Biotinylated cyclic naphthalene diimide as a searching tool for G4 sites on the genome.

A biotinyl cyclic naphthalene diimide (biotinyl cNDI) (1), in which biotin is introduced on the cyclic linker chain of cNDI with high G-quadruplex (G4) specificity, was synthesized. 1 was used for binding analysis to G4 DNAs such as c-myc, c-kit, CEGF, or TA-core. The results showed that 1 bind to G4 DNAs with high affinity and, especially, two molecules of 1 bind to c-myc DNA from top and bottom of G4 site at K = 3.9 × 10-6 M-1 without changing the G4 structure. As a pulldown assay, 1 and streptavidin magnetic beads could be used to recover a c-myc DNA or 120-mer DNA fragment having single c-myc sequence. The qPCR results for the 120-meric DNAs showed that more than 50% of genomic DNA fragments could be recovered by this pulldown assay. The results obtained here might allow the recovery of G4-containing DNA fragments from genomic DNA to analyze the true G4 present in the genome.

Cyclic anthraquinone derivatives, unique G-quadruplex binders, selectively induce cancer cell apoptosis and inhibit tumor growth.

Cyclic anthraquinone derivatives (cAQs), which link two side chains of 1,5-disubstituted anthraquinone as a threading DNA intercalator, have been developed as G-quartet (G4) DNA-specific ligands. Among the cAQs, cAQ-mBen linked through the 1,3-position of benzene had the strongest affinity for G4 recognition and stabilization in vitro and was confirmed to bind to the G4 structure in vivo, selectively inhibiting cancer cell proliferation in correlation with telomerase expression levels and triggering cell apoptosis. RNA-sequencing analysis further indicated that differentially expressed genes regulated by cAQ-mBen were profiled with more potential quadruplex-forming sequences. In the treatment of the tumor-bearing mouse model, cAQ-mBen could effectively reduce tumor tissue and had less adverse effects on healthy tissue. These results suggest that cAQ-mBen can be a potential cancer therapeutic agent as a G4 binder.

Fluorescence Imaging of Extracellular Potassium Ion Using Potassium Sensing Oligonucleotide.

Potassium-sensing oligonucleotide, PSO, a conjugate of a quadruplex structure-forming oligonucleotide with a peptide incorporating a Förster Resonance Energy Transfer (FRET) chromophore pair, has been developed for fluorescent detection of potassium ion (K+) in aqueous medium. PSO 1 could be introduced into cells for real-time imaging of cytoplasmic K+ concentrations. To perform fluorescent imaging of K+ on the cell surface, we synthesized twelve PSO derivatives with different types of peptide types and lengths, and oligonucleotide sequences including thrombin-binding aptamer (TBA) sequences with FAM and TAMRA as a FRET chromophore pair, and evaluated their performance. 1 was shown to respond selectively to K+, not to most ions present in vivo, and to show reciprocal fluorescence changes in response to K+ concentration. For the peptide chains and oligonucleotide sequences examined in this study, the PSO derivatives had K d values for K+ in the range of 5-30 mM. All PSO derivatives showed high K+ selectivity even in the presence of excess Na+. The PSO derivatives were successfully localized to the cell surface by biotinylated concanavalin A (ConA) or sulfo-NHS-biotin via streptavidin (StAv). Fluorescence imaging of extracellular K+ upon addition of apoptosis inducers was successfully achieved by 1 localized to the cell surface.

Naphthalene Diimides Carrying Two ß-Cyclodextrins Prefer Telomere RNA G-Quadruplex Recognition.

Newly synthesized naphthalene diimide carrying two ß-cyclodextrins (NDI-ß-CyDs) showed improved specificity for the parallel G-quadruplex structure alongside the hybrid G-quadruplex structure. Specifically, the highest binding affinity of NDI-ß-CyDs for the telomere RNA G-quadruplex was observed. The binding simulation indicated that ß-cyclodextrins might be available for loop nucleobase inclusion under its complex.

Replication Control of Human Telomere G-Quadruplex DNA by G-Quadruplex Ligands Dependent on Solution Environment.

The human telomere region is known to contain guanine-rich repeats and form a guanine-quadruplex (G4) structure. As telomeres play a role in the regulation of cancer progression, ligands that specifically bind and stabilize G4 have potential therapeutic applications. However, as the human telomere sequence can form G4 with various topologies due to direct interaction by ligands and indirect interaction by the solution environment, it is of great interest to study the topology-dependent control of replication by ligands. In the present study, a DNA replication assay of a template with a human telomere G4 sequence in the presence of various ligands was performed. Cyclic naphthalene diimides (cNDI1 and cNDI2) efficiently increased the replication stall of the template DNA at G4 with an anti-parallel topology. This inhibition was stability-dependent and topology-selective, as the replication of templates with hybrid or parallel G4 structures was not affected by the cNDI and cNDI2. Moreover, the G4 ligand fisetin repressed replication with selectivity for anti-parallel and hybrid G4 structures without stabilization. Finally, the method used, referred to as quantitative study of topology-dependent replication (QSTR), was adopted to evaluate the correlation between the replication kinetics and the stability of G4. Compared to previous results obtained using a modified human telomere sequence, the relationship between the stability of G4 and the effect on the topology-dependent replication varied. Our results suggest that native human telomere G4 is more flexible than the modified sequence for interacting with ligands. These findings indicate that the modification of the human telomeric sequence forces G4 to rigidly form a specific structure of G4, which can restrict the change in topology-dependent replication by some ligands.

Cyclic ferrocenylnaphthalene diimides as a probe for electrochemical telomerase assay.

Novel cyclic naphthalene diimides, 8 and 12, containing ferrocene in the cyclic linker were synthesized as G-quartet (G4) specific electrochemical ligands via the reaction of 1,1'-ferrocenedipropanoic acid and the terminal amine moieties of naphthalene diimides with varying linker lengths. The redox potentials of 8 and 12 were ca. 0.2 V (vs. Ag/AgCl), and the background current in an electrolyte was successfully suppressed. Both 8 and 12 bound to TA-core, representing human telomere G4, with K = 4.4 and 38 × 105 M-1, respectively. The current response of 12 to an electrode immobilized with G4 was the highest among the acyclic derivatives, suggesting its potential application in electrochemical telomerase assays.

Chemical Modulation of DNA Replication along G-Quadruplex Based on Topology-Dependent Ligand Binding.

Ligands that bind to and stabilize guanine-quadruplex (G4) structures to regulate DNA replication have therapeutic potential for cancer and neurodegenerative diseases. Because there are several G4 topologies, ligands that bind to their specific types may have the ability to preferentially regulate the replication of only certain genes. Here, we demonstrated that binding ligands stalled the replication of template DNA at G4, depending on different topologies. For example, naphthalene diimide derivatives bound to the G-quartet of G4 with an additional interaction between the ligand and the loop region of a hybrid G4 type from human telomeres, which efficiently repressed the replication of the G4. Thus, these inhibitory effects were not only stability-dependent but also topology-selective based on the manner in which G4 structures interacted with G4 ligands. Our original method, referred to as a quantitative study of topology-dependent replication (QSTR), was developed to evaluate correlations between replication rate and G4 stability. QSTR enabled the systematic categorization of ligands based on topology-dependent binding. It also demonstrated accuracy in determining quantitatively how G4 ligands control the intermediate state of replication and the kinetics of G4 unwinding. Hence, the QSTR index would facilitate the design of new drugs capable of controlling the topology-dependent regulation of gene expression.

Substituent effects of cyclic naphthalene diimide on G-quadruplex binding and the inhibition of cancer cell growth.

Interaction of cyclic naphthalene diimide derivatives (cNDIs), 1-4, with TA-core and c-myc as G-quartet (G4) DNA was studied under dilute or molecular crowding condition. Binding study for TA-core based on an isothermal titration calorimetry showed that 1-4 has 106 M-1 order of binding affinity with the following order: 1 > 4 > 2 > 3 under both conditions. Meting temperature (Tm) of TA-core obtained from the temperature dependence of circular dichroism spectra shows that TA-core was most stabilized by 4, which is in agreement with the result of PCR stop assay and the stabilization effect for 1-3 was correlated with their binding affinity under dilute condition. 3 showed specific growth inhibition of cancer cell line Ca9-22 at <0.03 µM of IC50, with no inhibitory effect against normal bone marrow cells. 3, which has highest value of ΔH/ΔG, shows the highest inhibition ability for Ca9-22, carrying a highest expression level of telomerase mRNA.

Detection of Tetraplex DNA and Detection by Tetraplex DNA.

G-quardruplex (G4) DNA forms through the gathering together of G-quartet planes formed with four guanine (G) bases. G4 DNA stabilizes with potassium ions (K+) by coordination with the G-quartet center. Fluorometric G4 DNA carrying the fluorescence resonance energy transfer (FRET) chromophore pair at both termini has been applied for the fluorometric sensing or imaging of K+ under a homogeneous aqueous medium. This system has realized non-conventional K+ selectivity over the sodium ion (Na+). The selectivity of the fluorescence G4 was converted to Na+ from K+ with a modification of its sequence. On the other hand, G4 DNA detection has been achieved in terms of cancer diagnosis because of a strong relationship of G4 DNA and cancer development. Ligands interacting with G4 are expected to have anti-cancer potential. In addition, fluorometric G4 ligands have been developed and tested as tools for the dynamic monitoring of G4 in living cells. Moreover, fluorometric G4 DNA has been utilized to evaluate the G4 ligand performance.

Exploring Providencia rettgeri for application to eco-friendly paper based microbial fuel cell.

We report results of the studies relating to improved stability (40 days) of small sized microbial fuel cell (MFC) fabricated using agarose embedded paper-based proton exchange membrane. A fermentative bacterium Providencia rettgeri was isolated from rotten potato slurry and identified by 16S rRNA sequencing. The electroactivity of the bacteria was monitored via chronoamperometric and cyclic voltammetric studies using a three-electrode system which indicated the presence of bacterial redox mediator. The Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) and UV-Vis absorption spectroscopy provided the evidence that Providencia rettgeri synthesized folate (vitamin B9) during fermentation that was found to act for the first time as a redox mediator in an MFC. The paper based designed MFC fed with Providencia rettgeri yielded open circuit voltage of 787.9 mV with power and current density of 5.02 W/m3 and 11.26 A/m3, respectively when measured across 10 kΩ. The microbial re-chargeable battery comprising of an assembly of parallelly aligned four units of MFCs when connected in series (total 16 MFCs), generated 1.5 V that was used for powering a red-light emitting device (LED).

The Interaction of Cyclic Naphthalene Diimide with G-Quadruplex under Molecular Crowding Condition.

G-quadruplex specific targeting molecules, also termed as G4 ligands, are attracting increasing attention for their ability to recognize and stabilize G-quadruplex and high potentiality for biological regulation. However, G4 ligands recognizing G-quadruplex were generally investigated within a dilute condition, which might be interfered with under a cellular crowding environment. Here, we designed and synthesized several new cyclic naphthalene diimide (cNDI) derivatives, and investigated their interaction with G-quadruplex under molecular crowding condition (40% v/v polyethylene glycol (PEG)200) to mimic the cellular condition. The results indicated that, under molecular crowding conditions, cNDI derivatives were still able to recognize and stabilize G-quadruplex structures based on circular dichroism measurement. The binding affinities were slightly decreased but still comparatively high upon determination by isothermal titration calorimetry and UV-vis absorbance spectroscopy. More interestingly, cNDI derivatives were observed with preference to induce a telomere sequence to form a hybrid G-quadruplex under cation-deficient molecular crowding conditions.

Electrochemical sensory detection of Sus scrofa mtDNA for food adulteration using hybrid ferrocenylnaphthalene diimide intercalator as a hybridization indicator.

In this study, an electrochemical DNA biosensor was developed based on the fabrication of silicon nanowires/platinum nanoparticles (SiNWs/PtNPs) on a screen-printed carbon electrode (SPCE) for the detection of Sus scrofa mitochondrial DNA (mtDNA) in food utilizing a new hybrid indicator, ferrocenylnaphthalene diimide (FND). The morphology and elemental composition of the SiNWs/PtNPs-modified SPCE was analyzed by field emission scanning electron microscopy (FESEM) combined with energy dispersive X-ray spectroscopy (EDX). Cyclic voltammetry (CV) was used to study the electrical contact between the PtNPs and the screen-printed working electrode through SiNWs, while electrochemical impedance spectroscopy (EIS) was used to measure the charge transfer resistance of the modified electrode. The results clearly showed that the SiNWs/PtNPs were successfully coated onto the electrode and the effective surface area for the SiNWs/PtNPs-modified SPCE was increased 16.8 times as compared with that of the bare SPCE. Differential pulse voltammetry used for the detection of porcine DNA with FND as an intercalator confirmed its specific binding to the double-stranded DNA (dsDNA) sequences. The developed biosensor showed a selective response towards complementary target DNA and was able to distinguish non-complementary and mismatched DNA oligonucleotides. The SiNWs/PtNPs-modified SPCE that was fortified with DNA hybridization demonstrated good linearity in the range of 3 × 10-9 M to 3 × 10-5 M (R 2 = 0.96) with a detection limit of 2.4 × 10-9 M. A cross-reactivity study against various types of meat and processed food showed good reliability for porcine samples.

Cyclic Naphthalene Diimide with a Ferrocene Moiety as a Redox-Active Tetraplex-DNA Ligand.

Cyclic naphthalene diimides (cNDIs), with a ferrocene moiety (cFNDs) and different linker lengths between the ferrocene and cNDI moieties, were designed and synthesized as redox-active, tetraplex-DNA ligands. Intramolecular stacking was observed between ferrocene and the NDI planes, which could affect the binding properties for G-quadruplexes. Interestingly, the circular dichroism spectrum of one of these compounds clearly shows new Cotton effects around 320-380 and 240 nm, which can be considered a direct evidence of intramolecular stacking of ferrocene and the NDI. Regarding recognition of hybrid G-quadruplexes, the less rigid structures (longer linkers) show higher binding affinity (106 m-1 order of magnitude). All new compounds show higher selectivity for G4 during electrochemical detection than noncyclic FND derivatives, which further identifies the redox-active potentiality of the cFNDs. Two of the three compounds tested even show preferential inhibition of cell growth in cancer cells over normal cells in a low concentration range, highlighting the potential for bioapplications of these cFNDs.

Cyclic Naphthalene Diimide Dimer with a Strengthened Ability to Stabilize Dimeric G-Quadruplex.

A new type of dimeric cyclic naphthalene diimide derivatives (cNDI-dimers) carrying varied linker length were designed and synthesized to recognize dimeric G-quadruplex structures. All of the cNDI-dimers exhibited a high preference for recognizing G-quadruplex structures, and significantly enhanced the thermal stability of the dimeric G-quadruplex structure over the cNDI monomer by increasing the melting temperature by more than 23 °C, which indicated the strengthened ability of cNDI dimers for stabilizing dimeric G-quadruplex. cNDI dimers also showed a stronger ability to inhibit telomerase activity and stop telomere DNA elongation than cNDI monomer, which showed an improved anticancer potentiality for further therapeutic application.

Synthesis of a Peptide-Human Telomere DNA Conjugate as a Fluorometric Imaging Reagent for Biological Sodium Ion.

A peptide-oligonucleotide conjugate (1) was synthesized by the attachment of FAM, TAMRA, and biotin moieties to a telomere DNA sequence of 5'-TAG GGT TAG GGT TAG GGT TAG GG-3'. This conjugate was induced to be an anti-parallel structure in the presence of sodium ion (Na+), whereas a hybrid one was formed under potassium ion (K+) as a monitoring by circular dichromic spectra. The conformation change of this conjugate gave an effective FRET signal change upon the addition of NaCl, compared with the case of KCl. Under 5 mM KCl as an extracellular condition, a FRET change was observed upon addition of NaCl and quantitative FRET change was observed in 0 - 250 mM NaCl. This conjugate was immobilized on the cell surface through a sugar chain on the cell, biotinyl concanavallin A and streptavidin. This conjugate was utilized for Na+ sensing based on anti-parallel tetraplex formation with Na+.

Membrane-Based Microwave-Mediated Electrochemical Immunoassay for the In Vitro, Highly Sensitive Detection of Osteoporosis-Related Biomarkers.

Highly sensitive and multiplexed in vitro detection of osteoporosis-related biochemical markers were carried out based on the membrane-based microwave-mediated electrochemical immunoassay (MMeEIA), where we can dramatically reduce the sample preparation time by shortening the incubation time of conjugation to obtain sensitive detection based on three dimensional conjugation of antibodies with target antigens in nylon membrane disk. C-terminal cross-linked telopeptide of type I collagen (CTx), Osteocalcin (OC), parathyroid hormone (PTH), and N-terminal propeptide of type I collagen (P1NP), which can be utilized to monitor the progress of osteoporosis, were quantified using their corresponding antibody immobilized in membranes. Coefficient of variations in this intra- and inter-assays were within 8.0% for all markers. When compared with data obtained from clinically used standard equipment (Roche modular E170), their coefficients of determination, R² values, are mostly more than 0.9. They show that the results obtained from MMeEIA are in good agreement with that from the conventional clinical instruments.

Cyclic perylene diimide: Selective ligand for tetraplex DNA binding over double stranded DNA.

Synthesized cyclic perylene diimide, cPDI, showed the binding constant of 6.3 × 106 M-1 with binding number of n = 2 with TA-core as a tetraplex DNA in 50 mM Tris-HCl buffer (pH = 7.4) containing 100 mM KCl using Schatchard analysis and showed a higher preference for tetraplex DNA than for double stranded DNA with over 103 times. CD spectra showed that TA-core induced its antiparallel conformation upon addition of cPDI in the absence or presence of K+ or Na+ ions. The cPDI inhibits the telomerase activity with IC50 of 0.3 µM using TRAP assay which is potential anti-cancer drug with low side effect.

The methylation status and expression of human telomerase reverse transcriptase is significantly high in oral carcinogenesis.

Telomerase activity is present in most cancers and is tightly regulated by the expression of human telomerase reverse transcriptase (hTERT). Hypermethylation in the promoter region of hTERT contributes to the regulation of hTERT expression. In this study, we investigated the methylation and expression of hTERT in oral squamous cell carcinoma (OSCC), oral leukoplakia, and normal oral mucosa. Furthermore, we investigated the significance of hTERT to the clinicopathological findings of OSCC. 35 OSCC, 50 oral leukoplakia (epithelial dysplasia n = 25, squamous cell hyperplasia n = 25), and 10 normal oral mucosa samples were investigated through methylation-specific PCR. Immunohistochemistry was analyzed in 35 OSCC, 50 oral leukoplakia, and 4 normal oral mucosa samples. The methylation and expression of hTERT increased from normal oral mucosa to oral leukoplakia to OSCC. In OSCC, all samples were methylated. However, partial methylation (20%) or unmethylation (80%), but never complete methylation, was observed in normal oral mucosa. Additionally, hTERT expression correlated with cervical lymph node metastasis. These results suggested that the methylation and expression of hTERT is high in oral carcinogenesis and may play an important role in oral cancer. hTERT expression may also be predictive of cervical lymph node metastasis.

Cyclic ferrocenylnaphthalene diimide derivative as a new class of G-quadruplex DNA binding ligand.

To identify an effective ligand that binds to a G-quadruplex structure but not a double-stranded DNA (dsDNA), a set of biophysical and biochemical experiments were carried out using newly synthesized cyclic ferrocenylnaphthalene diimide (cFNDI, 1) or the non-cyclic derivative (2) with various structures of G-quadruplex DNAs and dsDNA. Compound 1 bound strongly to G-quadruplexes DNAs (106M-1 order) with diminished binding to dsDNA (104M-1 order) in 100mM AcOH-AcOK buffer (pH 5.5) containing 100mM KCl. Interestingly, 1 showed an approximately 50-fold higher selectivity to mixed hybrid-type telomeric G-quadruplex DNA (K=3.4×106M-1 and a 2:1 stoichiometry) than dsDNA (K=7.5×104M-1) did. Furthermore, 1 showed higher thermal stability to G-quadruplex DNAs than it did to dsDNA with a preference for c-kit and c-myc G-quadruplex DNAs over telomeric and thrombin binding aptamers. Additionally, 1 exhibited telomerase inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.4µM. Compound 2 showed a preference for G-quadruplex; however, the binding affinity magnitude and preference were improved in 1 because the former had a cyclic structure.