Guanine Tetraplexes Research Articles

Silver(I)-Mediated 2D DNA Nanostructures.

Structural DNA nanotechnology enables the self-organization of matter at the nanometer scale, but approaches to expand the inorganic and electrical functionality of these scaffolds remain limited. Developments in nucleic acid metallics have enabled the incorporation of site-specific metal ions in DNA duplexes and provide a means of functionalizing the double helix with atomistic precision. Here a class of 2D DNA nanostructures that incorporate the cytosine-Ag+-cytosine (dC:Ag+:dC) base pair as a chemical trigger for self-assembly is described. It is demonstrated that Ag+-functionalized DNA can undergo programmable assembly into large arrays and rings, and can be further coassembled with guanine tetraplexes (G4). It is shown that 2D DNA lattices can be assembled with a variety of embedded nanowires at tunable spacing. These results serve as a foundation for further development of self-assembled, metalated DNA nanostructures, with potential for high-precision DNA nanoelectronics with nanometer pitch.

Conformational diversity of single-stranded DNA from bacterial repetitive extragenic palindromes: Implications for the DNA recognition elements of transposases.

Repetitive extragenic palindrome (REP)—associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5′-G and 3′-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 585–596, 2015.

Iso‐guanine quintet complexes coordinated by mono valent cations (Na+, K+, Rb+, and Cs+)

The geometric structures, the interaction energies, the vibrational characteristics, and the electronic structures of the complexes of the isoguanine (isoG) quintet coordinated with mono valent cations (Na(+), K(+), Rb(+), and Cs(+)) have been studied based on the nonplanar models. The geometry of the local minimum structure of the Na(+)-isoG quintet complex deviates significantly from the planar structure. The geometric characteristics of the Na(+)-isoG quintet complex support the experimental findings that Na(+) is unlikely to induce the formation of the isoG quintet-based pentaplexes. Similar to the guanine tetraplexes, the ionic selectivity of the isoG quintet-based pentaplexes is largely dominated by the hydration energy of the cations. After hydration correction, the positive value of the free energy difference for the formation of the Na(+)-isoG quintet complex (DeltaG(f)) suggests that the isoG quintet is unable to capture the hydrated Na(+). The negative values of DeltaG(f) for the K(+) and Rb(+) complexes implies that both ions have the tendency to be inserted into the isoG pentaplexes. This study suggests that, to elucidate the high Cs(+) selectivity of isoG pentaplexes, it is necessary to extend the model from the isoG quintet to the isoG decamer.

Theoretical and gas‐phase studies of specific cationized purine base quartet

Guanine tetraplexes are biological non-covalent systems stabilized by alkali cations. Thus, self-clustering of guanine, xanthine and hypoxanthine with alkali cations (Na(+), K(+) and Li(+)) is investigated by electrospray ionization mass spectrometry (ESI-MS) in order to provide new insights into G-quartets, hydrogen-bonded complexes. ESI assays displayed magic numbers of tetramer adducts with Na(+), Li(+) and K(+), not only for guanine, but also for xanthine bases. The optimized structures of guanine and xanthine quartets have been determined by B3LYP hybrid density functional theory calculations. Complexes of metal ions with quartets are classified into different structure types. The optimized structures obtained for each quartet explain the gas-phase results. The gas-phase binding sequence between the monovalent cations and the xanthine quartet follows the order Li(+) > Na(+) > K(+), which is consistent with that obtained for the guanine quartet in the literature. The smallest stabilization energy of K(+) and its position versus the other alkali metal ions in guanine and xanthine quartets is consistent with the fact that the potassium cation can be located between two guanine or xanthine quartets, for providing a [gua(or (xan))(8)+K](+) octamer adduct. Even if an abundant octamer adduct with K(+) for xanthine was detected by ESI-MS, it was not the case for guanine.

Linker chain effect of ferrocenylnaphthalene diimide derivatives on a tetraplex DNA binding

Spectrophotometric binding studies of a series of the naphthalene diimide derivatives, 1-7, carrying different chains with a human telomere oligonucleotide, d(TTAGG)(4) was carried out in 0.1 M AcOK-AcOH buffer (pH 5.6) and 0.1 M KCl. Under this condition, this DNA could exist as the mixture of two-type tetraplex structures and these derivatives could bind to this DNA with strong affinity of 10(6) M(-1). The effect of the linker chain is not so large in those binding affinity, but the ligand 5 having piperazine skeleton in the linker chain had relative higher affinity for this tetraplex DNA than other derivatives. Large hypochromic effect of these derivatives upon binding to the tetraplex DNA suggested that the binding mode of these derivatives might contribute the stacking interaction between the naphthalene diimide and guanine tetraplex planes.

The thrombin binding aptamer GGTTGGTGTGGTTGG forms a bimolecular guanine tetraplex

In the literature, the thrombin binding aptamer GGTTGGTGTGGTTGG is generally taken as a prototype of an intramolecular guanine tetraplex of DNA. Our results, however, show that this notion is not true in aqueous solutions. This conclusion is based on a dependence of the CD spectra on aptamer concentration, migration of the aptamer in polyacrylamide gels, and the Ferguson analysis of the gel migration data. The presented data document that the aptamer forms a bimolecular tetraplex. We furthermore show that only an extension of the aptamer by a sequence containing further guanines, or an elongation of loop regions, causes that its tetraplex folding is intramolecular.

Ethanol is a better inducer of DNA guanine tetraplexes than potassium cations

Guanine tetraplexes are a biologically relevant alternative of the Watson and Crick duplex of DNA. It is thought that potassium or other cations present in the cavity between consecutive guanine tetrads are an integral part of the tetraplexes. Here we show using CD spectroscopy that ethanol induces the guanine tetraplexes like or even better than potassium cations. We present examples of ethanol stabilizing guanine tetraplexes even in cases when potassium cations fail to do so. Hence, besides the A-form or Z-form, ethanol stabilizes another conformation of DNA, i.e., the guanine tetraplexes. We discuss the mechanism of the stabilization. Use of ethanol will permit studies of guanine tetraplexes that cannot be induced by potassium cations or other tetraplex-promoting agents. This work demonstrates that a still broader spectrum of nucleotide sequences can fold into guanine tetraplexes than has previously been thought. Aqueous ethanol may better simulate conditions existing in vivo than the aqueous solutions.

Guanine tetraplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats

Secondary structures of the G-rich strand of human telomere DNA fragments G3(TTAG3)n, n = 1–16, have been studied by means of circular dichroism spectroscopy and PAGE, in solutions of physiological potassium cation concentrations. It has been found that folding of these fragments into tetraplexes as well as tetraplex thermostabilities and enthalpy values depend on the number of TTAG3 repeats. The suggested topologies include, e.g. antiparallel and parallel bimolecular tetraplexes, an intramolecular antiparallel tetraplex, a tetraplex consisting of three parallel chains and one antiparallel chain, a poorly stable parallel intramolecular tetraplex, and both parallel and antiparallel tetramolecular tetraplexes. G3(TTAG3)3 folds into a single, stable and very compact intramolecular antiparallel tetraplex. With an increasing repeat number, the fragment tetraplexes surprisingly are ever less thermostable and their migration and enthalpy decrease indicate increasing irregularities or domain splitting in their arrangements. Reduced stability and different topology of lengthy telomeric tails could contribute to the stepwise telomere shortening process.

Conformational properties of DNA containing (CCA) n and (TGG) n trinucleotide repeats

We have used CD spectroscopy, polyacrylamide gel electrophoresis, and UV absorption spectroscopy to study conformational properties of DNA fragments containing (CCA) n and (TGG) n repeats, which are the most length-polymorphic microsatellite sequences of the human genome. The (CCA) n fragments are random single strands at neutral and alkaline pH but they fold into intramolecular intercalated cytosine tetraplexes at mildly acid pH values. More acid values stabilize intermolecular tetraplex formation. The behavior of (TGG) n repeats is more complex. They form hairpins or antiparallel homoduplexes in low salt solutions which, however, are transformed into parallel-stranded guanine tetraplexes at physiological KCl concentrations. Their molecularity depends on the repeat number: (TGG) 4 associates into an octameric complex, (TGG) 8 forms tetramolecular complexes. (TGG) n with odd repeat numbers (5, 7, and 9) generate bimolecular and tetramolecular tetraplexes. The only (TGG) 7 folds into an intramolecular tetraplex at low KCl concentrations, which is antiparallel-stranded. Moreover, the (TGG) n fragments provide various mutually slipped conformers whose population increases with salt concentration and with the increasing repeat number. However, the self-structures of both strands disappear in the presence of the complementary strand because both (TGG) n and (CCA) n prefer to associate into the classical heteroduplex. We suppose that the extreme conformational variability of the DNA strands stands behind the length polymorphism which the (CCA) n /(TGG) n repeats exhibit in the human genome.

PROSIT, AN ONLINE SERVICE TO CALCULATE PSEUDOROTATIONAL PARAMETERS OF NUCLEOSIDES AND NUCLEOTIDES

The online service PROSIT (Pseudo-Rotational Online Service and Interactive Tool) is a free service available at http://cactus.nci.nih.gov/prosit/ that performs pseudorotational analysis of nucleosides(tides). PROSIT reads the 3D coordinates of nucleosides and returns the pseudorotational phase angle P, puckering amplitude νmax, and other related information. As examples, the sugar conformations in a parallel-stranded guanine tetraplex and a four-way Holliday junction are presented here.

The guanine-rich fragile X chromosome repeats are reluctant to form tetraplexes.

Using circular dichroism spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we studied conformational properties of guanine-rich DNA strands of the fragile X chromosome repeats d(GGC)n, d(GCG)n and d(CGG)n, with n = 2, 4, 8 and 16. These strands are generally considered in the literature to form guanine tetraplexes responsible for the repeat expansion. However, we show in this paper that the repeats are reluctant to form tetraplexes. At physiological concentrations of either Na+ or K+ ions, the hexamers and dodecamers associate to form homoduplexes and the longer repeats generate homoduplexes and hairpins. The tetraplexes are rarely observed being relatively most stable with d(GGC)n and least stable with d(GCG)n. The tetraplexes are exclusively formed in the presence of K+ ions, at salt concentrations higher than physiological, more easily at higher than physiological temperatures, and they arise with extremely long kinetics (even days). Moreover, the capability to form tetraplexes sharply diminishes with the oligonucleotide length. These facts make the concept of the tetraplex appearance in this motif in vivo very improbable. Rather, a hairpin of the fragile X repeats, whose stability increases with the repeat length, is the probable structure responsible for the repeat expansion in genomes.

Crystal Structure of an RNA Purine-Rich Tetraplex Containing Adenine Tetrads: Implications for Specific Binding in RNA Tetraplexes

Purine-rich regions in DNA and RNA may contain both guanines and adenines, which have various biological functions. Here we report the crystal structure of an RNA purine-rich fragment containing both guanine and adenine at 1.4 Å resolution. Adenines form an adenine tetrad in the N6-H…N7 conformation. Substitution of an adenine tetrad in the guanine tetraplex does not change the global conformation but introduces irregularity in both the hydrogen bonding interaction pattern in the groove and the metal ion binding pattern in the central cavity of the tetraplex. The irregularity in groove binding may be critical for specific binding in tetraplexes. The formation of G-U octads provides a mechanism for interaction in the groove. Ba 2+ ions prefer to bind guanine tetrads, and adenine tetrads can only be bound by Na + ions, illustrating the binding selectivity of metal ions for the tetraplex.

Circular dichroism spectroscopy of conformers of (guanine + adenine) repeat strands of DNA.

(Guanine+adenine) strands of DNA are known to associate into guanine tetraplexes, homodimerize into parallel or antiparallel duplexes, and fold into a cooperatively melting single strand resembling the protein alpha helix. Using CD spectroscopy and other methods, we studied how this conformational polymorphism depended on the primary structure of DNA. The study showed that d(GGGA)(5) and d(GGA)(7) associated into homoduplexes at low salt or in the presence of LiCl but were prone to guanine tetraplex formation, especially in the presence of KCl. In addition, they yielded essentially the same CD spectrum in the presence of ethanol as observed with the ordered single strand of d(GA)(10). Strands of d(GA)(10), d(GGAA)(5), d(GAA)(7), and d(GAAA)(5) associated into homoduplexes in both LiCl and KCl solutions, but not into guanine tetraplexes. d(GAAA)(5) and d(GAA)(7) further failed to form the single-stranded conformer in aqueous ethanol. Adenine protonation, however, stabilized the single-stranded conformer even in these adenine-rich fragments. The ordered single strands, homoduplexes as well as the guanine tetraplexes, all provided strikingly similar CD spectra, indicating that all of the conformers shared similar base stacking geometries. The increasing adenine content only decreased the conformer thermostability.

Origin of Na+/K+ Selectivity of the Guanine Tetraplexes in Water: The Theoretical Rationale

Theoretical study of the “sandwich” models of the metal ion (Na+ and K+) G-tetrad complexes has been performed at the HF/6-311G(d,p)//HF6-31G(d,p) level. The distances between cations and the O6 atoms of guanines have been predicted to be 2.826 A for sodium and 2.951 A for potassium in the G4−M−G4(inter) model and 2.791 A for sodium and 2.927 A for potassium in the G4-M−G4(intra) model. These results are consistent with the Na+−O6 distance of 2.75 ± 0.02 A in the corresponding crystal structures. The binding energy between the cations and the G tetrad in the tetraplexes is greater for Na+ than for K+. This suggests the domination of the electrostatic interaction in the cation-tetraplexes systems. After the hydration energy correction, the stability sequence of monovalence cations in the guanine tetraplexes is found to be K+ > Na+. This finding gives the first direct theoretical evidence to support the hypothesis of Hud et al. that the preferred coordination of K+ over Na+ in G tetraplexes is actually domi...

X-ray analysis of an RNA tetraplex (UGGGGU) 4 with divalent Sr 2+ ions at subatomic resolution (0.61 Å)

Four-stranded guanine tetraplexes in RNA have been identified to be involved in crucial biological functions, such as dimerization of retroviral RNA, translational repression, and mRNA turnover. However, the structural basis for these biological processes is still largely unknown. Here we report the RNA tetraplex structure (UGGGGU)(4) at ultra-high resolution (0.61 A). The space group is P42(1)2, and cell constants are a = b = 36.16 A and c = 74.09 A. The structure was solved by the multiple-wavelength anomalous dispersion method using a set of three-wavelength data of the isomorphous bromo derivative (br)UGGGGU and refined to 0.61-A resolution. Each of the four strands in the asymmetric unit forms a parallel tetraplex with symmetry-related molecules. The tetraplex molecules stack on one another in opposite polarity (head-to-head or tail-to-tail) to form a pseudocontinuous column. All of the 5'-end uridines rotate around the backbone of G2, swing out, and form unique octaplexes with the neighboring G tetraplexes, whereas the 3'-end uridines are stacked-in and form uridine tetrads. All of the bases are anti, and the riboses are in the mixed C2'- and C3'-puckering mode. Strontium ions are observed in every other guanine tetrad plane, sitting on the fourfold axis and associated to the eight O6 atoms of neighboring guanine bases in a bipyramidal-antiprism geometry. The hydrogens are clearly observed in the structure.

A Remarkable Alteration in the Bonding Pattern: An HF and DFT Study of the Interactions between the Metal Cations and the Hoogsteen Hydrogen-Bonded G-Tetrad

The HF and DFT levels of theory were applied to study the interaction between monovalent cations and guanine tetrads. The calculations reveal that cation−guanine-tetrad complexes adopt the normal four-stranded Hoogsteen-bonded G-tetrad structure, and no bifurcated hydrogen bonds which stabilize the noninteracting G-tetrads were found. The gas-phase binding sequence between the monovalent cations and the G-tetrad complexes follows the order Li+ > Na+ > K+. After the hydration correction, the stability sequence of the monovalent cation−guanine-tetrad complexes in aqueous solutions follows the trend K+ > Na+ > Li +. The preferential binding of potassium over sodium and sodium over lithium in water solutions reproduces the experimental ion selectivity of the guanine tetraplex. In addition, the weak stabilization energy of the K+−G-tetrad in the coplanar form is consistent with the fact that the potassium cation tends to locate between two successive tetrads. The results of this study justify the conclusion of...

Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd

The single-stranded DNA sequence d(GT5G4CT4C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU5G4CU4C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 °C in the presence of Na+ ions but the structure is stabilized up to 75 °C in the presence of K+ ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 °C), and its stability is further enhanced in the presence of K+ ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets.

Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd

The single-stranded DNA sequence d(GT5G4CT4C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU5G4CU4C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 degrees C in the presence of Na+ ions but the structure is stabilized up to 75 degrees C in the presence of K+ ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 degrees C), and its stability is further enhanced in the presence of K+ ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets.

The crystal structure of a parallel-stranded guanine tetraplex at 0.95Å resolution

In both DNA and RNA, stretches of guanine bases can form stable four-stranded helices in the presence of sodium or potassium ions. Sequences with a propensity to form guanine tetraplexes have been found in chromosomal telomers, immunoglobulin switch regions, and recombination sites. We report the crystal structure at 0.95 Å resolution of a parallel-stranded tetraplex formed by the hexanucleotide d(TG 4T) in the presence of sodium ions. The four strands form a right-handed helix that is stabilized by hydrogen-bonding tetrads of co-planar guanine bases. Well-resolved sodium ions are found between and, at defined points, within tetrad planes and are coordinated with the guanine O6 groups. Nine calcium ions have been identified, each with a well-defined hepta-coordinate hydration shell. Hydrogen-bonding water patterns are observed within the tetraplex’s helical grooves and clustered about the phosphate groups. Water molecules in the groove may form a hydrogen bond with the O4′, and may affect the stacking behavior of guanine. Two distinct stacking arrangements are noted for the guanine tetrads. The thymine bases do not contribute to the four-stranded conformation, but instead stack to stabilize the crystal lattice. We present evidence that the sugar conformation is strained and propose that this originates from forces that optimize guanine base stacking. Discrete conformational disorder is observed at several places in the phosphodiester backbone, which results from a simple crankshaft rotation that requires no net change in the sugar conformation.

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.