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.     

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.     

Tuning the Stereoselectivity of a DNA‐Catalyzed Michael Addition through Covalent Modification

Complexes of G‐quadruplex DNA and Cu^II ions have previously been applied as catalysts in asymmetric reactions, but the largely unspecific and noncovalent nature of the interaction has impeded understanding of the structural basis of catalysis. To better control the formation of a catalytically competent species, DNA quadruplexes were derivatized with linker‐bpy‐Cu^II complexes in a site‐specific manner and applied in asymmetric aqueous Michael additions. These modified quadruplexes exhibited high rate acceleration and stereoselectivity. Different factors were found to be important for the catalytic performance of the modified G‐quadruplexes, among them, the position of modification, the topology of the quadruplex, the nature of the ligand, and the length of the linker between the ligand and DNA. Moving the same ligand by just two nucleotides inverted the stereochemical outcome: quadruplexes modified at position 10 formed the (?)‐enantiomer with up to 92 % ee, while DNA derivatized at position 12 formed the (+)‐enantiomer with up to 75 % ee. This stereopreference was maintained when applied to structurally different Michael acceptors. This work demonstrates a new and simple way to tune the stereoselectivity in DNA‐based asymmetric catalysis.     

Sugar–Edge Interactions in a DNA–RNA G‐Quadruplex: Evidence of Sequential C−H⋅⋅⋅O Hydrogen Bonds Contributing to RNA Quadruplex Folding

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.     

Binding Behaviors for Different Types of DNA G‐Quadruplexes: Enantiomers of [Ru(bpy)2(L)]2+ (L=dppz,dppz‐idzo)

Polymorphic DNA G‐quadruplex recognition has attracted great interest in recent years. The strong binding affinity and potential enantioselectivity of chiral [Ru(bpy)₂(L)]²⁺ (L=dipyrido[3,2‐a:2′,3′‐c]phenazine, dppz‐10,11‐imidazolone; bpy=2,2′‐bipyridine) prompted this investigation as to whether the two enantiomers, Δ and Λ, can show different effects on diverse structures with a range of parallel, antiparallel and mixed parallel/antiparallel G‐quadruplexes. These studies provide a striking example of chiral‐selective recognition of DNA G‐quadruplexes. As for antiparallel (tel‐Na⁺) basket G‐quadruplex, the Λ enantiomers bind stronger than the Δ enantiomers. Moreover, the behavior reported here for both enantiomers stands in sharp contrast to B‐DNA binding. The chiral selectivity toward mixed parallel/antiparallel (tel‐K⁺) G‐quadruplex of both compounds is weak. Different loop arrangements can change chiral complex selectivity for both antiparallel and mixed parallel/antiparallel G‐quadruplex. Whereas both Δ and Λ isomers bind to parallel G‐quadruplexes with comparable affinity, no appreciable stereoselective G‐quadruplex binding of the isomers was observed. In addition, different binding stoichiometries and binding modes for Δ and Λ enantiomers were confirmed. The results presented here indicate that chiral selective G‐quadruplex binding is not only related to G‐quadruplex topology, but also to the sequence and the loop constitution.     

The Effect of DNA Sequence Directionality on G‐Quadruplex Folding

Sequence inversion in G‐rich DNA from 5′→3′ to 3′→5′ exerts a substantial effect on the number of structures formed, while the type of G‐quadruplex fold is in fact determined by the presence of K⁺ or Na⁺ ions. The melting temperatures of G‐quadruplexes adopted by oligonucleotides with sequences in the 5′→3′ direction are higher than those of their 3′→5′ counterparts with both KCl and NaCl. CD, UV, and NMR spectroscopy demonstrates the importance of primary sequence for the structural diversity of G‐quadruplexes. The changes introduced by mere sequence reversal of the G‐rich DNA segment have a substantial impact on the polymorphic nature of the resulting G‐quadruplexes and their potential physiological roles. The insights resulting from this study should enable extension of the empirical rules for the prediction of G‐quadruplex topology.     

G‐Quadruplex Secondary Structure Obtained from Circular Dichroism Spectroscopy

A curated library of circular dichroism spectra of 23 G‐quadruplexes of known structure was built and analyzed. The goal of this study was to use this reference library to develop an algorithm to derive quantitative estimates of the secondary structure content of quadruplexes from their experimental CD spectra. Principal component analysis and singular value decomposition were used to characterize the reference spectral library. CD spectra were successfully fit to obtain estimates of the amounts of base steps in anti–anti, syn–anti or anti–syn conformations, in diagonal or lateral loops, or in other conformations. The results show that CD spectra of nucleic acids can be analyzed to obtain quantitative structural information about secondary structure content in an analogous way to methods used to analyze protein CD spectra.     

Molecular Recognition and Visual Detection of G‐Quadruplexes by a Dicarbocyanine Dye

The interactions of a dicarbocyanine dye 3,3′‐diethylthiadicarbocyanine, DiSC₂(5) , with DNA G‐quadruplexes were studied by means of a combination of various spectroscopic techniques. Aggregation of excess dye as a result of its positive charge is promoted by the presence of the polyanionic quadruplex structure. Specific high‐affinity binding to the parallel quadruplex of the MYC promoter sequence involves stacking of DiSC₂(5) on the external G‐tetrads; the 5′‐terminal tetrad is the favored binding site. Significant energy transfer between DNA and the dye in the UV spectral region is observed upon DiSC₂(5) binding. The transfer efficiency strongly depends on the DNA secondary structure as well as on the G‐quadruplex topology. These photophysical features enable the selective detection of DNA quadruplexes through sensitized DiSC₂(5) fluorescence in the visible region.     

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.     

Carbohydrate–DNA Interactions at G‐Quadruplexes: Folding and Stability Changes by Attaching Sugars at the 5′‐End

Quadruplex DNA structures are attracting an enormous interest in many areas of chemistry, ranging from chemical biology, supramolecular chemistry to nanoscience. We have prepared carbohydrate–DNA conjugates containing the oligonucleotide sequences of G‐quadruplexes (thrombin binding aptamer (TBA) and human telomere (TEL)), measured their thermal stability and studied their structure in solution by using NMR and molecular dynamics. The solution structure of a fucose–TBA conjugate shows stacking interactions between the carbohydrate and the DNA G‐tetrad in addition to hydrogen bonding and hydrophobic contacts. We have also shown that attaching carbohydrates at the 5′‐end of a quadruplex telomeric sequence can alter its folding topology. These results suggest the possibility of modulating the folding of the G‐quadruplex by linking carbohydrates and have clear implications in molecular recognition and the design of new G‐quadruplex ligands.     

Copper‐Induced Topology Switching and Thrombin Inhibition with Telomeric DNA G‐Quadruplexes

The topological diversity of DNA G‐quadruplexes may play a crucial role in its biological function. Reversible control over a specific folding topology was achieved by the synthesis of a chiral, glycol‐based pyridine ligand and its fourfold incorporation into human telomeric DNA by solid‐phase synthesis. Square‐planar coordination to a Cu^II ion led to the formation of a highly stabilizing intramolecular metal–base tetrad, substituting one G‐tetrad in the parent unimolecular G‐quadruplex. For the Tetrahymena telomeric repeat, Cu^II‐triggered switching from a hybrid‐dominated conformer mixture to an antiparallel topology was observed. Cu^II‐dependent control over a protein–G‐quadruplex interaction was shown for the thrombin–tba pair (tba=thrombin‐binding aptamer).     

Structural Properties of Hachimoji Nucleic Acids and Their Building Blocks: Comparison of Genetic Systems with Four,Six and Eight Alphabets

Expansion of the genetic alphabet is an ambitious goal. A recent breakthrough has led to the eight-base (hachimoji) genetics having canonical and unnatural bases. However, very little is known on the molecular-level features that facilitate the candidature of unnatural bases as genetic alphabets. Here we amalgamated DFT calculations and MD simulations to analyse the properties of the constituents of hachimoji DNA and RNA. DFT reveals the dominant syn conformation for isolated unnatural deoxyribonucleosides and at the 5′-end of oligonucleotides, although an anti/syn mixture is predicted at the nonterminal and 3′-terminal positions. However, isolated ribonucleotides prefer an anti/syn mixture, but mostly prefer anti conformation at the nonterminal positions. Further, the canonical base pairing combinations reveals significant strength, which may facilitate replication of hachimoji DNA. We also identify noncanonical base pairs that can better tolerate the substitution of unnatural pairs in RNA. Stacking strengths of 51 dimers reveals higher average stacking stabilization of dimers of hachimoji bases than canonical bases, which provides clues for choosing energetically stable sequences. A total of 14.4 μs MD simulations reveal the influence of solvent on the properties of hachimoji oligonucleotides and point to the likely fidelity of replication of hachimoji DNA. Our results pinpoint the features that explain the experimentally observed stability of hachimoji DNA.     

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.     

The adamantane rearrangement of syn- and anti-Tricyclo[4.2.1.1.12,5] decane. Part II. Rearrangements initiated by regioselective formation of carbocations at C(3) and C(9)

The endo- and exo-alcohols 5–12 of syn-( 1 ) and anti-tricyclo[4.2.1. 1^2.5]decane ( 2 ) were treated with BF₃/Et₃SiH (ionic hydrogenation) in order to study the behaviour of the corresponding regioselectively generated carbocations at C(3) ( a (syn), b (anti)) and C(9) ( c (syn), d (anti)). The anti-hydrocarbon 2 is practically the sole product obtained starting with the four 3-alcohols (via a → b from 5 and 6 (syn) and via b from 9 and 10 (anti)). The four 9-alcohols in each case yield a mixture of 2-endo, 3-endo- ( 3 ) and 2-exo,3-exo-trimethylene-8,9,10-trinorbornane (4) (via c → e from 7 and 8 (syn) and via d → f from 11 and 12 (anti)), but no hydrocarbon 2 , i.e. none of the 1,3-H shifts c → a and d → b is involved.     

Photocrosslinking of G‐Quadruplex‐Forming Sequences found in Human Promoters

While is it well known that human telomeric DNA sequences can adopt G‐quadruplex structures, some promoters sequences have also been found to form G‐quadruplexes, and over 40% of promoters contain putative G‐quadruplex‐forming sequences. Because UV light has been shown to crosslink human telomeric G‐quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G‐quadruplexes in promoters. To investigate this possibility, 15 potentially UV‐crosslinkable G‐quadruplex‐forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G‐quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non‐B DNA structures, such as G‐quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.