Quadruplex Conformation Research Articles

The Interaction of a Gemini Surfactant with a DNA Quadruplex.

DNA secondary structures are stabilized by mono- and divalent cations. To examine the stability of the DNA quadruplex formed from (TTAGGG)4, its interaction with a dicationic Gemini surfactant in standard phosphate buffer was investigated. The Gemini surfactant begins to form micelles in buffer at a cmc (critical micelle concentration) of 1.5 mM. In this study, solutions of DNA were prepared in buffer with surfactant concentrations ranging from 0.0 to 3.0 mM, i.e., above and below the cmc of the surfactant. In all samples of DNA and surfactant, a precipitate formed. The fraction of DNA precipitated depends upon both the initial DNA concentration and the initial concentration of the surfactant. In those samples where the DNA did not totally precipitate, the residual DNA assumed a quadruplex conformation. It was determined that two surfactant molecules per DNA phosphate are needed to completely precipitate all of the DNA in a particular sample. An estimated apparent K sp for the DNA:surfactant complex was determined.

Tumor acidic microenvironment activatable DNA nanostructure for precise cancer cell targeting and inhibition

Precise tumor targeting and therapy is a major trend in cancer treatment. Herein, we designed a tumor acidic microenvironment activatable drug loaded DNA nanostructure, in which, we make a clever use of the sequences of AS1411 and i-motif, which can partially hybridization, and designed a simple while robust DNA d-strand nanostructure, in which, i-motif sequence was designed to regulate the binding ability of the AS1411 aptamer to target tumor, in the normal physiological environment, i-motif inhibits the targeting ability of AS1411. In the acidic tumor microenvironment, i-motif forms a quadruplex conformation and dissociates from AS1411, restoring the targeting ability of AS1411, only when acidic condition and tumor cell receptor are present, this nanostructure can be internalized by the tumor cells. Moreover, the structure change of this nanostructure can realize the release of loaded drug. This drug loaded A-I-Duplex DNA structure showed cancer cell and spheroid targeting and inhibition ability, which is promising in the clinical cancer therapy.

Thermally Induced Transitions of d(G4T4G3) Quadruplexes Can Be Described as Kinetically Driven Processes.

DNA sequences that are rich in guanines and can form four-stranded structures are called G-quadruplexes. Due to the growing evidence that they may play an important role in several key biological processes, the G-quadruplexes have captured the interest of several researchers. G-quadruplexes may form in the presence of different metal cations as polymorphic structures formed in kinetically governed processes. Here we investigate a complex polymorphism of d(G4T4G3) quadruplexes at different K+ concentrations. We show that population size of different d(G4T4G3) quadruplex conformations can be manipulated by cooling rate and/or K+ concentration. We use a kinetic model to describe data obtained from DSC, CD and UV spectroscopy and PAGE experiments. Our model is able to describe the observed thermally induced conformational transitions of d(G4T4G3) quadruplexes at different K+ concentrations.

Human DNA Telomeres in Presence of Oxidative Lesions: The Crucial Role of Electrostatic Interactions on the Stability of Guanine Quadruplexes.

By using all atom molecular dynamics simulations, we studied the behavior of human DNA telomere sequences in guanine quadruplex (G4) conformation and in the presence of oxidative lesions, namely abasic sites. In particular, we evidenced that while removing one guanine base induces a significant alteration and destabilization of the involved leaflet, human telomere oligomers tend, in most cases, to maintain at least a partial quadruplex structure, eventually by replacing the empty site with undamaged guanines of different leaflets. This study shows that (i) the disruption of the quadruplex leaflets induces the release of at least one of the potassium cations embedded in the quadruplex channel and that (ii) the electrostatic interactions of the DNA sequence with the aforementioned cations are fundamental to the maintenance of the global quadruplex structure.

Fluorimetric Detection of G-Quadruplex DNA in Solution and Adsorbed on Surfaces with a Selective Trinuclear Cyanine Dye.

Quadruplex DNA, which is a relevant target for anticancer therapies, may alter its conformation because of interactions with interfaces. In pursuit of a versatile methodology to probe adsorption-induced conformational changes, the interaction between a fluorescent [2.2.2]heptamethinecyanine dye and quadruplex DNA (G4-DNA) was studied in solution and on surfaces. In solution, the cyanine dye exhibits a strong light-up effect upon the association with G4-DNA without interference from double-stranded DNA. In addition, a terminal π-stacking as a binding mode between the cyanine dye and G4-DNA is concluded using NMR spectroscopy. To unravel the effects of adsorption on the conformation of quadruplex-DNA, G4-DNA, and double-stranded and single-stranded DNA were adsorbed to positively charged poly(allylamine) hydrochloride (PAH) surfaces, both in planar and in constrained 55 nm diameter aluminum oxide nanopore formats. All DNA forms showed a very strong affinity to the PAH surfaces as shown by surface plasmon resonance and reflectometric interference spectroscopy. The significant increase of the fluorescence emission intensity of the cyanine light-up probe observed exclusively for surface immobilized G4-DNA affords evidence for the adsorption of G4-DNA on PAH with retained quadruplex conformation.

A topological transition from bimolecular quadruplex to G-triplex/tri-G-quadruplex exhibited by truncated double repeats of human telomere.

Human telomeric G-rich sequences can fold back into various conformations depending upon the salt (Na+ or K+) at physiological pH. On the basis of results obtained by native PAGE electrophoresis, circular dichroism, and UV-melting experiments, we report here that truncated sequences of human telomere (d-GGGTTAGGG; GM9, d-AGGGTTAGGG; GM10, d-TAGGGTTAGGG; GM11) adopt a varied range of quadruplex conformations as a function of the cation present. By correlating CD and gel electrophoresis experiments; it was concluded that the GM9 oligonucleotide can self-associate to form a tetramer quadruplex (antiparallel; AP) in Na+ solution and a mixture of G-triplex (AP) or tri-G-quadruplex (parallel; P) along with a tetramer G-quadruplex structure (AP) in K+. The GM10 oligonucleotide formed a bimolecular G-quadruplex in both Na+ and K+ solutions, while GM11 associated to form a bimolecular G-quadruplex (AP) structure in Na+ solution and a mixture of bimolecular G-quadruplex (AP) and bimolecular G-quadruplex (P) along with parallel G-triplex or antiparallel tri-G-quadruplex in K+. All the UV-melting profiles, thermal difference spectra, and CD melting curves suggested the formation of a variety of G-quadruplex conformations by the DNA sequences studied in Na+ and K+ ions. Hypothetical models for different conformations adopted by these DNA molecules have also been proposed, which may further enhance our knowledge about the divergent topologies of guanine quadruplexes.

Mercury ion-DNA specificity triggers a distinctive photoluminescence depression in organic semiconductor probes guided with a thymine-rich oligonucleotide sequence.

DNA strands have been recently found to play a role in crystallizing organic semiconductors as a substitute for conventional surfactants. Such DNA-guided organic semiconductor particles possessed the recognition ability to complementary target DNAs, resulting in "enhanced luminescence" due to the lesser degree of non-radiative dissipation. Apart from this, in this study we developed selective recognition of mercury ions by utilizing DNA probes having ion-specific thymine-rich motifs. Strikingly, the specific ion-DNA interaction triggered rather distinctive "depressed luminescence" emitting from the particles. The mercury ions were found to be present both at the surface and the inner regions, which were discovered to relate to the drastic morphological distortion of the particles as evidenced by elemental, electron microscopy, and confocal fluorescence microscopy analyses. This novel phenomenon discovered would expand the technological values of organic semiconductors conjugated with oligonucleotides toward a wider range of target-specific applications.

Spectroscopic insights into quadruplexes of five-repeat telomere DNA sequences upon G-block damage

BackgroundThe DNA lesions, resulting from oxidative damage, were shown to destabilize human telomere four-repeat quadruplex and to alter its structure. Long telomere DNA, as a repetitive sequence, offers, however, other mechanisms of dealing with the lesion: extrusion of the damaged repeat into loop or shifting the quadruplex position by one repeat. MethodsUsing circular dichroism and UV absorption spectroscopy and polyacrylamide electrophoresis, we studied consequences of lesions at different positions of the model five-repeat human telomere DNA sequences on the structure and stability of their quadruplexes in sodium and in potassium. ResultsThe repeats affected by lesion are preferentially positioned as terminal overhangs of the core quadruplex structurally similar to the four-repeat one. Forced affecting of the inner repeats leads to presence of variety of more parallel folds in potassium. In sodium the designed models form mixture of two dominant antiparallel quadruplexes whose population varies with the position of the affected repeat. The shapes of quadruplex CD spectra, namely the height of dominant peaks, significantly correlate with melting temperatures. ConclusionLesion in one guanine tract of a more than four repeats long human telomere DNA sequence may cause re-positioning of its quadruplex arrangement associated with a shift of the structure to less common quadruplex conformations. The type of the quadruplex depends on the loop position and external conditions. General significanceThe telomere DNA quadruplexes are quite resistant to the effect of point mutations due to the telomere DNA repetitive nature, although their structure and, consequently, function might be altered.

Observation of a Dynamic G-Tetrad Flip in Intramolecular G-Quadruplexes.

A MYC sequence forming an intramolecular G-quadruplex with a parallel topology was modified by the incorporation of 8-bromoguanosine (BrG) analogues in one of its outer G-tetrads. The propensity of the BrG analogues to adopt a syn glycosidic torsion angle results in an exceptional monomolecular quadruplex conformation featuring a complete flip of one tetrad while keeping a parallel orientation of all G-tracts as shown by circular dichroism and nuclear magnetic resonance spectroscopic studies. When substituting three of the four G-tetrad residues with BrG analogues, two coexisting quadruplex conformational isomers with an all-syn and all-anti outer G-quartet are approximately equally populated in solution. A dynamic interconversion of the two quadruplexes with an exchange rate (kex) of 0.2 s-1 is demonstrated through the observation of exchange crosspeaks in rotating frame Overhauser effect spectroscopy and nuclear Overhauser effect spectroscopy experiments at 50 °C. The kinetic properties suggest disruption of the corresponding outer G-tetrad but not of the whole quadruplex core during the tetrad flip. Conformational syn-anti isomers with homopolar and heteropolar stacking interactions are nearly isoenergetic with a transition enthalpy of 18.2 kJ/mol in favor of the all-syn isomer.

Separation of Quadruplex Polymorphism in DNA Sequences by Reversed-Phase Chromatography.

This unit describes a method for the separation of a mixture of quadruplex conformations formed from the same parent sequence via reversed-phase chromatography (RPC). Polymorphism is inherent to quadruplex formation and even relatively simple quadruplex-forming sequences can fold into a cornucopia of possible conformations and topologies. Isolation of a specific conformation for study can be problematic. This is especially true for conformations of the human telomere sequence d(GGG(TTAGGG)3). High performance liquid chromatography (HPLC), especially reversed-phase chromatography, has been a mainstay of nucleic acid research and purification for many decades. We have successfully applied this method to the problem of separating individual quadruplex species in the ensemble from the same parent sequence.

G-quadruplex formation between G-rich PNA and homologous sequences in oligonucleotides and supercoiled plasmid DNA.

Guanine (G)-rich DNA sequences can adopt four-stranded quadruplex conformations that may play a role in the regulation of genetic processes. To explore the possibility of targeted molecular recognition of DNA sequences with short G-rich peptide nucleic acids (PNA) and to assess the strand arrangement in such complexes, we used PNA and DNA with the Oxytricha nova telomeric sequence d(G4T4G4) as a model. PNA probes were complexed with DNA targets in the following forms: single-stranded oligonucleotides, a loop of DNA in a hairpin conformation, and as supercoiled plasmid with the (G4T4G4)/(C4A4C4) insert. Gel-shift mobility assays demonstrated formation of stable hybrid complexes between the homologous G4T4G4 PNA and DNA with multiple modes of binding. Chemical and enzymatic probing revealed sequence-specific and G-quadruplex dependent binding of G4T4G4 PNA to dsDNA. Spectroscopic and electrophoretic analysis of the complex formed between PNA and the synthetic DNA hairpin containing the G4T4G4 loop showed that the stoichiometry of a prevailing complex is three PNA strands per one DNA strand. We speculate how this new PNA-DNA complex architecture can help to design more selective, quadruplex-specific PNA probes.

G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3′-untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3′ UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson–Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site.

Conformational switch of insulin-binding aptamer into G-quadruplex induced by K+ and Na+: an experimental and theoretical approach

Guanine-rich sequences can form the G-quadruplex structure in the presence of specific metal ions. Here, circular dichroism, UV–vis absorption, fluorescence, and molecular dynamics simulation studies revealed that insulin-binding aptamer (IBA) could form an intramolecular G-quadruplex structure after binding K+. Circular dichroism (CD) spectra demonstrated that IBA could fold into a parallel G-quadruplex with a strong positive peak at 263 nm. Analysis of equilibrium titration data revealed that cation binding was cooperative with the Hill coefficient of 2.01 in K+ and 1.90 in Na+. Thermal denaturation assays indicated that K+-induced G-quadruplex is more stable than Na+-induced structure. Folding of IBA into G-quadruplex leading to the contact quenching occurs as a result of the formation of a nonfluorescent complex between donor and acceptor. Based on fluorescence quenching of IBA folding, a potassium-sensing aptasensor in the range of 0–1.4 mM was proposed. Since the quenching process was predominantly static, the binding constant and the number of binding sites were determined. In this research, based on the experimental data, the initial model of IBA G-quadruplex was constructed by molecular modeling method. The modeling structure of IBA is an intramolecular parallel-strand quadruplex conformation with two guanine tetrads. The extended molecular dynamics simulation for the model indicated that the G-quadruplex maintains its structure very well in aqueous solution in presence of K+ in the central cavity. In contrast, it was demonstrated that the G-quadruplex structure of model in the water collapses in absence of this cation.

Design, synthesis and properties of artificial nucleic acids from (R)-4-amino-butane-1,3-diol

A new artificial nucleic acid analogue, (R)-Am-BuNA, was developed with a simplified acyclic (R)-4-amino-butane-1,3-diol phosphodiester backbone. Phosphoramidite monomers of (R)-Am-BuNA were incorporated into DNA oligonucleotides (ODNs) and G-quadruplexes. Their thermal stability, conformation change and biological stability were further investigated using UV-melting, circular dichroism (CD) and gel electrophoresis. The results suggested that thermal stability of the duplexes of (R)-Am-BuNA modified ODNs and their complementary ODN is highly dependent on the substitution position. Substitution of thymidine at the 7th position in a thrombin-binding DNA aptamer (TBA) results in a slight increase in Tm with no effect on quadruplex conformation on the CD spectrum in comparison to that of the natural G-quadruplex. Further enzymatic experiments with fetal bovine serum (FBS) and snake venom phosphodiesterase (SVPDE) indicated that only single replacement of a (R)-Am-BuNA modified nucleobase greatly inhibited oligonucleotide degradation, which shows their promising applications as capping nucleotides in nucleic acid drugs.

Polyethylene glycol binding alters human telomere G-quadruplex structure by conformational selection

Polyethylene glycols (PEGs) are widely used to perturb the conformations of nucleic acids, including G-quadruplexes. The mechanism by which PEG alters G-quadruplex conformation is poorly understood. We describe here studies designed to determine how PEG and other co-solutes affect the conformation of the human telomeric quadruplex. Osmotic stress studies using acetonitrile and ethylene glycol show that conversion of the ‘hybrid’ conformation to an all-parallel ‘propeller’ conformation is accompanied by the release of about 17 water molecules per quadruplex and is energetically unfavorable in pure aqueous solutions. Sedimentation velocity experiments show that the propeller form is hydrodynamically larger than hybrid forms, ruling out a crowding mechanism for the conversion by PEG. PEGs do not alter water activity sufficiently to perturb quadruplex hydration by osmotic stress. PEG titration experiments are most consistent with a conformational selection mechanism in which PEG binds more strongly to the propeller conformation, and binding is coupled to the conformational transition between forms. Molecular dynamics simulations show that PEG binding to the propeller form is sterically feasible and energetically favorable. We conclude that PEG does not act by crowding and is a poor mimic of the intranuclear environment, keeping open the question of the physiologically relevant quadruplex conformation.

Stable label-free fluorescent sensing of biothiols based on ThT direct inducing conformation-specific G-quadruplex

In this work, a new, label-free, turn-on fluorescent sensor for biothiols detection based on ThT direct inducing conformation-specific G-quadruplex is developed. The sensing approach is based on a conformational switch of oligonucleotide controlled by Hg2+ and a commercially available water-soluble fluorescent dye, Thioflavin T (ThT). A noticeable fluorescence light-up in ThT on binding to the G-quadruplex grants the sensor excellent sensitivity. The specific quadruplex conformation induced directly by ThT and pronounced structural selectivity of ThT for G-quadruplexes could generate more stable luminescence and make sure high specificity in complex biological samples. The present assay allows for the selective determination of cysteine and glutathione in the range of 2.0×10−8–2.5×10−6M and 3.0×10−8–2.0×10−6M with a detection limit of 8.4nM and 13.9nM respectively. The diagnostic capability and potential in practical applications of this method have been demonstrated by detecting biothiols in human blood serum.

Conformational Switching of Human G-Quadruplexed DNA with Ligand Binding under Molecular Crowding Conditions

Formation of quadruplexed DNA (qDNA) within telomeric DNA has the potential to effectively inhibit the activity of telomerase, a key enzyme in tumorigenesis. The effects of molecular crowding (MC) within the cell can affect the folding of qDNA and modulate telomerase activity. Furthermore, MC can affect the ability of ligand binding to qDNA in promotion of the folded quadruplex conformation. Thus it is important that quadruplex-ligand binding be evaluated under more physiologically relevant conditions. We have examined the effects of MC on the association of N-methyl mesoporphyrin IX (NMM), a ligand which selectively binds to G-quadruplexed DNA, on the conformation of (TTAGGG)4 (HT4) and (TTAGGG)8 (HT8) human telomeric sequences. We and others have shown that in K+ solutions, HT4 forms a hybrid conformation, whereas the HT8 sequence forms two consecutive hybrid quadruplexes. With addition of crowding mimetics (Ficoll 70; Ficoll 400; and PEG 200 (20 and 40% w/v)) to the HT4 sequence, CD studies show a conformational switch to an antiparallel structure in Ficoll 400 (40% w/v) and a parallel conformation in PEG 200 (40% w/v), with no conformational changes observed in Ficoll 70. For HT8, a similar effect was observed, but in addition an anti-parallel conformation was obtained in Ficoll 70 (40% w/v), which suggests an enhanced sensitivity to dehydration for the HT8 sequence. Additionally, thermal CD melting studies showed stabilization of HT8 conformation in Ficoll compared with HT4. With binding of NMM to HT4 in 20% (w/v) crowding mimetics a shift to the parallel conformation was observed. However, the antiparallel (more dehydrated) conformation for HT8 is promoted in Ficoll 400. We plan more detailed environmental studies of NMM binding to qDNA under crowding conditions using fluorescence spectroscopy.(Supported by NIH 5SC3 GM095437)

Stability and free energy calculation of LNA modified quadruplex: a molecular dynamics study

Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, which are the fundamental in protecting the cell from recombination and degradation. Telomeric DNA sequences can form four stranded quadruplex structures, which are involved in the structure of telomere ends. The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadrulex as an attractive target for cancer therapeutic intervention. Molecular dynamic simulation offers the prospects of detailed description of the dynamical structure with ion and water at molecular level. In this work we have taken a oligomeric part of human telomeric DNA, d(TAGGGT) to form different monomeric quadruplex structures d(TAGGGT)₄. Here we report the relative stabilities of these structures under K⁺ ion conditions and binding interaction between the strands, as determined by molecular dynamic simulations followed by energy calculation. We have taken locked nucleic acid (LNA) in this study. The free energy molecular mechanics Poission Boltzman surface area calculations are performed for the determination of most stable complex structure between all modified structures. We calculated binding free energy for the combination of different strands as the ligand and receptor for all structures. The energetic study shows that, a mixed hybrid type quadruplex conformation in which two parallel strands are bind with other two antiparallel strands, are more stable than other conformations. The possible mechanism for the inhibition of the cancerous growth has been discussed. Such studies may be helpful for the rational drug designing.

Conformations Consistent with Charge Migration Observed in DNA and RNA X-ray Structures

Electron holes are known to migrate along the DNA or RNA duplexes and to localize preferentially on successive guanines. The stationary point conformations of Gua pairs that can trap or let pass these holes have been characterized by quantum chemistry calculations. Here we show their recurrent occurrence in DNA and RNA X-ray structures, often in quadruplex conformations or in interaction with proteins, ligands or metal ions. These findings give support to the biological, possibly regulatory, roles of charge migration in cell functioning.

Optical Nanosensor Architecture for Cell-Signaling Molecules Using DNA Aptamer-Coated Carbon Nanotubes

We report a novel optical biosensor platform using near-infrared fluorescent single-walled carbon nanotubes (SWNTs) functionalized with target-recognizing aptamer DNA for noninvasively detecting cell-signaling molecules in real time. Photoluminescence (PL) emission of aptamer-coated SWNTs is modulated upon selectively binding to target molecules, which is exploited to detect insulin using an insulin-binding aptamer (IBA) as a molecular recognition element. We find that nanotube PL quenches upon insulin recognition via a photoinduced charge transfer mechanism with a quenching rate of k(q) = 5.85 × 10(14) M(-1) s(-1) and a diffusion-reaction rate of k(r) = 0.129 s(-1). Circular dichroism spectra reveal for the first time that IBA strands retain a four-stranded, parallel guanine quadruplex conformation on the nanotubes, ensuring target selectivity. We demonstrate that these IBA-functionalized SWNT sensors incorporated in a collagen extracellular matrix (ECM) can be regenerated by removing bound analytes through enzymatic proteolysis. As proof-of-concept, we show that the SWNT sensors embedded in the ECM promptly detect insulin secreted by cultured pancreatic INS-1 cells stimulated by glucose influx and report a gradient contour of insulin secretion profile. This novel design enables new types of label-free assays and noninvasive, in situ, real-time detection schemes for cell-signaling molecules.