Quadruplex DNA Research Articles

PhenDV, a turn-off fluorescent quadruplex DNA probe for improving the sensitivity of drug screening assays.

We report a new turn-off fluorescent probe, PhenDV, for the identification of high affinity quadruplex (G4) DNA ligands. This push-pull fluorophore displays a high fluorescence quantum yield in water (ΦF = 0.21) and is a selective and strong quadruplex DNA binder. We describe its use as a fluorescent indicator for the G4 Fluorescent Intercalator Displacement (FID) assay as its fluorescence is strongly quenched when bound to G4 DNA and fully restored when displaced by ligand. This probe improves the sensitivity of the G4-FID assay, as the read out relies on increased fluorescence instead of quenching observed with classical on/off probes.

Specifically targeting mixed-type dimeric G-quadruplexes using berberine dimers.

Three polyether-tethered berberine dimers (1a-c) were studied for their binding affinity, selectivity and thermal stabilization towards human telomeric dimeric quadruplex DNA (G2T1). Compound 1a with the shortest polyether linker showed the highest affinity (Ka > 108 M-1) and 76-508-fold higher selectivity for mixed-type G2T1 over antiparallel G2T1 and three monomeric G-quadruplexes, which are human telomeric monomeric quadruplex G1, c-kit 1 and c-kit 2. Compound 1a induced the formation of quadruplex structures and showed higher thermal stabilization for mixed-type G2T1 than for anti-parallel G2T1, G1 and ds DNA. Spectroscopic studies suggest that compound 1a could bind to mixed-type G2T1 via end-stacking and external binding modes. These results suggest that the polyether linkers in these compounds play an important role in regulating the binding affinity and selectivity towards mixed-type G2T1 and that compound 1a could target mixed-type G2T1 at other genome regions with antiparallel G2T1 and monomeric G-quadruplexes. These results may provide useful guidance for the rational design of selective multimeric G-quadruplex binders and potential anticancer agents.

Combining a Ru(II) "Building Block" and Rapid Screening Approach to Identify DNA Structure-Selective "Light Switch" Compounds.

A chemically reactive Ru(II) "building block", able to undergo condensation reactions with substituted diamines, was utilized to create a small library of luminescent "light switch" dipyrido-[3,2-a:2',3'-c] phenazine (dppz) complexes. The impact of substituent identity, position, and the number of substituents on the light switch effect was investigated. An unbiased, parallel screening approach was used to evaluate the selectivity of the compounds for a variety of different biomolecules, including protein, nucleosides, single stranded DNA, duplex DNA, triplex DNA, and G-quadruplex DNA. Combining these two approaches allowed for the identification of hit molecules that showed different selectivities for biologically relevant DNA structures, particularly triplex and quadruplex DNA.

Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP)

The development of quadruplex-directed molecular diagnostic and therapy rely on mechanistic insights gained at both cellular and tissue levels by fluorescence imaging. This technique is based on fluorescent reporters that label cellular DNA and RNA quadruplexes to spatiotemporally address their complex cell biology. The photophysical characteristics of quadruplex probes usually dictate the modality of cell imaging by governing the selection of the light source (lamp, LED, laser), the optical light filters and the detection modality. Here, we report the characterizations of prototype from a new generation of quadruplex dye termed G4-REP (for quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality (owing to the versatility gained through the red-edge effect), thus allowing for diverse applications and most imaging facilities. This is demonstrated by cell images (and associated quantifications) collected through confocal and multiphoton microscopy as well as through real-time live-cell imaging system over extended period, monitoring both non-cancerous and cancerous human cell lines. Our results promote a new way of designing versatile, efficient and convenient quadruplex-reporting dyes for tracking these higher-order nucleic acid structures in living human cells. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

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.

Targeting human telomeric and c-myc G-quadruplexes with alkynylplatinum(II) terpyridine complexes under molecular crowding conditions

The interactions between alkynylplatinum(II) terpyridine complexes 1–3 and the G-quadruplex DNA, including c-myc and telomeric quadruplex DNA, are investigated both in dilute solution and under molecular crowding conditions. The UV–vis absorption spectroscopy, circular dichroism and molecular docking studies suggest that 1–3 associate with telomeric and c-myc G-quadruplexes via groove binding, and electrostatic interactions. Experimental studies indicate that under molecular crowding conditions (in the presence of 40wt% PEG 200), 1–2 show weak affinity for c-myc, while 3 still displays high affinity and selectivity for c-myc. On the other hand, 1–3 act as efficient and selective ligand for telomeric quadruplex DNA under molecular crowding conditions. The complex 3 exhibits excellent cytotoxicity against A549, K562 and SGC-7901, with IC50 values that are 35.0-fold, 10.0-fold, and 12.1-fold lower than the values of cisplatin under the same conditions, respectively.

Interaction of hemin with quadruplex DNA.

A DNA enzyme with peroxidase activity is a G-quadruplex-based DNAzyme formed by hemin and G-quadruplex DNA. Activity of peroxide DNAzymes can be influenced by the structure of quadruplex DNA. In this investigation, the interaction of hemin with T30695 G-quadruplex DNA is evaluated. Molecular dynamic simulation indicates that the binding mode of hemin to G-quadruplex DNA is end-stacking, which is consistent with absorption spectroscopy. Based on fluorescence spectroscopy, hemin ejects thiazole orange from bases of four-strand DNA. Circular dichroism spectra showed that no alteration occurs in this type of DNA structure. Graphical Abstract Peroxidase DNAzyme is formed by hemin and G-quadruplex DNA.

Binding of Telomestatin to a Telomeric G-Quadruplex DNA Probed by All-Atom Molecular Dynamics Simulations with Explicit Solvent.

Telomestatin, a natural product isolated from Streptomyces anulatus, stabilizes telomeric DNA G-quadruplexes. Treatment with this ligand induces apoptosis of various cancer cells with a relatively low effect on somatic cells because of its high selectivity toward G-quadruplex over duplex DNA. A high-resolution structure of a G-quadruplex in complex with telomestatin does not yet exist because of its low solubility, and the binding nature of this ligand remains elusive. In this study, we utilized molecular binding simulations and MMGBSA binding energy analysis to decipher the nature of the binding of telomestatin to a telomeric G-quadruplex. We identified three major binding poses: bottom intercalation, top stacking, and groove binding. The top stacking mode resembles the pose observed in an NMR complex of the same G-quadruplex with the telomestatin analogue L2H. The bottom intercalation and groove binding poses were not observed in the previous studies of L2H. The bottom intercalation mode exhibited the most favorable binding energy among the three modes, while also partially intercalating into the telomeric quadruplex. The dynamic and energetic properties of these three binding modes are thoroughly examined. "Flip insertion" and "slide insertion" were observed in the bottom intercalation mode. Our findings also provide insight into the design of more selective DNA quadruplex ligands as anticancer agents in the future.

Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method.

Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities.

Molecular Dynamics Simulation Study of Parallel Telomeric DNA Quadruplexes at Different Ionic Strengths: Evaluation of Water and Ion Models.

Most molecular dynamics (MD) simulations of DNA quadruplexes have been performed under minimal salt conditions using the Åqvist potential parameters for the cation with the TIP3P water model. Recently, this combination of parameters has been reported to be problematic for the stability of quadruplex DNA, especially caused by the ion interactions inside or near the quadruplex channel. Here, we verify how the choice of ion parameters and water model can affect the quadruplex structural stability and the interactions with the ions outside the channel. We have performed a series of MD simulations of the human full-parallel telomeric quadruplex by neutralizing its negative charge with K(+) ions. Three combinations of different cation potential parameters and water models have been used: (a) Åqvist ion parameters, TIP3P water model; (b) Joung and Cheatham ion parameters, TIP3P water model; and (c) Joung and Cheatham ion parameters, TIP4Pew water model. For the combinations (b) and (c), the effect of the ionic strength has been evaluated by adding increasing amounts of KCl salt (50, 100, and 200 mM). Two independent simulations using the Åqvist parameters with the TIP3P model show that this combination is clearly less suited for the studied quadruplex with K(+) as counterions. In both simulations, one ion escapes from the channel, followed by significant deformation of the structure, leading to deviating conformation compared to that in the reference crystallographic data. For the other combinations of ion and water potentials, no tendency is observed for the channel ions to escape from the quadruplex channel. In addition, the internal mobility of the three loops, torsion angles, and counterion affinity have been investigated at varied salt concentrations. In summary, the selection of ion and water models is crucial as it can affect both the structure and dynamics as well as the interactions of the quadruplex with its counterions. The results obtained with the TIP4Pew model are found to be closest to the experimental data at all of the studied ion concentrations.

Biological oscillations: Fluorescence monitoring by confocal microscopy

Fluctuations play a vital role in biological systems. Single molecule spectroscopy has recently revealed many new kinds of fluctuations in biological molecules. In this account, we focus on structural fluctuations of an antigen-antibody complex, conformational dynamics of a DNA quadruplex, effects of taxol on dynamics of microtubules, intermittent red-ox oscillations at different organelles in a live cell (mitochondria, lipid droplets, endoplasmic reticulum and cell membrane) and stochastic resonance in gene silencing. We show that there are major differences in these dynamics between a cancer cell and the corresponding non-cancer cell.

Specific Stabilization of c-MYC and c-KIT G-Quadruplex DNA Structures by Indolylmethyleneindanone Scaffolds.

Stabilization of G-quadruplex DNA structures by small molecules has emerged as a promising strategy for the development of anticancer drugs. Since G-quadruplex structures can adopt various topologies, attaining specific stabilization of a G-quadruplex topology to halt a particular biological process is daunting. To achieve this, we have designed and synthesized simple structural scaffolds based on an indolylmethyleneindanone pharmacophore, which can specifically stabilize the parallel topology of promoter quadruplex DNAs (c-MYC, c-KIT1, and c-KIT2), when compared to various topologies of telomeric and duplex DNAs. The lead ligands (InEt2 and InPr2) are water-soluble and meet a number of desirable criteria for a small molecule drug. Highly specific induction and stabilization of the c-MYC and c-KIT quadruplex DNAs (ΔT1/2 up to 24 °C) over telomeric and duplex DNAs (ΔT1/2 ∼ 3.2 °C) by these ligands were further validated by isothermal titration calorimetry and electrospray ionization mass spectrometry experiments (Ka ∼ 10(5) to 10(6) M(-1)). Low IC50 (∼2 μM) values were emerged for these ligands from a Taq DNA polymerase stop assay with the c-MYC quadruplex forming template, whereas the telomeric DNA template showed IC50 values >120 μM. Molecular modeling and dynamics studies demonstrated the 5'- and 3'-end stacking modes for these ligands. Overall, these results demonstrate that among the >1000 quadruplex stabilizing ligands reported so far, the indolylmethyleneindanone scaffolds stand out in terms of target specificity and structural simplicity and therefore offer a new paradigm in topology specific G-quadruplex targeting for potential therapeutic and diagnostic applications.

A 4:1 stoichiometric binding and stabilization of mitoxantrone-parallel stranded G-quadruplex complex established by spectroscopy techniques

Small molecule ligands which specifically bind and stabilize G-quadruplex structures in telomeric ends inhibit the activity of telomerase enzyme, an important marker for cancer. Understanding of the binding mode of ligand-G quadruplex complex is important for evaluating relative efficacy of anti-tumor drugs. The present study is focused on interaction of anti-tumor drug mitoxantrone (MTX) with tetra-molecular parallel stranded G-quadruplex sequence d-TTGGGGT using absorbance, fluorescence and circular dichroism spectroscopy techniques. Absorbance of mitoxantrone shows hypochromism up to MTX (D)/DNA quadruplex (N) ratio ~5, followed by hyperchromism up to D/N=0.21 accompanied by a red shift of 15nm. The fluorescence emission of MTX shows decrease up to D/N ~5 and then increases with red shift of 8nm. The two observed fluorescent lifetimes, 0.17ns (91%) and 0.44ns (9%), indicate dual binding mode. Absence of isobestic and isoemissive point indicates presence of multiple complexes. Circular Dichroism spectra showing positive induced band at 645nm and two exciton bands centered at 619 and 664nm suggest binding of mitoxantrone as a dimer. Proton NMR studies show intermolecular MTX-MTX short contacts confirming existence of stacked dimer of MTX. Thermal melting transitions of DNA saturate at D/N=4 with ΔTm=25°C. The results establish highly specific external groove binding of 4 molecules of mitoxantrone as two dimers at two distinct sites of DNA.

IFI16 Preferentially Binds to DNA with Quadruplex Structure and Enhances DNA Quadruplex Formation.

Interferon-inducible protein 16 (IFI16) is a member of the HIN-200 protein family, containing two HIN domains and one PYRIN domain. IFI16 acts as a sensor of viral and bacterial DNA and is important for innate immune responses. IFI16 binds DNA and binding has been described to be DNA length-dependent, but a preference for supercoiled DNA has also been demonstrated. Here we report a specific preference of IFI16 for binding to quadruplex DNA compared to other DNA structures. IFI16 binds to quadruplex DNA with significantly higher affinity than to the same sequence in double stranded DNA. By circular dichroism (CD) spectroscopy we also demonstrated the ability of IFI16 to stabilize quadruplex structures with quadruplex-forming oligonucleotides derived from human telomere (HTEL) sequences and the MYC promotor. A novel H/D exchange mass spectrometry approach was developed to assess protein interactions with quadruplex DNA. Quadruplex DNA changed the IFI16 deuteration profile in parts of the PYRIN domain (aa 0–80) and in structurally identical parts of both HIN domains (aa 271–302 and aa 586–617) compared to single stranded or double stranded DNAs, supporting the preferential affinity of IFI16 for structured DNA. Our results reveal the importance of quadruplex DNA structure in IFI16 binding and improve our understanding of how IFI16 senses DNA. IFI16 selectivity for quadruplex structure provides a mechanistic framework for IFI16 in immunity and cellular processes including DNA damage responses and cell proliferation.

Strong preference of BRCA1 protein to topologically constrained non-B DNA structures.

BackgroundThe breast and ovarian cancer susceptibility gene BRCA1 encodes a multifunctional tumor suppressor protein BRCA1, which is involved in regulating cellular processes such as cell cycle, transcription, DNA repair, DNA damage response and chromatin remodeling. BRCA1 protein, located primarily in cell nuclei, interacts with multiple proteins and various DNA targets. It has been demonstrated that BRCA1 protein binds to damaged DNA and plays a role in the transcriptional regulation of downstream target genes. As a key protein in the repair of DNA double-strand breaks, the BRCA1-DNA binding properties, however, have not been reported in detail.ResultsIn this study, we provided detailed analyses of BRCA1 protein (DNA-binding domain, amino acid residues 444–1057) binding to topologically constrained non-B DNA structures (e.g. cruciform, triplex and quadruplex). Using electrophoretic retardation assay, atomic force microscopy and DNA binding competition assay, we showed the greatest preference of the BRCA1 DNA-binding domain to cruciform structure, followed by DNA quadruplex, with the weakest affinity to double stranded B-DNA and single stranded DNA. While preference of the BRCA1 protein to cruciform structures has been reported previously, our observations demonstrated for the first time a preferential binding of the BRCA1 protein also to triplex and quadruplex DNAs, including its visualization by atomic force microscopy.ConclusionsOur discovery highlights a direct BRCA1 protein interaction with DNA. When compared to double stranded DNA, such a strong preference of the BRCA1 protein to cruciform and quadruplex structures suggests its importance in biology and may thus shed insight into the role of these interactions in cell regulation and maintenance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-016-0068-6) contains supplementary material, which is available to authorized users.

Steady-State and Time-Resolved Studies into the Origin of the Intrinsic Fluorescence of G-Quadruplexes.

Stretches of guanines in DNA and RNA can fold into guanine quadruplex structures (GQSs). These structures protect telomeres in DNA and regulate gene expression in RNA. GQSs have an intrinsic fluorescence that is sensitive to different parameters, including loop sequence and length. However, the dependence of GQS fluorescence on solution and sequence parameters and the origin of this fluorescence are poorly understood. Herein we examine effects of dangling nucleotides and cosolute conditions on GQS fluorescence using both steady-state and time-resolved fluorescence spectroscopy. The quantum yield of dGGGTGGGTGGGTGGG, termed "dG3T", is found to be modest at ∼2 × 10(-3). Nevertheless, dG3T and its variants are significantly brighter than the common nucleic acid fluorophore 2-aminopurine (2AP) largely due to their sizable extinction coefficients. Dangling 5'-end nucleotides generally reduce emission and blue-shift the resultant spectrum, whereas dangling 3'-end nucleotides slightly enhance fluorescence, particularly on the red side of the emission band. Time-resolved fluorescence decays are broadly distributed in time and require three exponential components for accurate fits. Time-resolved emission spectra suggest the presence of two emitting populations centered at ∼330 and ∼390 nm, with the redder component being a well-defined long-lived (∼1 ns) entity. Insights into GQS fluorescence obtained here should be useful in designing brighter intrinsic RNA and DNA quadruplexes for use in label-free biotechnological applications.

Model of cooperative interaction of TMPyP4 porphyrine with DNA quadruplex

Intrinsic binding constants of the TMPyP4 porphyrin with G-quadruplex of DNA (ckit2) have been calculated; they can be applied as reference for quantitative evaluation of the possibility of such structures participation in biochemical processes. The conclusion about existence of two different sites of the TMPyP4 binding to ckit2 binding made earlier on the basis of the stepwise complex formation model has been revised using the model of mutual influence (cooperativity) of the ligands bound to the oligonucleotide. The model has been verified using principal component analysis and factor analysis methods. The ckit2 biomolecule can be considered as an oligonucleotide with equivalent mutually affecting binding sites.

Synthesis of New Benzimidazole and Benzothiazole Disulfide Metal Complexes as G-quadruplex Binding Ligands.

Compounds that can bind and stabilize non-canonical DNA structures are named quadruplex and are of interest in anticancer drug design due to their selective inhibitions of telomerase and consequent effects on cell proliferation. In this study, we report novel Co/Cu [II] complex compounds as G-quadruplex DNA binding ligands. The results from the preliminary assay indicated that the introduction of a positively charged 6-membered tail to the aromatic terminal group of benzimidazole significantly enhanced the binding affinity with the quadruplex and exhibited anti-telomerase activity. These derivatives showed significant selectivities for the telomeric quadruplex over duplex nucleic acids. The stabilization of non-canonical forms estimated with the FRET DNA technology using different sequences, such as F21T, c-kit1 and c-kit2, in cancer cell lines were assessed. Three members of this family showed to be very selective in stabilizing one particular G-quadruplex.

Playing Around with the Size and Shape of Quinolizinium ­Derivatives: Versatile Ligands for Duplex, Triplex, Quadruplex and Abasic Site-Containing DNA

The association of heteroaromatic ligands with DNA is an important and biologically relevant process, because it may have a strong influence on the function of the nucleic acid. As a result, efficient and selective DNA-targeting ligands are considered as promising lead structures for drugs. In this context, we established the quinolizinium ion as a versatile building block for the design of DNA-binding ligands, with the long-term goal to evaluate and understand the structural parameters that govern the association of cationic hetarenes with DNA. In this account we demonstrate that annelated quinolizinium derivatives are easily available and that their structure and substitution pattern are highly variable. Most notably, the availability of several derivatives with different size and shape enables the assessment of structure–property relationships regarding their DNA-binding properties. It is shown with exemplary case studies that the systematic variation of the ligand structure, along with analysis of the binding parameters, can be employed to analyze the structural requirements of a ligand to bind to different DNA forms, such as triplex, quadruplex and abasic site-containing DNA. 1 Introduction 2 Association of Quinolizinium Derivatives with Double-Stranded DNA 3 Association of Quinolizinium Derivatives with Triplex DNA 4 Association of Quinolizinium Derivatives with Quadruplex DNA 5 Association of Quinolizinium Derivatives with Abasic Positions in DNA 6 Conclusion

Dihydrochelerythrine and its derivatives: Synthesis and their application as potential G-quadruplex DNA stabilizing agents

A convenient route was envisaged toward the synthesis of dihydrochelerythrine (DHCHL), 4 by intramolecular Suzuki coupling of 2-bromo-N-(2-bromobenzyl)-naphthalen-1-amine derivative 5 via in situ generated arylborane. This compound was converted to (±)-6-acetonyldihydrochelerythrine (ADC), 3 which was then resolved by chiral prep-HPLC. Efficiency of DHCHL for the stabilization of promoter quadruplex DNA structures and a comparison study with the parent natural alkaloid chelerythrine (CHL), 1 was performed. A thorough investigation was carried out to assess the quadruplex binding affinity by using various biophysical and biochemical studies and the binding mode was explained by using molecular modeling and dynamics studies. Results clearly indicate that DHCHL is a strong G-quadruplex stabilizer with affinity similar to that of the parent alkaloid CHL. Compounds ADC and DHCHL were also screened against different human cancer cell lines. Among the cancer cells, (±)-ADC and its enantiomers showed varied (15–48%) inhibition against human colorectal cell line HCT116 and breast cancer cell line MDA-MB-231 albeit low enantio-specificity in the inhibitory effect; whereas DHCHL showed 30% inhibition against A431 cell line only, suggesting the compounds are indeed cancer tissue specific.