Guanine-rich DNA Sequences Research Articles

Towards Metal-Mediated G-Quartet Analogues: 1,2,4-Triazole Nucleotides

We proposed that metal-coordinating nucleotides could be used to control the assembly of G-quadruplexes through the formation of an artificial metal-centered quartet. Several guanine-rich DNA sequences containing 1,2,4-triazole-functionalized nucleotides were investigated. These oligonucleotides were designed to form quartets mediated by metal-triazole bonding both on the surface of and within the G-quadruplex core. In contrast to duplex studies in which 1,2,4-triazole nucleosides serve as a mimic of Watson-Crick base-pairs, our results show that these nucleosides are not suitable components of an artificial metal-centered quartet.

P6.08 - Cationic porphyrin-phenazine conjugates as quadruplex DNA ligands and potential agents for anticancer therapy

ABSTRACT Introduction: Telomeres are guanine-rich DNA sequences at the ends of chromosomes. Telomeric DNA forms G-quadruplexes (G4-DNA), and stabilization of these structures with small molecules can inhibit telomerase, an enzyme responsible for telomere elongation upon cellular division. It is highly active at majority of tumor cells, so G4-DNA and telomerase are considered as targets for the development of novel anticancer drugs. We present porphyrin conjugate with phenazine derivative and its metal complexes as selective G4-DNA ligands and potential antitumor agents. Materials and methods: Conjugates of tricationic porphyrin TMP3 and its Zn(II) and Mn(III) complexes with imidazo[4, 5-b]phenazine (ImPhz) intercalating agent were synthesized as previously reported [ 1 ]. Their binding affinity to G4 and dsDNA was studied using FID (Fluorescent Interacalator Displacement) method. The level of telomerase inhibition was determined by TRAP (Telomeric Repeat Amplification Protocol) in vitro assay. Antiproliferative activity of conjugates was tested on a mouse Lewis lung carcinoma cell culture. Results: All compounds show strong affinity to Tel22 G4-DNA. Dissociation constants for non-metalated conjugate, its Zn and Mn complex are 20.1, 46.3 and 3.8 µM, respectively. All ligands have a good selectivity for quadruplex over duplex DNA. The ratio between G4 and dsDNA constants is 5 for TMP3-ImPhz and 8 and 10 for its Zn and Mn complexes. High affinity of conjugates for G4-DNA is associated with a good ability to inhibit telomerase. The most efficient Zn complex completely inhibits the enzyme at 2.5 µM concentration being several times more active than non-conjugated porphyrins. Non-metalated conjugate shows inhibition effect at 5 µM, whereas Mn derivative is less active. Compounds display antiproliferative activity in vitro in micromolar range, with EC50 values for TMP3-ImPhz, its Mn and Zn complex to be 21.8, 11.2 and 5.9 µM, respectively. Conclusion: All studied compounds have strong binding affinity and selectivity for quadruplex DNA. They are efficient telomerase inhibitors and demonstrate good antiproliferative activity. Thus, these conjugates can be considered as potential agents for anticancer therapy.

DNA methyltransferase activity detection based on fluorescent silver nanocluster hairpin-shaped DNA probe with 5’-C-rich/G-rich-3’ tails

DNA methylation has received a large amount of attention due to its close relationship to a wide range of biological phenomena, such as gene activation, gene imprinting, and chromatin stability. Herein, we have designed a hairpin-shaped DNA probe with 5’-C-rich/G-rich-3’ tails and developed a simple and reliable fluorescence turn-off assay for DNA adenine methylation (Dam) methyltransferase (MTase) detection combining site recognition and the fluorescence enhancement of DNA-templated silver nanoclusters (DNA–AgNCs) by guanine-rich DNA sequences. A designed hairpin probe with 5’ CCCTTACCCC and 3’ GGGTGGGGTGGGGTGGGG displays a bright red emission after reacting with AgNO3 and NaBH4. In the presence of Dam MTase, the methylation-sensitive restriction endonuclease Dpn I which has the same recognition site with the Dam MTase can split the probe, freeing the G-rich sequence from the C-rich sequence, thus quenching the fluorescence of DNA–AgNCs. Compared to traditional fluorescent-based methods, this strategy is simple and inexpensive. A linear response to concentrations of Dam MTase which range from 1U/mL to 100U/mL and a detection limit of 1U/mL are obtained without any amplification steps. In addition, we also demonstrate the method can be used for evaluation and screening of inhibitors for Dam MTase.

A new label-free and turn-on fluorescence probe for hydrogen peroxide and glucose detection based on DNA–silver nanoclusters

A label-free and turn-on strategy for H2O2 and glucose detection based on the cleavage of ssDNA by ˙OH and the fluorescence enhancement effect when guanine-rich (G-rich) DNA sequences are in proximity to DNA–silver nanoclusters (DNA–Ag NCs).

High-Affinity Fluorescence Sensing of G-Quadruplexes

Guanine-rich DNA and RNA sequences can adopt highly polymorphic four-stranded structures, so-called G-quadruplexes (GQs), which play an important role in different biological processes such as telomere maintenance, abortive transcription and gene regulation. Their biological relevance has increased the interest in the development of probes to sense GQs and the study of these compounds as potential anticancer agents. Although a wide variety of fluorescent sensors have been reported for GQ detection so far, none is a ratiometric probe. Ratiometric probes are powerful sensors for which the relative variation between two emission peaks constitutes an absolute observable, avoiding the unwanted photobleaching and background effects.In the present work, we introduce a couple of selective sensors. A compound for ratiometric studies that undergoes excited-state intramolecular proton transfer (ESIPT) and a naturally occurring compound that lights up when bound to GQs. The interaction of both fluorophores with several GQ-forming sequences was tested using steady-state and lifetime binding assays. Single-, double- and triple-stranded DNAs were used as controls.We demonstrate that ESIPT probes can be selective and quantitative ratiometric GQ sensors, thanks to the presence of two different emission peaks with good spectral separation. Additionally, some natural probes and their derivatives can act as fluorescence light-up probes in the determination of GQs with a remarkably high affinity when compared with most of the popular sensors. Our natural compound presents most of these characteristics making it a prominent candidate to develop next generation of GQ sensors.

ATRX promotes gene expression by facilitating transcriptional elongation through guanine-rich coding regions.

ATRX is a chromatin remodeling protein involved in deposition of the histone variant H3.3 at telomeres and pericentromeric heterochromatin. It also influences the expression level of specific genes; however, deposition of H3.3 at transcribed genes is currently thought to occur independently of ATRX. We focused on a set of genes, including the autism susceptibility gene Neuroligin 4 (Nlgn4), that exhibit decreased expression in ATRX-null cells to investigate the mechanisms used by ATRX to promote gene transcription. Overall TERRA levels, as well as DNA methylation and histone modifications at ATRX target genes are not altered and thus cannot explain transcriptional dysregulation. We found that ATRX does not associate with the promoter of these genes, but rather binds within regions of the gene body corresponding to high H3.3 occupancy. These intragenic regions consist of guanine-rich DNA sequences predicted to form non-B DNA structures called G-quadruplexes during transcriptional elongation. We demonstrate that ATRX deficiency corresponds to reduced H3.3 incorporation and stalling of RNA polymerase II at these G-rich intragenic sites. These findings suggest that ATRX promotes the incorporation of histone H3.3 at particular transcribed genes and facilitates transcriptional elongation through G-rich sequences. The inability to transcribe genes such as Nlgn4 could cause deficits in neuronal connectivity and cognition associated with ATRX mutations in humans.

Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization

In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0–160.0 and 6–240nM, can be linearly detected with the detection limits of 2.1 and 3.4nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs.

Aptamer/Au nanoparticles/cobalt sulfide nanosheets biosensor for 17β-estradiol detection using a guanine-rich complementary DNA sequence for signal amplification.

We have developed a sensitive sensing platform for 17β-estradiol by combining the aptamer probe and hybridization reaction. In this assay, 2-dimensional cobalt sulfide nanosheet (CoS) was synthesized by a simple hydrothermal method with L-cysteine as sulfur donor. An electrochemical aptamer biosensor was constructed by assembling a thiol group tagged 17β-estradiol aptamer on CoS and gold nanoparticles (AuNPs) modified electrode. Methylene blue was applied as a tracer and a guanine-rich complementary DNA sequence was designed to bind with the unbound 17β-estradiol aptamer for signal amplification. The binding of guanine-rich DNA to the aptamer was inhibited when the aptamer captured 17β-estradiol. Using guanine-rich DNA in the assay greatly amplified the redox signal of methylene blue bound to the detection probe. The CoS/AuNPs film formed on the biosensor surface appeared to be a good conductor for accelerating the electron transfer. The method demonstrated a high sensitivity of detection with the dynamic concentration range spanning from 1.0×10(-9) to 1.0×10(-12) M and a detection limit of 7.0×10(-13) M. Besides, the fabricated biosensor exhibited good selectivity toward 17β-estradiol even when interferents were presented at 100-fold concentrations. Our attempt will extend the application of the CoS nanosheet and this signal amplification assay to biosensing areas.

A new label-free and turn-on strategy for endonuclease detection using a DNA–silver nanocluster probe

Endonuclease plays a vital role in a variety of biological processes and the assay of endonuclease activity and inhibitors is of high importance in the fields ranging from biotechnology to pharmacology. Howerer, traditional techniques usually suffer from time intensive, laborious, and cost-expensive. This work aims to develop a facile and sensitive method for endonuclease activity assay by making use of the fluorescence enhancement effect when DNA–silver nanoclusters (DNA–Ag NCs) are in proximity to guanine-rich DNA sequences. The system mainly consists of block DNA (B-DNA), G-DNA and Ag-DNA. B-DNA serves as the substrate of the endonuclease (S1 nuclease as the model enzyme). G-DNA, which is predesigned entirely complementary to B strand, contains a guanine-rich overhang sequence and hybridization part at the 5′-end. Ag–DNA involves a sequence for Ag NCs synthesis and a sequence complementary to the hybridization part of the G-DNA. In the “off” state, B-DNA plays the role as a blocker that inhibit the proximity between Ag NCs and guanine-rich DNA sequences, resulting in a low fluorescence readout. However, if S1 nuclease is introduced into the system, B-DNA was cleaved into mono- or short-oligonucleotides fragments, which could not hybridize with G-DNA. As a result, the subsequent addition of DNA–Ag NCs could bring guanine-rich DNA sequences close to the Ag NCs, accompanied by a significant fluorescence enhancement. Therefore, endonuclease activity could be successfully quantified by monitoring the variation in fluorescence intensity. In addition, this approach can also be applied for inhibitor screening of endonuclease. This label-free and turn-on fluorescent assays employing the mechanism proposed here for the detection of nuclease and inhibitors turn out to be sensitive, selective, and convenient.

Cover Picture: A New Pathway of DNA G‐Quadruplex Formation (Angew. Chem. Int. Ed. 19/2014)

Guanine-rich DNA sequences can self-assemble into specific pre-organized DNA structures, where two-by-two guanine residues shown as “duets” are involved in GG N1-carbonyl symmetric base pairs. In their Communication on page 4881 ff., J. Lah, J. Plavec, and co-workers demonstrate the transition of these “duets” into guanine-quartets, presented as a “string quartet” when interacting with cations, such as potassium ions. This rearrangement consequently leads to formation of G-quadruplex structures.

N‐methylmesoporphyrin IX fluorescence as a reporter of strand orientation in guanine quadruplexes

Guanine quadruplexes (GQ) are four-stranded DNA structures formed by guanine-rich DNA sequences. The formation of GQs inhibits cancer cell growth, although the detection of GQs in vivo has proven difficult, in part because of their structural diversity. The development of GQ-selective fluorescent reporters would enhance our ability to quantify the number and location of GQs, ultimately advancing biological studies of quadruplex relevance and function. N-methylmesoporphyrin IX (NMM) interacts selectively with parallel-stranded GQs; in addition, its fluorescence is sensitive to the presence of DNA, making this ligand a possible candidate for a quadruplex probe. In the present study, we investigated the effect of DNA secondary structure on NMM fluorescence. We found that NMM fluorescence increases by about 60-fold in the presence of parallel-stranded GQs and by about 40-fold in the presence of hybrid GQs. Antiparallel GQs lead to lower than 10-fold increases in NMM fluorescence. Single-stranded DNA, duplex, or i-motif, induce no change in NMM fluorescence. We conclude that NMM shows promise as a 'turn-on' fluorescent probe for detecting quadruplex structures, as well as for differentiating them on the basis of strand orientation.

The immunostimulatory effect of CpG oligodeoxynucleotides on peripheral blood mononuclear cells of healthy dogs and dogs with atopic dermatitis

Synthetic oligodeoxynucleotides containing cytosine phosphatidyl guanine-rich DNA sequences (CpG ODN) can promote T-helper type 1 (Th1) responses, reduce T-helper type 2 (Th2) responses and/or favour regulatory T cell (Treg) responses in vitro and in vivo in humans and animals, by acting via Toll-like receptor 9 (TLR9). Since CpG ODN can be used as immune-modulators for canine atopic dermatitis (AD), the aim of the current study was to investigate their immunostimulatory potential on peripheral blood mononuclear cells (PBMC) and their subsets, from AD and healthy dogs. Expression of TLR9 and cytokine mRNA in CpG ODN-stimulated and unstimulated cells was assessed by real-time quantitative PCR.Stimulation of PBMC with CpG class C ODN upregulated mRNA expression of interleukin (IL)-6, interferon (IFN)-γ and IL-12p40 in AD dogs (P<0.05). It also stimulated IFN-γ protein secretion by PBMC of atopic and healthy dogs as measured by ELISA. In healthy dogs only, CpG class C ODN stimulated IFN-α mRNA production by CD21+ cells, and IL-10, IL-13 and IFN-γ mRNA production by CD3+ cells. Increased expression of TLR9 mRNA was only observed in CD3+ cells from AD dogs. No significantly increased gene expression was found in the CD11c+ subset upon stimulation, for those genes evaluated. The results indicate that PBMC of healthy and atopic dogs are sensitive to stimulation with CpG ODN class C, with a resulting Th1 cytokine response in AD dogs and a mixed Th1/Th2/Treg cytokine response in healthy dogs. From this study, little evidence was found to support the use of CpG ODN class C for therapeutic purposes in dogs affected with AD.

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

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

G-Quadruplexes as Sensing Probes

Guanine-rich sequences of DNA are able to create tetrastranded structures known as G-quadruplexes; they are formed by the stacking of planar G-quartets composed of four guanines paired by Hoogsteen hydrogen bonding. G-quadruplexes act as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplexes form a complex with anionic porphyrin hemin and exhibit peroxidase-like activity. This review focuses on overview of sensing techniques based on G-quadruplex complexes with anionic porphyrins for detection of various analytes, including metal ions such as K+, Ca2+, Ag+, Hg2+, Cu2+, Pb2+, Sr2+, organic molecules, nucleic acids, and proteins. Principles of G-quadruplex-based detection methods involve DNA conformational change caused by the presence of analyte which leads to a decrease or an increase in peroxidase activity, fluorescence, or electrochemical signal of the used probe. The advantages of various detection techniques are also discussed.

Label-Free and Turn-on Aptamer Strategy for Cancer Cells Detection Based on a DNA–Silver Nanocluster Fluorescence upon Recognition-Induced Hybridization

We present here a label-free and turn-on aptamer strategy for cancer cell detection based on the recognition-induced conformation alteration of aptamer and hybridization-induced fluorescence enhancement effect of DNA-silver nanoclusters (DNA-Ag NCs) in proximity of guanine-rich DNA sequences. In this strategy, two tailored DNA probes were involved. One is designed as a hairpin-shaped structure consisting of a target specific aptamer sequence at the 3'-end, a guanine-rich DNA sequence, and an arm segment at the 5'-end (denote as recognition probe). The other, serving as a signal probe, contains a sequence for Ag NCs templated synthesis and a link sequence complementary to the arm segment of the recognition probe. Recognizing and binding of the aptamer to cancer cells enforces the recognition probe to undergo a conformational alteration and then initiates hybridization between the arm segment of the recognition probe and the link sequence of the signal probe. The Ag NCs are then close to the guanine-rich DNA, leading to an enhanced fluorescence readout. As proof-of-concept, the CCRF-CEM cancer cell detection were performed by using the specific aptamer, sgc8c. It was demonstrated that this strategy could specially image the CCRF-CEM cells. Determination by flow cytometry allowed for detection of as low as 150 CCRF-CEM cells in 200 μL binding buffer. The general applicability of the strategy is also achieved in the successful detection of Ramos cells. These results implied that this strategy holds considerable potential for simple, sensitive, universal, and specific cancer cell detection with no required washing and separation steps.

Adenosine Triphosphate Sensing by Electrocatalysis with DNAzyme

Electrocatalysis of redox enzymes shows wide application for biosensing. DNAzymes exhibiting specific catalytic activities have aroused great interest recently. However, there are few studies on the electrocatalysis between DNAzyme and electron mediator. In this paper, based on the electrocatalysis of methylene blue (MB) and horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme), an amplified electrochemical biosensor for the detection of adenosine triphosphate (ATP) was designed. In the present system, by means of the ATP-aptamer interaction, two guanine-rich DNA sequences, one of which was labeled with MB at the 5′ end, were assembled on the gold electrode. In the presence of K+ and hemin, the guanine-rich DNA sequences transferred to HRP-DNAzyme. The conformational change of the structure resulted in the approaching of MB and HRP-DNAzyme which made the electrocatalytic process between MB and HRP-DNAzyme possible. We used cyclic voltammetry and electrochemical impedance spectroscopy to study the electrocatalytic process. The system was therefore utilized for amplified detection of ATP without imposing any new constraints to the platform which showed satisfactory result.

A label-free DNA-templated silver nanocluster probe for fluorescence on–off detection of endonuclease activity and inhibition

Endonuclease cleavage of DNA plays an important role in biological and medicinal chemistry. This study aimed to develop a reliable and sensitive method for nuclease activity assay by combining the high specificity of DNA cleavage reactions with ultrahigh fluorescence turn-on abilities of guanine-rich (G-rich) DNA sequences in proximity to silver nanoclusters (Ag NCs). The DNA-templated Ag NC (DNA-Ag NC) probe with endonuclease recognition sequence consists of NC and a G-rich probe. The NC probe was designed by adding Ag NC nucleation sequence at the 5′-end. The G-rich probe is the complementary DNA sequence modified by adding a G-rich overhang sequence at the 3′-end. Thus, the fluorescence of DNA-Ag NC probe was activated because of DNA hybridization. When these DNA-Ag NC probes were exposed to the targeted endonucleases, specific DNA cleavages occurred, and pieces of G-rich DNA fragments separated from Ag NCs, resulting in fluorescence turn-off. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. Detection was demonstrated by assaying EcoRI activity. Under optimized conditions, the fluorescence reduction efficiency was linear with the EcoRI concentration in the range of 5.0×10−4UμL−1 to 3.0×10−3UμL−1, with a detection limit of 3.5×10−4UμL−1, which is much better than or at least comparable with that in previous reports. The potential application of the proposed method for screening endonuclease inhibitors was also demonstrated. The presented assay protocol proved to be convenient, effective, sensitive, and easy in preparing the fluorescent probe.

Electrochemical potassium ion sensor based on DNA G-quadruplex conformation and gold nanoparticle amplification

A simple, rapid, highly sensitive electrochemical sensor for potassium ion (K+) based on the conformational change of DNA sequence containing guanine-rich segments is presented. In the presence of K+, guanine-rich DNA sequence folds to G-quadruplex structure, allowing a ferrocene tag to transfer electrons to the electrode. Gold nanoparticles (AuNPs), which are self-assembled on the surface of a bare gold electrode by using 4-aminothiophenol as a medium, offer a big surface area to immobilize a large number of aptamers and improve the sensitivity of the sensor. The square-wave voltammetry peak current increases with K+ concentration. The plots of peak current against K+ concentration and the logarithm of K+ concentration are linear over the range from 0.1 to 1.0 mmol·L−1 and from 1 to 30 mmol·L−1, respectively. A lower detection limit of 0.1 mmol·L−1 K+ is obtained for AuNPs-modified sensor, which greatly surpasses that (100 mmol·L−1) of the sensor without AuNPs modification by three orders of magnitude. Thus, the sensor with AuNPs amplification is expected to open new opportunities for highly sensitive detection of other biomolecules in the future.

Excimer Formation by Stacking G‐Quadruplex Blocks

A special stack: In the presence of K+ ions, a specific guanine-rich DNA sequence forms a stacked G-quadruplex dimer giving rise to excimer emission in the 400 nm region. This emission with nanosecond lifetime occurs only for a particular base overlap pattern between two stacked G-quadruplex blocks and can be manipulated by changing DNA sequence and external conditions.

Recognition of intermolecular G-quadruplexes by full length nucleophosmin. Effect of a leukaemia-associated mutation

Nucleophosmin (NPM) is a nucleolar protein involved in ribosome biogenesis. NPM1 gene is frequently mutated in acute myeloid leukaemia (AML), correlating with aberrant cytoplasmic localization of the protein. NPM attachment to the nucleolus in physiological conditions probably depends on binding to nucleic acids, and this recognition could be altered in AML. NPM associates to guanine-rich DNA sequences, able to fold as “G-quadruplexes”. We have analyzed the interaction of pentameric, full length NPM with G-rich oligonucleotides, finding that the protein binds preferentially high-order G-quadruplexes. AML-associated mutation significantly hampers DNA binding, pointing to a possible mechanism contributing to pathological mislocalization of NPM.