Structure,properties, and biological relevance of the DNA and RNA G-quadruplexes: Overview 50 years after their discovery

G-quadruplexes (G4s), which are known to have important roles in regulation of key biological processes in both normal and pathological cells, are the most actively studied non-canonical structures of nucleic acids. In this review, we summarize the results of studies published in recent years that change significantly scientific views on various aspects of our understanding of quadruplexes. Modern notions on the polymorphism of DNA quadruplexes, on factors affecting thermodynamics and kinetics of G4 folding–unfolding, on structural organization of multiquadruplex systems, and on conformational features of RNA G4s and hybrid DNA–RNA G4s are discussed. Here we report the data on location of G4 sequence motifs in the genomes of eukaryotes, bacteria, and viruses, characterize G4-specific small-molecule ligands and proteins, as well as the mechanisms of their interactions with quadruplexes. New information on the structure and stability of G4s in telomeric DNA and oncogene promoters is discussed as well as proof being provided on the occurrence of G-quadruplexes in cells. Prominence is given to novel experimental techniques (single molecule manipulations, optical and magnetic tweezers, original chemical approaches, G4 detection in situ, in-cell NMR spectroscopy) that facilitate breakthroughs in the investigation of the structure and functions of G-quadruplexes.     

Quadruplex DNA: sequence, topology and structure

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes.     

Direct experimental evidence for quadruplex–quadruplex interaction within the human ILPR

Here we report the analysis of dual G-quadruplexes formed in the four repeats of the consensus sequence from the insulin-linked polymorphic region (ACAGGGGTGTGGGG; ILPR_n=4). Mobilities of ILPR_n=4 in nondenaturing gel and circular dichroism (CD) studies confirmed the formation of two intramolecular G-quadruplexes in the sequence. Both CD and single molecule studies using optical tweezers showed that the two quadruplexes in the ILPR_n=4 most likely adopt a hybrid G-quadruplex structure that was entirely different from the mixture of parallel and antiparallel conformers previously observed in the single G-quadruplex forming sequence (ILPR_n=2). These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem. Furthermore, mechanical pulling of the ILPR_n=4 at the single molecule level suggests that the two quadruplexes are unfolded cooperatively, perhaps due to a quadruplex–quadruplex interaction (QQI) between them. Additional evidence for the QQI was provided by DMS footprinting on the ILPR_n=4 that identified specific guanines only protected in the presence of a neighboring G-quadruplex. There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR.     

The four repeat Giardia lamblia telomere forms tetramolecular G-quadruplex with antiparallel topology

Abstract

Guanine rich DNA sequences of regulatory genomic regions form secondary structures known as G-quadruplexes usually stabilized by tetrads of Hoogsteen hydrogen bonded guanines. The in vivo existence of G-quadruplexes ascertains their biological roles. Human telomeric repeats are the most studied G-rich sequences. The four repeat Giardia telomeric sequence (TAGGG)₄ differs from its human counterpart (TTAGGG)_4, by deletion of one T at the G-tract intervening site of each repeat. We show here that whilst the two repeat Giardia telomeric sequence (TAGGG)₂ forms parallel and antiparallel quadruplexes with tetramolecular topology exclusively, the four repeat version (TAGGG)₄ forms a tetramolecular (antiparallel) and unimolecular (parallel) quadruplexes in Na⁺. The tetramolecular (antiparallel) G-quadruplex formed by four repeats of Giardia telomeric sequence is stabilized by the additional Watson-Crick bonding between its intervening TA bases aligned in antiparallel fashion. Four stranded antiparallel quadruplex for four repeats of any telomeric sequence have not been characterized till date. We hypothesize that telomeric association in antiparallel fashion, (via G-overhangs to form tetramolecular quadruplex) could be a biologically relevant molecular event. Further, coexistence of Hoogsteen as well as Watson-Crick base pairing might give insight for recognition of conformationally diverse DNA structures by ligands.

Communicated by Ramaswamy H. Sarma     

The flanking sequence contributes to the immobilisation of spermine at the G-quadruplex in the NHE (nuclease hypersensitivity element) III1 of the c-Myc promoter

Defining the molecular basis of the DNA sequence selectivity of polyamine binding is central to understanding polyamine-dependent gene expression. We have studied, by selective NMR experiments, the variation of spermine mobility and conformation in the presence of G-quadruplexes formed by sequences of the purine-rich strand of the c-Myc promoter, nuclease hypersensitivity element III₁ (NHE III₁). All the NHE quadruplexes restrict spermine mobility and induce a spermine conformational change but the most effective immobilisation occurs when all five G-tracts of the NHE III₁ are present. This suggests structure within the nucleotides flanking the G-quadruplex has a role in immobilising spermine.     

Re-evaluation of G-quadruplex propensity with G4Hunter

Critical evidence for the biological relevance of G-quadruplexes (G4) has recently been obtained in seminal studies performed in a variety of organisms. Four-stranded G-quadruplex DNA structures are promising drug targets as these non-canonical structures appear to be involved in a number of key biological processes. Given the growing interest for G4, accurate tools to predict G-quadruplex propensity of a given DNA or RNA sequence are needed. Several algorithms such as Quadparser predict quadruplex forming propensity. However, a number of studies have established that sequences that are not detected by these tools do form G4 structures (false negatives) and that other sequences predicted to form G4 structures do not (false positives). Here we report development and testing of a radically different algorithm, G4Hunter that takes into account G-richness and G-skewness of a given sequence and gives a quadruplex propensity score as output. To validate this model, we tested it on a large dataset of 392 published sequences and experimentally evaluated quadruplex forming potential of 209 sequences using a combination of biophysical methods to assess quadruplex formation in vitro. We experimentally validated the G4Hunter algorithm on a short complete genome, that of the human mitochondria (16.6 kb), because of its relatively high GC content and GC skewness as well as the biological relevance of these quadruplexes near instability hotspots. We then applied the algorithm to genomes of a number of species, including humans, allowing us to conclude that the number of sequences capable of forming stable quadruplexes (at least in vitro) in the human genome is significantly higher, by a factor of 2–10, than previously thought.     

Quadruplex ligands may act as molecular chaperones for tetramolecular quadruplex formation

G-quadruplexes are a family of four-stranded DNA structures, stabilized by G-quartets, that form in the presence of monovalent cations. Efforts are currently being made to identify ligands that selectively bind to G-quadruplex motifs as these compounds may interfere with the telomere structure, telomere elongation/replication and proliferation of cancer cells. The kinetics of quadruplex–ligands interactions are poorly understood: it is not clear whether quadruplex ligands lock into the preformed structure (i.e. increase the lifetime of the structure by lowering the dissociation constant, k_off) or whether ligands actively promote the formation of the complex and act as quadruplex chaperones by increasing the association constant, k_on. We studied the effect of a selective quadruplex ligand, a bisquinolinium pyridine dicarboxamide compound called 360A, to distinguish these two possibilities. We demonstrated that, in addition to binding to and locking into preformed quadruplexes, this molecule acted as a chaperone for tetramolecular complexes by acting on k_on. This observation has implications for in vitro and in vivo applications of quadruplexes and should be taken into account when evaluating the cellular responses to these agents.     

Effect of sequence and metal ions on UVB-induced anti cyclobutane pyrimidine dimer formation in human telomeric DNA sequences

Irradiation of G-quadruplex forming human telomeric DNA with ultraviolet B (UVB) light results in the formation of anti cyclobutane pyrimidine dimers (CPDs) between loop 1 and loop 3 in the presence of potassium ions but not sodium ions. This was unexpected because the sequences involved favor the nonphotoreactive hybrid conformations in K⁺ solution, whereas a potentially photoreactive basket conformation is favored in Na⁺ solution. To account for these contradictory results, it was proposed that the loops are too far apart in the basket conformation in Na⁺ solution but close enough in a two G-tetrad basket-like form 3 conformation that can form in K⁺ solution. In the current study, Na⁺ was still found to inhibit anti CPD formation in sequences designed to stabilize the form 3 conformation. Furthermore, anti CPD formation in K⁺ solution was slower for the sequence previously shown to exist primarily in the proposed photoreactive form 3 conformation than the sequence shown to exist primarily in a nonphotoreactive hybrid conformation. These results suggest that the form 3 conformation is not the principal photoreactive conformation, and that G-quadruplexes in K⁺ solution are dynamic and able to access photoreactive conformations more easily than in Na⁺ solution.     

Structural dynamics of possible late-stage intermediates in folding of quadruplex DNA studied by molecular simulations

Explicit solvent molecular dynamics simulations have been used to complement preceding experimental and computational studies of folding of guanine quadruplexes (G-DNA). We initiate early stages of unfolding of several G-DNAs by simulating them under no-salt conditions and then try to fold them back using standard excess salt simulations. There is a significant difference between G-DNAs with all-anti parallel stranded stems and those with stems containing mixtures of syn and anti guanosines. The most natural rearrangement for all-anti stems is a vertical mutual slippage of the strands. This leads to stems with reduced numbers of tetrads during unfolding and a reduction of strand slippage during refolding. The presence of syn nucleotides prevents mutual strand slippage; therefore, the antiparallel and hybrid quadruplexes initiate unfolding via separation of the individual strands. The simulations confirm the capability of G-DNA molecules to adopt numerous stable locally and globally misfolded structures. The key point for a proper individual folding attempt appears to be correct prior distribution of syn and anti nucleotides in all four G-strands. The results suggest that at the level of individual molecules, G-DNA folding is an extremely multi-pathway process that is slowed by numerous misfolding arrangements stabilized on highly variable timescales.     

Heme activation by DNA: isoguanine pentaplexes,but not quadruplexes,bind heme and enhance its oxidative activity

Guanine-rich, single-stranded, DNAs and RNAs are able to fold to form G-quadruplexes that are held together by guanine base quartets. G-quadruplexes are known to bind ferric heme [Fe(III)-protoporphyrin IX] and to strongly activate such bound hemes toward peroxidase (1-electron oxidation) as well as oxygenase/peroxygenase (2-electron oxidation) activities. However, much remains unknown about how such activation is effected. Herein, we investigated whether G-quadruplexes were strictly required for heme activation or whether related multi-stranded DNA/RNA structures such as isoguanine (iG) quadruplexes and pentaplexes could also bind and activate heme. We found that iG-pentaplexes did indeed bind and activate heme comparably to G-quadruplexes; however, iG-quadruplexes did neither. Earlier structural and computational studies had suggested that while the geometry of backbone-unconstrained iG-quintets templated by cations such as Na⁺ or NH₄⁺ was planar, that of iG-quartets deviated from planarity. We hypothesize that the binding as well as activation of heme by DNA or RNA is strongly supported by the planarity of the nucleobase quartet or quintet that interacts directly with the heme.     

High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element

Guanine-rich DNA and RNA sequences are known to fold into secondary structures known as G-quadruplexes. Recent biochemical evidence along with the discovery of an increasing number of sequences in functionally important regions of the genome capable of forming G-quadruplexes strongly indicates important biological roles for these structures. Thus, molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes as well as potential therapeutic agents. Guanine-rich peptide nucleic acids have been previously shown to hybridize to homologous DNA or RNA sequences forming PNA-DNA (or RNA) quadruplexes. For this paper we studied the hybridization of an eight-mer G-rich PNA to a quadruplex-forming sequence derived from the promoter region of the MYC proto-oncogene. UV melting analysis, fluorescence assays, and surface plasmon resonance experiments reveal that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 10(8) M(-1) or Kd = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed.     

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.     

Sequence effects in single-base loops for quadruplexes

Intramolecular G-quadruplexes formed by a single DNA strand have attracted much interest due to the possibility that they may form in telomeres, oncogene promoter sequences and other biologically relevant regions of the genome. Therefore, it is important to understand the rules that govern the formation of these intramolecular structures and to determine their stabilities. We compared here 27 different sequences containing four tracts of three guanines with a medium (3) or relatively long (6-9 bases) central loop and two loops composed of a single nucleotide H (A, T or C) corresponding to the GGGHGGGN3-9GGGHGGG motif. These sequences are similar to sequence motifs that can be found in repeated and promoter sequences. Several conclusions were reached: (i) all sequences are prone to form very stable quadruplexes in potassium (Tm between 55 degrees C and 83 degrees C); (ii) these quadruplexes also form in sodium but with a strongly decreased Tm, with a 21 to 36 degrees C difference in melting temperature between Na+ and K+; (iii) a long (9 bases) central loop had a minimal deleterious impact on the stability of the quadruplex; (iv) pyrimidines are preferred over adenine in both single-base loops; (v) the folding topology is relatively robust in potassium: the CD spectra of all oligonucleotides matched the one of all-parallel stranded reference quadruplexes; (vi) conversely, in sodium the folding is diverse and sequence-dependent, as judged from CD and electrophoresis results.     

A structure-activity relationship of a thrombin-binding aptamer containing LNA in novel sites

In this report, structural characterization, aptamer stability and thrombin of a new modified thrombin-ligand complex binding aptamer (TBA) containing anti-guanine bases and a loop position locked nucleic acid (LNA) are presented. NMR, circular dichroic spectroscopy and molecular modeling were used to characterize the three-dimensional structure of two G-quadruplexes. LNA-modification of the anti-guanosines yields G-quadruplexes that show affinity and inhibitory activity toward thrombin, whereas LNA-modification of a thymine nucleotide in the TGT loop increases the thermal stability of TBA. As assessed by denatured PAGE electrophoresis, all modified aptamers display an increase in environmental stability. The prothrombin time assay and fibrinogen assay showed that the aptamers still had good inhibitory activity, and 15 of them had the longest PT time. Therefore, the LNA modification is well suited to improve the physicochemical and biological properties of the native thrombin-binding aptamer.