The solution structure of d(G(4)T(4)G(3))(2): a bimolecular G-quadruplex with a novel fold

The G-rich 11-mer oligonucleotide d(G(4)T(4)G(3)) forms a bimolecular G-quadruplex in the presence of sodium ions with a topology that is distinct from the folds of the closely related and well-characterized sequences d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)). The solution structure of d(G(4)T(4)G(3))(2) has been determined using a combination of NMR spectroscopy and restrained molecular dynamics calculations. d(G(4)T(4)G(3))(2) forms an asymmetric dimeric fold-back structure consisting of three stacked G-quartets. The two T(4) loops that span diagonally across the outer faces of the G-quartets assume different conformations. The glycosidic torsion angle conformations of the guanine bases are 5'-syn-anti-syn-anti-(T(4) loop)-anti-syn-anti in one strand and 5'-syn-anti-syn-anti-(T(4) loop)-syn-anti-syn in the other strand. The guanine bases of the two outer G-quartets exhibit a clockwise donor-acceptor hydrogen-bonding directionality, while those of the middle G-quartet exhibit the anti-clockwise directionality. The topology of this G-quadruplex, like other bimolecular fold-back structures with diagonal loops, places each strand of the G-quartet region next to a neighboring parallel and an anti-parallel strand. The two guanine residues not involved in G-quartet formation, G4 and G12 (i.e. the fourth guanine base of one strand and the first guanine base of the other strand), adopt distinct conformations. G4 is stacked on top of an adjacent G-quartet, and this base-stacking continues along with the bases of the loop residues T5 and T6. G12 is orientated away from the core of G-quartets; stacked on the T7 base and apparently involved in hydrogen-bonding interactions with the phosphodiester group of this same residue. The cation-dependent folding of the d(G(4)T(4)G(3))(2) quadruplex structure is distinct from that observed for similar sequences. While both d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)) form bimolecular, diagonally looped G-quadruplex structures in the presence of Na(+), K(+) and NH(4)(+), we have observed this folding to be favored for d(G(4)T(4)G(3)) in the presence of Na(+), but not in the presence of K(+) or NH(4)(+). The structure of d(G(4)T(4)G(3))(2) exhibits a "slipped-loop" element that is similar to what has been proposed for structural intermediates in the folding pathway of some G-quadruplexes, and therefore provides support for the feasibility of these proposed transient structures in G-quadruplex formation.     

A sequence-independent analysis of the loop length dependence of intramolecular RNA G-quadruplex stability and topology

G-Quadruplexes are noncanonical nucleic acid secondary structures based on guanine association that are readily adopted by G-rich RNA and DNA sequences. Naturally occurring genomic G-quadruplex-forming sequences have functional roles in biology that are mediated through structure. To appreciate how this is achieved, an understanding of the likelihood of G-quadruplex formation and the structural features of the folded species under a defined set of conditions is informative. We previously systematically investigated the thermodynamic stability and folding topology of DNA G-quadruplexes and found a strong dependence of these properties on loop length and loop arrangement [Bugaut, A., and Balasubramanian, S. (2008) Biochemistry 47, 689-697]. Here we report on a complementary analysis of RNA G-quadruplexes using UV melting and circular dichroism spectroscopy that also serves as a comparison to the equivalent DNA G-quadruplex-forming sequences. We found that the thermodynamic stability of G-quadruplex RNA can be modulated by loop length while the overall structure is largely unaffected. The systematic design of our study also revealed subtle loop length dependencies in RNA G-quadruplex structure.     

Solution structure of a DNA quadruplex containing ALS and FTD related GGGGCC repeat stabilized by 8-bromodeoxyguanosine substitution

A prolonged expansion of GGGGCC repeat within non-coding region of C9orf72 gene has been identified as the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are devastating neurodegenerative disorders. Formation of unusual secondary structures within expanded GGGGCC repeat, including DNA and RNA G-quadruplexes and R-loops was proposed to drive ALS and FTD pathogenesis. Initial NMR investigation on DNA oligonucleotides with four repeat units as the shortest model with the ability to form an unimolecular G-quadruplex indicated their folding into multiple G-quadruplex structures in the presence of K⁺ ions. Single dG to 8Br-dG substitution at position 21 in oligonucleotide d[(G₄C₂)₃G₄] and careful optimization of folding conditions enabled formation of mostly a single G-quadruplex species, which enabled determination of a high-resolution structure with NMR. G-quadruplex structure adopted by d[(G₄C₂)₃GG^BrGG] is composed of four G-quartets, which are connected by three edgewise C-C loops. All four strands adopt antiparallel orientation to one another and have alternating syn-anti progression of glycosidic conformation of guanine residues. One of the cytosines in every loop is stacked upon the G-quartet contributing to a very compact and stable structure.     

47 Incorporation of alternative nucleic acid chemistries into G-quadruplex structure

Nucleic acids that form G-quadruplex (G4) structure have found applications in a host of research and technology regimes. Numerous G4 based aptamer drugs have been identified with pharmacological activity against cancer, HIV, prions, and blood coagulation (1). In the field of nanotechnology, G4 based sensors and nano-machines have also received much attention. The ability to synthesize nucleic acid ex-vivo allows for the site-specific incorporation of non-natural chemistries into nucleic acids that can be used to tune their physical and pharmacological properties. We summarize the results of a series of studies investigating the effective incorporation of alternative nucleic acid chemistries into G4 DNA. These modified chemistries include C8-modified guanine bases, as well as 2′-F, 2′-F-ANA, and Locked nucleic acid (LNA) modifications to the ribose sugar. We report primarily on the effect of these modifications on G-quadruplex folding topology, thermal stability, and structure. The substitution of LNA-guanosine into the core guanine tetrads disrupts structure in specific structural environments. On the other hand, 2′-F- and 2′-F-ANA guanosine can generally be incorporated without disrupting the structure when substituted into guanine bases in certain structural conformations. We find that 2′-F-ANA-guanosine and 2′-F-guanosine are powerful tools for controling the conformation of G4 structures (2). Functionalization at the C8 of the guanine base stabilizes in a manner dependent on the glycosidic conformation of the base, with different modification chemistries stabilizing to varying extents (3). The results of these studies provide useful insight on how to effectively incorporate some useful chemical tools from the growing toolbox of modified nucleic acid chemistries into G-quadruplex nucleic acid.     

d(G3T4G4) forms unusual dimeric G-quadruplex structure with the same general fold in the presence of K+, Na+ or NH4+ ions

We have recently communicated that DNA oligonucleotide d(G(3)T(4)G(4)) forms a dimeric G-quadruplex in the presence of K(+) ions [J. Am. Chem. Soc.2003, 125, 7866-7871]. The high-resolution NMR structure of d(G(3)T(4)G(4))(2) G-quadruplex exhibits G-quadruplex core consisting of three stacked G-quartets. The two overhanging G3 and G11 residues are located at the opposite sides of the end G-quartets and are not involved in G-quartet formation. d(G(3)T(4)G(4))(2) G-quadruplex represents the first bimolecular G-quadruplex where end G-quartets are spanned by diagonal (T4-T7) as well as edge-type loops (T15-T18). Three of the G-rich strands are parallel while one is anti-parallel. The G12-G22 strand demonstrates a sharp reversal in strand direction between residues G19 and G20 that is accommodated with the leap over the middle G-quartet. The reversal in strand direction is achieved without any extra intervening residues. Here we furthermore examined the influence of different monovalent cations on the folding of d(G(3)T(4)G(4)). The resolved imino and aromatic proton resonances as well as (sequential) NOE connectivity patterns showed only minor differences in key intra- and interquartet NOE intensities in the presence of K(+), Na(+) and NH(4)(+) ions, which were consistent with subtle structural differences while retaining the same folding topology of d(G(3)T(4)G(4))(2) G-quadruplex.     

Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb)

The formation of G-quadruplex and i-motif structures in the 5′ end of the retinoblastoma (Rb) gene was examined using chemical modifications, circular dichroism (CD) and fluorescence spectroscopy. It was found that substitutions of 8-methylguanine at positions that show syn conformations in antiparallel G-quadruplexes stabilize the structure in the G-rich strand. The complementary C-rich 18mer forms an i-motif structure, as suggested by CD spectroscopy. Based on the C to T mutation experiments, C bases participated in the C–C⁺ base pair of the i-motif structure were determined. Experiments of 2-aminopurine (2-AP) substitution reveal that an increase of fluorescence in the G-quadruplex relative to duplex is attributed to unstacked 2-AP within the loop of G-quadruplex. The fluorescence experiments suggest that formation of the G-quadruplex and i-motif can compete with duplex formation. Furthermore, a polymerase arrest assay indicated that formation the G-quadruplex structure in the Rb gene acts as a barrier in DNA synthesis.     

Highly fluorescent guanosine mimics for folding and energy transfer studies

Guanosines with substituents at the 8-position can provide useful fluorescent probes that effectively mimic guanine residues even in highly demanding model systems such as polymorphic G-quadruplexes and duplex DNA. Here, we report the synthesis and photophysical properties of a small family of 8-substituted-2′-deoxyguanosines that have been incorporated into the human telomeric repeat sequence using phosphoramidite chemistry. These include 8-(2-pyridyl)-2′-deoxyguanosine (2PyG), 8-(2-phenylethenyl)-2′-deoxyguanosine (StG) and 8-[2-(pyrid-4-yl)-ethenyl]-2′-deoxyguanosine (4PVG). On DNA folding and stability, 8-substituted guanosines can exhibit context-dependent effects but were better tolerated by G-quadruplex and duplex structures than pyrimidine mismatches. In contrast to previously reported fluorescent guanine analogs, 8-substituted guanosines exhibit similar or even higher quantum yields upon their incorporation into nucleic acids (Φ = 0.02–0.45). We have used these highly emissive probes to quantify energy transfer efficiencies from unmodified DNA nucleobases to 8-substituted guanosines. The resulting DNA-to-probe energy transfer efficiencies (η_t) are highly structure selective, with η_t(duplex) < η_t(single-strand) < η_t(G-quadruplex). These trends were independent of the exact structural features and thermal stabilities of the G-quadruplexes or duplexes containing them. The combination of efficient energy transfer, high probe quantum yield, and high molar extinction coefficient of the DNA provides a highly sensitive and reliable readout of G-quadruplex formation even in highly diluted sample solutions of 0.25 nM.     

Physiological relevance of telomeric G-quadruplex formation: a potential drug target

The concept of a G-quartet, a unique structural arrangement intrinsic to guanine-rich DNA, was first introduced by Gellert and colleagues over 40 years ago. For decades, it has been uncertain whether the G-quartet and the structure that it gives rise to, the G-quadruplex, are purely in vitro phenomena. Nevertheless, the presence of signature G-rich motifs in the eukaryotic genome, and the plethora of proteins that bind to, modify or resolve this nucleic acid structure in vitro have provided circumstantial evidence for its physiological relevance. More recently, direct visualisation of G-quadruplex DNA at native telomeres was achieved, bolstering the evidence for its existence in the cell. Furthermore, G-quadruplex folded telomeric DNA has been found to perturb telomere function and to impede the action of telomerase, an enzyme overexpressed in >85% of human cancers, hence opening up a novel avenue for cancer therapy in the form of G-quadruplex stabilising agents.     

G-quadruplex preferentially forms at the very 3' end of vertebrate telomeric DNA

Human chromosome ends are protected with kilobases repeats of TTAGGG. Telomere DNA shortens at replication. This shortening in most tumor cells is compensated by telomerase that adds telomere repeats to the 3′ end of the G-rich telomere strand. Four TTAGGG repeats can fold into G-quadruplex that is a poor substrate for telomerase. This property has been suggested to regulate telomerase activity in vivo and telomerase inhibition via G-quadruplex stabilization is considered a therapeutic strategy against cancer. Theoretically G-quadruplex can form anywhere along the long G-rich strand. Where G-quadruplex forms determines whether the 3′ telomere end is accessible to telomerase and may have implications in other functions telomere plays. We investigated G-quadruplex formation at different positions by DMS footprinting and exonuclease hydrolysis. We show that G-quadruplex preferentially forms at the very 3′ end than at internal positions. This property provides a molecular basis for telomerase inhibition by G-quadruplex formation. Moreover, it may also regulate those processes that depend on the structure of the very 3′ telomere end, for instance, the alternative lengthening of telomere mechanism, telomere T-loop formation, telomere end protection and the replication of bulky telomere DNA. Therefore, targeting telomere G-quadruplex may influence more telomere functions than simply inhibiting telomerase.     

Evidence of the formation of G-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene

The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates.     

Conformation and Stability of Intramolecular Telomeric G-Quadruplexes: Sequence Effects in the Loops

Telomeres are guanine-rich sequences that protect the ends of chromosomes. These regions can fold into G-quadruplex structures and their stabilization by G-quadruplex ligands has been employed as an anticancer strategy. Genetic analysis in human telomeres revealed extensive allelic variation restricted to loop bases, indicating that the variant telomeric sequences maintain the ability to fold into G-quadruplex. To assess the effect of mutations in loop bases on G-quadruplex folding and stability, we performed a comprehensive analysis of mutant telomeric sequences by spectroscopic techniques, molecular dynamics simulations and gel electrophoresis. We found that when the first position in the loop was mutated from T to C or A the resulting structure adopted a less stable antiparallel topology; when the second position was mutated to C or A, lower thermal stability and no evident conformational change were observed; in contrast, substitution of the third position from A to C induced a more stable and original hybrid conformation, while mutation to T did not significantly affect G-quadruplex topology and stability. Our results indicate that allelic variations generate G-quadruplex telomeric structures with variable conformation and stability. This aspect needs to be taken into account when designing new potential anticancer molecules.     

Telomeric repeat mutagenicity in human somatic cells is modulated by repeat orientation and G-quadruplex stability

Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]₁₀ repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]₄ and [TTTTGGGG]₄ are thermally more stable compared to human [TTAGGG]₄. Consistent with this, replication of vectors with ciliate [TTGGGG]₁₀ repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]₁₀ vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.     

Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation

We report the NMR solution structure of the intramolecular G-quadruplex formed in human telomeric DNA in K⁺. The hybrid-type telomeric G-quadruplex consists of three G-tetrads linked with mixed parallel–antiparallel G-strands, with the bottom two G-tetrads having the same G-arrangement (anti:anti:syn:anti) and the top G-tetrad having the reversed G-arrangement (syn:syn:anti:syn). The three TTA loop segments adopt different conformations, with the first TTA assuming a double-chain-reversal loop conformation, and the second and third TTA assuming lateral loop conformations. The NMR structure is very well defined, including the three TTA loops and the two flanking sequences at 5′- and 3′-ends. Our study indicates that the three loop regions interact with the core G-tetrads in a specific way that defines and stabilizes the unique human telomeric G-quadruplex structure in K⁺. Significantly, a novel adenine triple platform is formed with three naturally occurring adenine residues, A21, A3 and A9, capping the top tetrad of the hybrid-type telomeric G-quadruplex. This adenine triple is likely to play an important role in the formation of a stable human telomeric G-quadruplex structure in K⁺. The unique human telomeric G-quadruplex structure formed in K⁺ suggests that it can be specifically targeted for anticancer drug design.     

Perylene side chains modulate G-quadruplex conformation in biologically relevant DNA sequences

The stabilisation of different G-quadruplex intra- and intermolecular structures by a number of perylene derivatives characterised by side chains ending with linear or cyclic amines was investigated by electrophoretic (EMSA) and spectroscopic (CD) techniques. The G-rich sequences included the biologically relevant human telomeric TTAGGG runs and the NHE region of the c-myc oncogene. The test compounds could be subdivided into two families: derivatives carrying a cyclic amine in the side chains, which show a reduced binding to the G-quadruplex form, and linear amine congeners, exhibiting enhanced affinity. The latter efficiently induce pairing of multiple DNA chains, while the former are not able to overcome the original folding of the nucleic acid sequence which is preserved in the complex. Remarkably, addition of the perylenes to G-rich sequences paired in a double helical form results in G-quadruplex induction by weak binders only. This is likely related to the ability of strong G-quadruplex binders, but not of weak G-quadruplex binders, to efficiently intercalate into the double-stranded arrangement, which becomes stabilised and is not prone to undergo denaturation and subsequent G-quadruplex folding essentially for kinetic reasons. Hence, two apparently conflicting requirements emerge from this work. In fact, linear alkylamino terminals in the perylene side chains are capable of strong and selective G-quadruplex recognition, but only cyclic amine end groups favour duplex-quadruplex transitions that are likely crucial to produce biological and pharmacological effects in living systems.