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.     

Bulges in left-handed G-quadruplexes

G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures.     

Internal sodium ions and water molecules in guanine quadruplexes: magnetic relaxation dispersion studies of [d(G3T4G3)]2 and [d(G4T4G4)]2

The structural stability of guanine quadruplexes depends critically on an unusual configuration of dehydrated Na (+) or K (+) ions, closely spaced along the central axis of the quadruplex. Crystallography and NMR spectroscopy indicate that these internal ions can be located between the G-quartet planes as well as in the thymine loops, but the precise ion coordination has been firmly established in only a few cases. Here, we examine the bimolecular diagonal-looped foldback quadruplexes [d(G 3T 4G 3)] 2 (Q3) and [d(G 4T 4G 4)] 2 (Q4) by (2)H, (17)O, and (23)Na magnetic relaxation dispersion (MRD). The MRD data indicate that both quadruplexes contain Na (+) ions between the T 4 loops and the terminal G-quartets and that these ions have one water ligand. These ions exchange with external ions on a time scale of 10-60 mus at 27 degrees C, while their highly ordered water ligands have residence times in the range 10 (-8)-10 (-6) s. The MRD data indicate that Q4 contains three Na (+) ions in the stem sites, in agreement with previous solid-state (23)Na NMR findings but contrary to the only crystal structure of this quadruplex. For Q3, the MRD data suggest a less symmetric coordination of the two stem ions. In both quadruplexes, the stem ions have residence times of 0.6-1.0 ms at 27 degrees C. The equilibrium constant for Na (+) --> K (+) exchange is approximately 4 for both loop and stem sites in Q3, in agreement with previous (1)H NMR findings.     

Specific DNA G‐quadruplexes bind to ethanolamines

A significant number of G‐quadruplex‐forming sequences have been revealed in human genome by bioinformatic searches, implying that G‐quadruplexes may be involved in important biological processes and may be new chemotherapeutic targets. Therefore, it is important to discover the potential interactions of G‐quadruplexes with other molecules or groups. Here we describe a class of G‐quadruplexes, which can bind to ethanolamine groups that widely exist in biomolecules and drug molecules. The specific interaction of these G‐quadruplexes with ethanolamine groups was identified by high performance affinity chromatography (HPAC) using immobilized ethanolamine and diethanolamine as stationary phase reagents. The circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE) show that these ethanolamine binding quadruplexes adopt an intramolecularly parallel structure. The relationship of ethanolamine binding and G‐quadruplexe structure provides new clues for the G‐quadruplex‐related studies as well as for the molecular designs of therapeutic reagents. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 874–883, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Synthesis of quadruplex‐forming tetra‐end‐linked oligonucleotides: Effects of the linker size on quadruplex topology and stability

G‐quadruplexes are characteristic structural arrangements of guanine‐rich DNA sequences that abound in regions with relevant biological significance. These structures are highly polymorphic differing in the number and polarity of the strands, loop composition, and conformation. Furthermore, the cation species present in solution strongly influence the topology of the G‐quadruplexes. Recently, we reported the synthesis and structural studies of new G‐quadruplex forming oligodeoxynucleotides (ODNs) in which the 3′‐ and/or the 5′‐ends of four ODN strands are linked together by a non‐nucleotidic tetra‐end‐linker (TEL). These TEL‐ODN analogs having the sequence TGGGGT are able to form parallel G‐quadruplexes characterized by a remarkable high thermal stability. We report here an investigation about the influence of the reduction of the TEL size on the molecularity, topology, and stability of the resulting TEL‐G‐quadruplexes using a combination of circular dichroism (CD), CD melting, ¹H NMR spectroscopy, gel electrophoresis, and molecular modeling data. We found that all TEL‐(TGGGGT)₄ analogs, regardless the TEL size and the structural orientation of the ODN branches, formed parallel TEL‐G‐quadruplexes. The molecular modeling studies appear to be consistent with the experimental CD and NMR data revealing that the G‐quadruplexes formed by TEL‐ODNs having the longer TEL (L 1 ‐ 4 ) are more stable than the corresponding G‐quadruplexes having the shorter TEL (S 1 ‐ 4 ). The relative stability of S 1 ‐ 4 was also reported. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 466–477, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

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.     

G-quadruplex motifs arranged in tandem occurring in telomeric repeats and the insulin-linked polymorphic region

To date, various G-quadruplex structures have been reported in the human genome. There are numerous studies focusing on quadruplex-forming sequences in general, but few studies have focused on two or more quadruplexes in the same molecule, which are most commonly found in telomeric DNA and other tandem repeats, e.g., insulin-linked polymorphic region (ILPR). Although the human telomere consists of a number of repeats, higher-order G-quadruplex structures are discussed less often because of the complexity of the structures. In this study, sequences consisting of 4-12 repeats of d(G(4)TGT), d(G(3)T(2)A), and/or d(G(4)T(2)A) have been studied by circular dichroism, ultraviolet spectroscopy, and temperature-gradient gel electrophoresis. These sequences serve as a model for the arrangement of quadruplexes in the telomere and ILPR in solution. Our major findings are as follows. (i) The number of G-rich repeats has a great influence on G-quadruplex stability. (ii) The evidence of quadruplex-quadruplex interaction is confirmed. (iii) For the first time, we directly observed the melting behavior of different conformers in a single experiment. Our results agree with other calorimetric and spectroscopic data and data obtained by single-molecule studies, atomic force microscopy, and mechanical unfolding by optical tweezers. We propose that the end of telomeres can be formed by only a few tandem quadruplexes (fewer than three). Our findings improve our understanding of the mechanism of G-quadruplex formation in long repeats in G-rich-regulating parts of genes and telomere ends.     

Guanine quadruplex formation by RNA/DNA hybrid analogs of Oxytricha telomere G(4)T(4)G(4) fragment

Using circular dichroism spectroscopy, gel electrophoresis, and ultraviolet absorption spectroscopy, we have studied quadruplex folding of RNA/DNA analogs of the Oxytricha telomere fragment, G(4)T(4)G(4), which forms the well-known basket-type, antiparallel quadruplex. We have substituted riboguanines (g) for deoxyriboguanines (G) in the positions G1, G9, G4, and G12; these positions form the terminal tetrads of the G(4)T(4)G(4) quadruplex and adopt syn, syn, anti, and anti glycosidic geometries, respectively. We show that substitution of a single sugar was able to change the quadruplex topology. With the exception of G(4)T(4)G(3)g, which adopted an antiparallel structure, all the RNA/DNA hybrid analogs formed parallel, bimolecular quadruplexes in concentrated solution at low salt. In dilute solutions ( approximately 0.1 mM nucleoside), the RNA/DNA hybrids substituted at positions 4 or 12 adopted antiparallel quadruplexes, which were especially stable in Na(+) solutions. The hybrids substituted at positions 1 and 9 preferably formed parallel quadruplexes, which were more stable than the nonmodified G(4)T(4)G(4) quadruplex in K(+) solutions. Substitutions near the 3'end of the molecule affected folding more than substitutions near the 5'end. The ability to control quadruplex folding will allow further studies of biophysical and biological properties of the various folding topologies. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 797-806, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Intramolecular higher order packing of parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad of GGA triplet repeat DNA

GGA triplet repeats are widely dispersed throughout eukaryotic genomes and are frequently located within biologically important regions such as gene regulatory regions and recombination hot spot sites. We determined the structure of d(GGA)4 (12-mer) under physiological conditions and founded the formation of an intramolecular parallel quadruplex for the first time. Later, a similar architecture to that of the intramolecular parallel quadruplex was found for a telomere DNA in the crystalline state. Here, we have determined the structure of d(GGA)8 (24-mer) under physiological conditions. Two intramolecular parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad are formed in d(GGA)8. These quadruplexes are packed in a tail-to-tail manner. This is the first demonstration of the intramolecular higher order packing of quadruplexes at atomic resolution. K+ ions, but not Na+ ones, are critically required for the formation of this unique structure. The elucidated structure suggests the mechanisms underlying the biological events related to the GGA triplet repeat. Furthermore, in the light of the structure, the mode of the higher order packing of the telomere DNA is discussed.     

End-stacking of copper cationic porphyrins on parallel-stranded guanine quadruplexes

Nucleic acids that contain multiple sequential guanines assemble into guanine quadruplexes (G-quadruplexes). Drugs that induce or stabilize G-quadruplexes are of interest because of their potential use as therapeutics. Previously, we reported on the interaction of the Cu(2+) derivative of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine (CuTMpyP4), with the parallel-stranded G-quadruplexes formed by d(T(4)G( n )T(4)) (n = 4 or 8) (Keating and Szalai in Biochemistry 43:15891-15900, 2004). Here we present further characterization of this system using a series of guanine-rich oligonucleotides: d(T(4)G( n )T(4)) (n = 5-10). Absorption titrations of CuTMpyP4 with all d(T(4)G( n )G(4)) quadruplexes produce approximately the same bathochromicity (8.3 +/- 2 nm) and hypochromicity (46.2-48.6%) of the porphyrin Soret band. Induced emission spectra of CuTMpyP4 with d(T(4)G( n )T(4))(4) quadruplexes indicate that the porphyrin is protected from solvent. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry revealed a maximum porphyrin to quadruplex stoichiometry of 2:1 for the shortest (n = 4) and longest (n = 10) quadruplexes. Electron paramagnetic resonance spectroscopy shows that bound CuTMpyP4 occupies magnetically noninteracting sites on the quadruplexes. Consistent with our previous model for d(T(4)G(4)T(4)), we propose that two CuTMpyP4 molecules are externally stacked at each end of the run of guanines in all d(T(4)G( n )T(4)) (n = 4-10) quadruplexes.     

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.     

A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes

Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5–18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (ΔG₃₇) ranging from −1.7 to −15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex–duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.     

Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding

Human telomeres are composed of duplex TTAGGG repeats and a 3' single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.     

Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target and in virus biology

We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.     

Biological aspects of DNA/RNA quadruplexes.

Among the many unusual conformations of DNA and RNA, quadruplex structures, based on the guanine quartet, possess several unique properties. These properties, along with the general features of guanine quadruplexes, are described in the context of possible roles for these structures in biological systems. A variety of experimental observations supporting the notion that quadruplexes are important in vivo is presented, including proteins known to specifically bind to quadruplex structures, guanine-rich DNA, and RNA sequences endowed with the potential for forming quartet-based structures in telomeres and regulatory regions, such as gene promoters, quadruplexes as DNA aptamer folding motifs arising from in vitro selection experiments, and potential chemotherapeutic, quadruplex-forming oligonucleotides. Taken together, all of these observations argue cogently not only for the presence of quadruplexes in biological systems but also for their significance in terms of their roles in various biological processes.     

ILPR repeats adopt diverse G‐quadruplex conformations that determine insulin binding

The insulin‐linked polymorphic region (ILPR) is a VNTR region located upstream of the insulin (INS) gene consisting of the repeat 5′‐ACAGGGGTGTGGGG (repeat a) and several less abundant sequence repeats (b–n). Here, we have investigated the structural polymorphism of G‐quadruplexes formed from the most common repeat sequences (a–c) and their effect on insulin protein binding. We first established that the ILPR repeats “b” and “c” can form quadruplex structures. Insulin has previously been shown to bind a G‐quadruplex formed by a dimer of the repeat “a”. Our findings show that insulin binds preferentially to the repeat “a” G‐quadruplex (K_d = 0.17 ± 0.03 μM) over G‐quadruplexes formed from other ILPR repeats that were tested (K_ds from 0.71 ± 0.15 to 1.07 ± 0.09 μM). Additionally, the Watson‐Crick complementary relationship between the loop regions of repeat “a” (ACA and TGT) seemingly play an important role in favoring a specific G‐quadruplex conformation, which based on our data is critical for insulin binding. Affinity for insulin is reduced in sequences lacking the putative WC complementarity, however upon engineered restoration of complementarity, insulin binding is recovered. A DMS footprinting assay on the repeat “a” G‐quadruplex in the presence of insulin, combined with binding affinities for ILPR mutants led to identification of a loop nucleotide critical for binding. Uniquely, insulin shows clear preference for binding to the G‐quadruplexes with the more antiparallel feature. Collectively, our results illustrate the specific nature of insulin binding to the ILPR G‐quadruplexes and begin to provide molecular details on such interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 21–31, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Folding thermodynamics of the hybrid‐1 type intramolecular human telomeric G‐quadruplex

Guanine‐rich DNA sequences that may form G‐quadruplexes are located in strategic DNA loci with the ability to regulate biological events. G‐quadruplexes have been under intensive scrutiny owing to their potential to serve as novel drug targets in emerging anticancer strategies. Thermodynamic characterization of G‐quadruplexes is an important and necessary step in developing predictive algorithms for evaluating the conformational preferences of G‐rich sequences in the presence or the absence of their complementary C‐rich strands. We use a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the 26‐meric human telomeric sequence d[A₃G₃(T₂AG₃)₃A₂]. In the presence of K⁺ ions, the latter adopts the hybrid‐1 G‐quadruplex conformation, a tightly packed structure with an unusually small number of solvent‐exposed atomic groups. The K⁺‐induced folding of the G‐quadruplex at room temperature is a slow process that involves significant accumulation of an intermediate at the early stages of the transition. The G‐quadruplex state of the oligomeric sequence is characterized by a larger volume and compressibility and a smaller expansibility than the coil state. These results are in qualitative agreement with each other all suggesting significant dehydration to accompany the G‐quadruplex formation. Based on our volume data, 432 ± 19 water molecules become released to the bulk upon the G‐quadruplex formation. This large number is consistent with a picture in which DNA dehydration is not limited to water molecules in direct contact with the regions that become buried but involves a general decrease in solute–solvent interactions all over the surface of the folded structure. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 216–227, 2014.     

Fluorescence-based duplex-quadruplex competition test to screen for telomerase RNA quadruplex ligands

RNA and DNA guanine-rich sequences can adopt unusual structures called Guanine quadruplexes (G4). A quadruplex-prone RNA sequence is present at the 5'-end of the 451-nt-long RNA component of telomerase, hTERC. As this quadruplex may interfere with P1 helix formation, a key structural element for this RNA, we are seeking molecules that would alter this RNA duplex-quadruplex equilibrium. In this work, we present a fluorescence-based test designed to identify G4 ligands specific for the hTERC G-rich motif and that can prevent P1 helix formation. From an initial panel of 169 different molecules, 11 were found to be excellent P1 duplex inhibitors. Interestingly, some of the compounds not only exhibit a strong selectivity for quadruplexes over duplexes, but also demonstrated a preference for G4-RNA over all other quadruplexes. This test may easily be adapted to almost any quadruplex-forming sequence and converted into HTS format.