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.     

Biophysical properties of quadruple helices of modified human telomeric DNA

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)n. The guanine rich strand can fold into four-stranded G-quadruplex structures, which have recently become attractive for biomedical research. Indeed, the aptamers based on the quadruplex motif may prove useful as tools aimed at binding and inhibiting particular proteins, catalyzing various biochemical reactions, or even serving as pharmaceutically active agents. The incorporation of modified bases into oligonucleotides can have profound effects on their folding and may produce useful changes in physical and biological properties of the resulting DNA fragments. In this work, the adenines of the human telomeric repeat oligonucleotide d(TAGGGT) and d(AGGGT) were substituted by 2'-deoxy-8-(propyn-1-yl)adenosine (A-->APr) or by 8-bromodeoxyadenosine (A-->ABr). The biophysical properties of the resulting quadruplex structures were compared with the unmodified quadruplexes. NMR and CD spectra of the studied sequences were characteristic of parallel-stranded, tetramolecular quadruplexes. The analysis of the equilibrium melting curves reveals that the modifications stabilize the quadruplex structure. The results are useful when considering the design of novel aptameric nucleic acids with diverse molecular recognition capabilities that would not be present using native RNA/DNA sequences.     

A comparison of DNA and RNA quadruplex structures and stabilities

Guanosine-rich sequences are prone to fold into four-stranded nucleic acid structures. Such quadruplex sequences have long been suspected to play important roles in regulatory processes within cells. Although DNA quadruplexes have been studied in great detail, four-stranded structures made up from RNA have received only minor attention, although it is known that RNA is able to form stable quadruplexes as well.Here, we compare quadruplex structures and stabilities of a variety of DNA and RNA sequences. We focus on well established DNA sequences and determine the topologies and stabilities of the corresponding RNA sequences by CD spectroscopy and CD thermal melting experiments. We find that the RNA sequences exclusively fold into the all-parallel conformation in contrast to the diverse topologies adopted by DNA quadruplexes. The thermal stabilities of the RNA structures rival those of the corresponding DNA sequences, often displaying enhanced stabilities compared to their DNA counterparts. Especially thermodynamically less stable sequences show a strong preference for potassium, with the RNA quadruplexes exhibiting much higher stabilities than the corresponding DNAs. The latter finding suggests that quadruplexes formed at critical positions in mRNAs might perturb gene expression to a larger extend than previously anticipated.     

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.     

Intramolecular and intermolecular guanine quadruplexes of DNA in aqueous salt and ethanol solutions

DNA guanine quadruplexes are all based on stacks of guanine tetrads, but they can be of many types differing by mutual strand orientation, topology, position and structure of loops, and the number of DNA molecules constituting their structure. Here we have studied a series of nine DNA fragments (G(3)Xn)(3)G(3), where X = A, C or T, and n = 1, 2 or 3, to find how the particular bases and their numbers enable folding of the molecule into quadruplex and what type of quadruplex is formed. We show that any single base between G(3) blocks gives rise to only four-molecular parallel-stranded quadruplexes in water solutions. In contrast to previous models, even two Ts in potential loops lead to tetramolecular parallel quadruplexes and only three consecutive Ts lead to an intramolecular quadruplex, which is antiparallel. Adenines make the DNA less prone to quadruplex formation. (G(3)A(2))(3)G(3) folds into an intramolecular antiparallel quadruplex. The same is true with (G(3)A(3))(3)G(3) but only in KCl. In NaCl or LiCl, (G(3)A(3))(3)G(3) prefers to generate homoduplexes. Cytosine still more interferes with the quadruplex, which only is generated by (G(3)C)(3)G(3), whereas (G(3)C(2))(3)G(3) and (G(3)C(3))(3)G(3) generate hairpins and/or homoduplexes. Ethanol is a more potent DNA guanine quadruplex inducer than are ions in water solutions. It promotes intramolecular folding and parallel orientation of quadruplex strands, which rather corresponds to quadruplex structures observed in crystals.     

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.     

Guanines are a quartet's best friend: impact of base substitutions on the kinetics and stability of tetramolecular quadruplexes

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well-defined non-guanine base quartets in a number of NMR and X-ray structures, our data suggest that most non-guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G-quartets. Two notable exceptions were found with 8-bromo-guanine (X) and 6-methyl-isoxanthopterin (P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 5' end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/dissociation properties.     

Stability and structure of telomeric DNA sequences forming quadruplexes containing four G-tetrads with different topological arrangements

Telomeres are DNA-protein structures at the ends of eukaryotic chromosomes, the DNA of which comprise noncoding repeats of guanine-rich sequences. Telomeric DNA plays a fundamental role in protecting the cell from recombination and degradation. Telomeric sequences can form quadruplex structures stabilized by guanine quartets. These structures can be constructed from one, two, or four oligonucleotidic strands. Here, we report the thermodynamic characterization of the stability, analyzed by differential scanning calorimetry, of three DNA quadruplexes of different molecularity, all containing four G-tetrads. The conformational properties of these quadruple helices were studied by circular dichroism. The investigated oligomers form well-defined G-quadruplex structures in the presence of sodium ions. Two have the truncated telomeric sequence from Oxytricha, d(TGGGGT) and d(GGGGTTTTGGGG), which form a tetramolecular and bimolecular quadruplex, respectively. The third sequence, d(GGGGTTGGGGTGTGGGGTTGGGG) was designed to form a unimolecular quadruplex. The thermodynamic parameters of these quadruplexes have been determined. The tetramolecular structure is thermodynamically more stable than the bimolecular one, which, in turn, is more stable than the unimolecular one. The experimental data were discussed in light of the molecular-modeling study.     

Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides.

Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA.     

Research progress of RNA quadruplex

RNA/DNA sequences rich in guanine (G) can form a 4-strand structure, G-quadruplex, which has been extensively researched and observed in mammalian, fungi, and plants, with in vivo existence in eukaryotic cells. Compared with DNA quadruplex, the potential existence of RNA quadruplex appears to be generally rare; however, it is believed by some researchers to be more inevitable in vivo and speculated to play an important role where it exists. Recently, researches concerning the function of G-quadruplexes in RNAs commence, making much progress. However, there is no available review particularly focusing on RNA quadruplex till now as we know. Therefore, we decide to give a review to comprehensively summarize research progress on it. This review highlights the diverse topologies for RNA quadruplex structure and its effect factors; outlines the current knowledge of RNA quadruplex's physiological functions in biological systems, especially in gene expression; and presents the prospects of RNA quadruplex.     

Insights into the RNA quadruplex binding specificity of DDX21

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2′OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2′OH of loop nucleotides.     

G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K(d) values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 +/- 6 and 77 +/- 6 Pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures.     

The high-resolution crystal structure of a parallel intermolecular DNA G-4 quadruplex/drug complex employing syn glycosyl linkages

We have determined the X-ray structure of the complex between the DNA quadruplex d(5′-GGGG-3′)₄ and daunomycin, as a potential model for studying drug–telomere interactions. The structure was solved at 1.08 Å by direct methods in space group I4. The asymmetric unit comprises a linear arrangement of one d(GGGG) strand, four daunomycin molecules, a second d(GGGG) strand facing in the opposite direction to the first, and Na and Mg cations. The crystallographic 4-fold axis generates the biological unit, which is a 12-layered structure comprising two sets of four guanine layers, with four layers each of four daunomycins stacked between the 5′ faces of the two quadruplexes. The daunomycin layers fall into two groups which are novel in their mode of self assembly. The only contacts between daunomycin molecules within any one of these layers are van der Waals interactions, however there is substantial π–π stacking between successive daunomycin layers and also with adjacent guanine layers. The structure differs significantly from all other parallel d(TGGGGT)₄ quadruplexes in that the 5′ guanine adopts the unusual syn glycosyl linkage, refuting the widespread belief that such conformations should all be anti. In contrast to the related d(TGGGGT)/daunomycin complex, there are no ligand–quadruplex groove insertion interactions.     

A deoxyribozyme with a novel guanine quartet-helix pseudoknot structure

Here we report a deoxyribozyme with a unique structure that contains a two-tiered guanine quadruplex interlinked to a Watson-Crick duplex. Through in vitro selection, sequence mutation, and methylation interference, we show the presence of both the two-tiered guanine-quadruplex and two helical regions contained in the active structure of this self-phosphorylating deoxyribozyme. Interestingly, one GG element of the quadruplex is part of a hairpin loop within one of the identified helical regions. Circular dichroism analysis showed that antiparallel quadruplex formation was dependent on this helix. To our knowledge, this is the first report of a pseudoknot nucleic acid structure that involves a guanine quadruplex. Our findings indicate that guanine quadruplexes can be part of complex structural arrangements, increasing the likelihood of finding more complex guanine quadruplex arrangements in biological systems.     

Substitution of adenine for guanine in the quadruplex-forming human telomere DNA sequence G(3)(T(2)AG(3))(3)

We have studied the formation and structural properties of quadruplexes of the human telomeric DNA sequence G(3)(T(2)AG(3))(3) and related sequences in which each guanine base was replaced by an adenine base. None of these single base substitutions hindered the formation of antiparallel quadruplexes, as shown by circular dichroism, gel electrophoresis, and UV thermal stability measurements in NaCl solutions. Effect of substitution did differ, however, depending on the position of the substituted base. The A-for-G substitution in the middle quartet of the antiparallel basket scaffold led to the most distorted and least stable structures and these sequences preferred to form bimolecular quadruplexes. Unlike G(3)(T(2)AG(3))(3), no structural transitions were observed for the A-containing analogs of G(3)(T(2)AG(3))(3) when sodium ions were replaced by potassium ions. The basic quadruplex topology remained the same for all sequences studied in both salts. As in vivo misincorporation of A for a G in the telomeric sequence is possible and potassium is a physiological salt, these findings may have biological relevance.     

Interactions of cryptolepine and neocryptolepine with unusual DNA structures

Cryptolepine, the main alkaloid present in the roots of Cryptolepis sanguinolenta, presents a large spectrum of biological properties. It has been reported to behave like a DNA intercalator with a preference for GC-rich sequences. In this study, dialysis competition assay and mass spectrometry experiments were used to determine the affinity of cryptolepine and neocryptolepine for DNA structures among duplexes, triplexes, quadruplexes and single strands. Our data confirm that cryptolepine and neocryptolepine prefer GC over AT-rich duplex sequences, but also recognize triplex and quadruplex structures. These compounds are weak telomerase inhibitors and exhibit a significant preference for triplexes over quadruplexes or duplexes.     

Binding properties of human telomeric quadruplex multimers: a new route for drug design

Human telomeric G-quadruplex structures are known to be promising targets for an anticancer therapy. In the past decade, several research groups have been focused on the design of new ligands trying to optimize the interactions between these small molecules and the G-quadruplex motif. In most of these studies, the target structures were the single quadruplex units formed by short human DNA telomeric sequences (typically 21-26 nt). However, the 3′-terminal single-stranded human telomeric DNA is actually 100-200 bases long and can form higher-order structures by clustering several consecutive quadruplex units (multimers). Despite the increasing number of structural information on longer DNA telomeric sequences, very few data are available on the binding properties of these sequences compared with the shorter DNA telomeric sequences.In this paper we use a combination of spectroscopic (CD, UV and fluorescence) and calorimetric techniques (ITC) to compare the binding properties of the (TTAGGG)₈TT structure formed by two adjacent quadruplex units with the binding properties of the (AG₃TT)₄ single quadruplex structure. The three side-chained triazatruxene derivative azatrux and TMPyP4 cationic porphyrin were used as quadruplex ligands. We found that, depending on the drug, the number of binding sites per quadruplex unit available in the multimer structure was smaller or greater than the one expected on the basis of the results obtained from individual quadruplex binding studies. This work suggests that the quadruplex units along a multimer structure do not behave as completely independent. The presence of adjacent quadruplexes results in a diverse binding ability not predictable from single quadruplex binding studies. The existence of quadruplex-quadruplex interfaces in the full length telomeric overhang may provide an advantageous factor in drug design to enhance both affinity and selectivity for DNA telomeric quadruplexes.