Branched Nanowire Based Guanine Rich Oligonucleotides

Abstract

Self-assembly and aggregation of guanine rich sequences can provide useful insights into DNA nanotechnology and telomeric structure and function. In this paper, we designed a guanine rich sequence d(GGCGTTTTGCGG). We found that it can form stable structure in appropriate condition and it exhibits an anomalous CD spectra. This structures can be imaged in ambient environment with a Nanoscope III AFM (Digital Instruments). We found it forms branch structure and long multistrand DNA nanowire after incubation at 37°C for 612 hours in 25 mM TE (pH=8.0) + 5 mM Mg²⁺ + 50 mM K⁺. The ability to self-assemble into branches and long wires not only clearly demonstrate its potential as scaffold structures for nanotechnology, but also give aids to understand telomeric structure further. We have proposed a model to explain how these structures formed.     

Chlamydomonas reinhardtii telomere repeats form unstable structures involving guanine-guanine base pairs.

Unusual DNA structures involving four guanines in a planar formation (guanine tetrads) are formed by guanine-rich (G-rich) telomere DNA and other G-rich sequences (reviewed in (1)) and may be important in the structure and function of telomeres. These structures result from intrastrand and/or interstrand Hoogsteen base pairs between the guanines. We used the telomeric repeat of Chlamydomonas reinhardtii, TTTTAGGG, which contains 3 guanines and has a long interguanine A + T tract, to determine whether these sequences can form intrastrand and interstrand guanine tetrads. We have found that ss (TTTTAGGG)4 can form intrastrand guanine tetrads that are less stable than those formed by more G-rich telomere sequences. They are not only more stable, but also more compact, they are more stable in the presence of K+ than they are in the presence of Na+. While ds oligonucleotides with ss 3' overhangs of (TTTTAGGG)2 can be observed to associate as dimers, formation of this interstrand guanine tetrad structure occurs to a very limited extent and requires very high G-strand concentration, high ionic strength, and at least 49 hours of incubation. Our results suggest that, if telomere dimerization occurs in vivo, it would require factors in addition to the TTTTAGGG telomere sequence.     

Monovalent cation induced structural transitions in telomeric DNAs: G-DNA folding intermediates

Telomeric DNA consists of G- and C-rich strands that are always polarized such that the G-rich strand extends past the 3' end of the duplex to form a 12-16-base overhang. These overhanging strands can self-associate in vitro to form intramolecular structures that have several unusual physical properties and at least one common feature, the presence of non-Watson-Crick G.G base pairs. The term "G-DNA" was coined for this class of structures (Cech, 1988). On the basis of gel electrophoresis, imino proton NMR, and circular dichroism (CD) results, we find that changing the counterions from sodium to potassium (in 20 mM phosphate buffers) specifically induces conformational transitions in the G-rich telomeric DNA from Tetrahymena, d(T2G4)4 (TET4), which results in a change from the intramolecular species to an apparent multistranded structure, accompanied by an increase in the melting temperature of the base pairs of greater than 25 degrees, as monitored by loss of the imino proton NMR signals. NMR semiselective spin-lattice relaxation rate measurements and HPLC size-exclusion chromatography studies show that in 20 mM potassium phosphate (pH 7) buffer (KP) TET4 is approximately twice the length of the form obtained in 20 mM sodium phosphate (pH 7) buffer (NaP) and that mixtures of Na+ and K+ produce mixtures of the two forms whose populations depend on the ratio of the cations. Since K+ and NH4+ are known to stabilize a parallel-stranded quadruplex structure of poly[r(I)4], we infer that the multistranded structure is a quadruplex. Our results indicate that specific differences in ionic interactions can result in a switch in telomeric DNAs between intramolecular hairpin-like or quadruplex-containing species and intermolecular quadruplex structures, all of which involve G.G base pairing interactions. We propose a model in which duplex or hairpin forms of G-DNA are folding intermediates in the formation of either 1-, 2-, or 4-stranded quadruplex structures. In this model monovalent cations stabilize the duplex and quadruplex forms via two distinct mechanisms, counterion condensation and octahedral coordination to the carbonyl groups in stacked planar guanine "quartet" base assemblies. Substituting one of the guanosine residues in each of the repeats of the Tetrahymena sequence to give the human telomeric DNA, d(T2AG3)4, results in less effective K(+)-dependent stabilization. Thus, the ion-dependent stabilization is attenuated by altering the sequence. Upon addition of the Watson-Crick (WC) complementary strand, only the Na(+)-stabilized structure dissociates quickly to form a WC double helix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs

Structural properties of DNA oligonucleotides corresponding to the single-stranded molecular terminus of telomeres from several organisms were analyzed. Based on physical studies including nondenaturing polyacrylamide gel electrophoresis, absorbance thermal denaturation analysis, and 1H and 31P nuclear magnetic resonance spectroscopy, we conclude that these molecules can self-associate by forming non-Watson-Crick, guanine.guanine based-paired, intramolecular structures. These structures form below 40 degrees C at moderate ionic strength and neutral pH and behave like hairpin duplexes in nondenaturing polyacrylamide gels. Detailed analysis of the hairpin structure formed by the telomeric sequence from Tetrahymena, (T2G4)4, shows that it is a unique structure stabilized by hydrogen bonds and contains G residues in the syn conformation. We propose that this novel form of DNA is important for telomere function and sets a precedent for the biological relevance of non-Watson-Crick base-paired DNA structures.     

Frustrated folding of guanine quadruplexes in telomeric DNA

Human chromosomes terminate in long, single-stranded, DNA overhangs of the repetitive sequence (TTAGGG)n. Sets of four adjacent TTAGGG repeats can fold into guanine quadruplexes (GQ), four-stranded structures that are implicated in telomere maintenance and cell immortalization and are targets in cancer therapy. Isolated GQs have been studied in detail, however much less is known about folding in long repeat sequences. Such chains adopt an enormous number of configurations containing various arrangements of GQs and unfolded gaps, leading to a highly frustrated energy landscape. To better understand this phenomenon, we used mutagenesis, thermal melting, and global analysis to determine stability, kinetic, and cooperativity parameters for GQ folding within chains containing 8–12 TTAGGG repeats. We then used these parameters to simulate the folding of 32-repeat chains, more representative of intact telomeres. We found that a combination of folding frustration and negative cooperativity between adjacent GQs increases TTAGGG unfolding by up to 40-fold, providing an abundance of unfolded gaps that are potential binding sites for telomeric proteins. This effect was most pronounced at the chain termini, which could promote telomere extension by telomerase. We conclude that folding frustration is an important and largely overlooked factor controlling the structure of telomeric DNA.     

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

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

Light-induced oxidation of the telomeric G4 DNA in complex with Zn(II) tetracarboxymethyl porphyrin

Structure-specific ligands are convenient tools for the recognition, targeting or probing of non-canonical DNA structures. Porphyrin derivatives exhibit a preference for interaction with G-quadruplex (G4) structures over canonical duplex DNA and are able to cause photoinducible damage to nucleic acids. Here, we show that Zn(II) 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (ZnP1) interacts with different conformations of the telomeric sequence d(TAGGG(TTAGGG)₃) at submicromolar concentrations without any detectible disturbance of the particular fold. Among different folds, potassium (3+1) hybrid G4-structure. reveal the highest affinity to ZnP1. The pattern of guanine oxidation is specific for each telomeric DNA conformation and may serve as an additional tool for probing the G4 topology. The potassium (3+1) and parallel G4 conformations are more susceptible to light-induced oxidation than the sodium G4 conformation or double helix of the telomeric DNA. The major products of the guanine modifications are spiroiminodihydantoin (Sp) and 8-oxoguanine (8-oxoG). ZnP1-induced oxidation of guanines results in the structural rearrangement of parallel and (3+1) G4 conformations yielding an antiparallel-like G4 conformation. The mechanism of the observed light-induced conformational changes is discussed.     

Guanine-rich telomeric sequences stimulate DNA polymerase activity in vitro

Guanine-rich oligonucleotides and short telomeric DNA sequences can self-associate into G-quartet stabilized complexes. We discovered that this self-association can occur in sequencing reactions and that higher-order structures stimulate DNA polymerase to synthesize extended DNA strands. Base analogues were used to identify Hoogsteen base pairings as stabilizing forces in these stimulatory DNA structures. Scanning force microscopy confirmed that quartet-DNA was formed from these oligomers and that these extended, four-stranded structures could be bound by DNA polymerase. Since guanine quartet-stabilized structures are proposed to exist in vivo, such structures may stimulate DNA polymerization in vivo.     

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.     

Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G₃(TTAG₃)₃ forms an antiparallel quadruplex of the same basket type in solution containing either K⁺ or Na⁺ ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K⁺-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G₃(TTAG₃)₃ motif. Both G₃(TTAG₃)₃ and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K⁺-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K⁺-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG₃(TTAG₃)₃ by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K⁺ ions.     

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.     

Differential association of linker histones H1 and H5 with telomeric nucleosomes in chicken erythrocytes.

Rat liver telomeric DNA is organised into nucleosomes characterised by a shorter and more homogeneous average nucleosomal repeat than bulk chromatin as shown by Makarov et al. (1). The latter authors were unable to detect the association of any linker histone with the telomeric DNA. We have confirmed these observations but show that in sharp contrast chicken erythrocyte telomeric DNA is organised into nucleosomes whose spacing length and heterogeneity are indistinguishable from those of bulk chromatin. We further show that chicken erythrocyte telomeric chromatin contains chromatosomes which are preferentially associated with histone H1 relative to histone H5. This contrasts with bulk chromatin where histone H5 is the more abundant species. This observation strongly suggests that telomeric DNA condensed into nucleosome core particles has a higher affinity for H1 than H5. We discuss the origin of the discrimination of the lysine rich histones in terms of DNA sequence preferences, telomere nucleosome preferences and particular constraints of the higher order chromatin structure of telomeres.     

Effect of monovalent cation-induced telomeric DNA structure on the binding of Oxytricha telomeric protein.

Oligonucleotides bearing 4 repeats of telomeric deoxyguanosine-rich sequence undergo a monovalent cation-induced transition to a folded conformation with G-G base pairs, modeled as a 'G-quartet' structure. We have now deduced the rates of folding and unfolding of d(TTTTGGGG)4, which has four repeats of the Oxytricha telomeric DNA sequence. The estimated average values of delta G for the folded form at 37 degrees C are -2.2 kcal/mol and -4.7 kcal/mol in 50 mM na+ and K+, respectively. The fully folded DNA is not recognized by the Oxytricha telomere-binding protein; the substrate for protein binding has properties consistent with its being partly or fully unfolded. In confirmation of this conclusion, prevention of DNA folding by methylation enables the protein to bind as rapidly in the presence of monovalent cations as in their absence. The slow unfolding (t1/2 = 4 hr and 18 hr at 37 degrees C in Na+ and K+, respectively) of the DNA suggests that such structures would be long-lived if they formed in vivo, unless they can be actively unfolded. The inability of the telomere-binding protein to bind the stable, folded form of the 4-repeat telomeric sequence is a problem that may be circumvented in vivo by avoiding four single-stranded repeats.     

Deletion of Ogg1 DNA glycosylase results in telomere base damage and length alteration in yeast

Telomeres consist of short guanine‐rich repeats. Guanine can be oxidized to 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) and 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FapyG). 8‐oxoguanine DNA glycosylase (Ogg1) repairs these oxidative guanine lesions through the base excision repair (BER) pathway. Here we show that in Saccharomyces cerevisiae ablation of Ogg1p leads to an increase in oxidized guanine level in telomeric DNA. The ogg1 deletion (ogg1Δ) strain shows telomere lengthening that is dependent on telomerase and/or Rad52p‐mediated homologous recombination. 8‐oxoG in telomeric repeats attenuates the binding of the telomere binding protein, Rap1p, to telomeric DNA in vitro. Moreover, the amount of telomere‐bound Rap1p and Rif2p is reduced in ogg1Δ strain. These results suggest that oxidized guanines may perturb telomere length equilibrium by attenuating telomere protein complex to function in telomeres, which in turn impedes their regulation of pathways engaged in telomere length maintenance. We propose that Ogg1p is critical in maintaining telomere length homoeostasis through telomere guanine damage repair, and that interfering with telomere length homoeostasis may be one of the mechanism(s) by which oxidative DNA damage inflicts the genome.     

Unusual salt-induced behaviour of guanine-rich natural DNA evidenced by dynamic light scattering

The appearance of the slow mode, revealed by dynamic light scattering (DLS) measurements in Micrococcus luteus DNA with high GC content, and the effect of guanine sequences on changes of DNA physical state and conformational transitions were investigated. We used two different spectroscopic approaches: DLS, to evidence the relatively slowly diffusing particles arising at high salt concentration, ascribable to the formation of large unspecific molecular aggregates, and circular dichroism spectroscopy, to identify these entities. Our results bring us to conclude that a peculiar, unconventional, structural transition, due to the presence of long guanine stretches, in a well-defined experimental condition, can occur. We comment on the biological implications to detect, by spectroscopic measurements, such an unusual structure involved in the stability, protection and replication maintenance along the human telomeric G-rich strand.     

What the papers say: Telomeric DNA binding proteins

The physical ends of eukaryotic chromosomes form a specialized nucleoprotein complex composed of DNA and DNA binding proteins. This nucleoprotein complex, termed the telomere, is essential for chromosome stability. In most organisms, the DNA portion of the nucleoprotein complex consists of simple tandem DNA repeats with one strand guanine rich. The protein portion of the complex is less well understood. The experiments presented in two recent papers^(1,2) represent different stages in the characterization of the telomeric DNA binding proteins. The first paper presents a structure-function study of the Oxytricha telomeric DNA binding proteins and the second paper shows the identification and initial characterization of a telomeric DNA binding activity from Xenopus laevis. These two reports provided valuable information in understanding the structure and function of telomeres.     

Polymorphism of human telomeric quadruplex structures

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). Compounds that can stabilize the intramolecular DNA G-quadruplexes formed in the human telomeric sequence have been shown to inhibit the activity of telomerase and telomere maintenance, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. Knowledge of intramolecular human telomeric G-quadruplex structure(s) formed under physiological conditions is important for structure-based rational drug design and thus has been the subject of intense investigation. This review will give an overview of recent progress on the intramolecular human telomeric G-quadruplex structures formed in K(+) solution. It will also give insight into the structure polymorphism of human telomeric sequences and its implications for drug targeting.