Stabilization of G-quadruplex structure on vascular endothelial growth factor gene promoter depends on CpG methylation site and cation type.

BACKGROUND: DNA methylation at the 5-position of cytosine is an epigenetic modification of CpG dinucleotides. In addition to CpG methylation, the G-quadruplex (G4) structure has been reported as a regulator of gene expression. The identification of G4 forming sequences in CpG islands suggests an involvement of CpG-methylated G4 structures in biological processes; however, few reports have addressed the effects of CpG methylation on G4 structure. METHODS: The thermostability of a methylated, 21-mer G4 structure located on the vascular endothelial growth factor (VEGF) gene promoter containing four CpG sites (C1, C6, C11, and C17) were investigated using circular dichroism (CD) spectral analysis. RESULTS: CD melting analysis revealed that VEGF G4 was stabilized by a single CpG methylation on C11 in the presence of Na+ and Mg2+. However, either C1 or C11 methylation enhanced VEGF G4 thermal stability in the presence of K+. CONCLUSIONS: Single CpG methylation appears to enhance VEGF G4 thermostability in a manner dependent on both the CpG methylation site and cation type. GENERAL SIGNIFICANCE: These results are expected to contribute to the elucidation of the roles of CpG methylation-stabilized G4 structures in biological processes.

Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands.

BACKGROUND: G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay. RESULTS: When G-quadruplex-forming sequences from the Dele and Cdc6 genes have been cloned in reporter vectors carrying a minimal promoter and the luciferase gene, luciferase expression is activated. This has also been detected in experiments applying a promoterless reporter vector. Mutational analysis reveals that guanine bases, which form the G-tetrads, are important in the activation. In addition, the activation has been found to decrease by the telomestatin derivative L1H1-7OTD which can bind to the G-quadruplex DNA. When Dele and Cdc6 CpG islands, containing the G-quadruplex-forming sequence, have been cloned in the promoterless reporter vector, the luciferase expression is activated. Mutational analysis reveals that the expression level is decreased by mutation on Dele G-quadruplex; however, increased by mutation on Cdc6 G-quadruplex. CONCLUSION: Dele and Cdc6 G-quadruplex formation is significant in the transcriptional regulation. Dele and Cdc6 G-quadruplex DNA alone possess enhancer and promotor function. When studied in more complex CpG islands Dele G-quadruplex also demonstrates promotor activity, whereas Cdc6 G-quadruplex may possess a dual function of transcriptional regulation.

Global DNA Methylation Detection System Using MBD-Fused Luciferase Based on Bioluminescence Resonance Energy Transfer Assay.

DNA methylation plays an important role in the regulation of gene expression. In normal cells, transposable elements that constitute approximately 45% of the human genome are highly methylated to silence their expression. In cancer cells, transposable elements are hypomethylated; therefore, global DNA methylation level is considered as a biomarker for cancer diagnostics. In this study, a homogeneous assay for measuring global DNA methylation level based on bioluminescence resonance energy transfer (BRET) was developed using methyl-CpG binding domain (MBD)-fused luciferase. In this assay, the MBD-luciferase recognizes methylated CpG, thus, BRET between the luciferase and fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the DNA methylation level of the target DNA. Moreover, the BRET signal was correlated with the LINE1 DNA methylation level on human genomic DNA, as determined by the bisulfite method. These results indicate that the global DNA methylation level of human genomic DNA could be detected simply by measuring the BRET signal.

Detection of DNA Methylation of G-Quadruplex and i-Motif-Forming Sequences by Measuring the Initial Elongation Efficiency of Polymerase Chain Reaction.

DNA methylation has been proposed as one of the promising biomarkers for cancer diagnosis. In this study, we developed a DNA methylation detection system utilizing G-quadruplex and i-motif-forming sequences that requires neither sodium bisulfite treatment nor methylated DNA ligands. We hypothesized that G-quadruplex and i-motif structures would be stabilized by DNA methylation and arrest DNA polymerase activity during quantitative polymerase chain reaction (qPCR). The PCR products from VEGF, RET G-quadruplex, and i-motif-forming sequences were used as templates and analyzed by qPCR. Our results indicated that the initial elongation efficiency of PCR decreased with increasing DNA methylation levels in the G-quadruplex and i-motif-forming sequences. Moreover, we demonstrated that the initial elongation efficiency of PCR decreased with increased DNA methylation of the VEGF region on genomic DNA. These results indicated that DNA methylation of the G-quadruplex and i-motif-forming sequences on genomic DNA can be detected by qPCR.

Novel automated pulse immunoassay for human alpha-fetoprotein.

BACKGROUND: To improve current alpha-fetoprotein (AFP) assays, which are expensive and time-consuming, a specific AFP reagent has been developed for practical use in our newly developed high-speed, highly sensitive pulse immunoassay (PIA) system, in which a latex immunoagglutination reaction is carried out under a high-frequency pulse voltage, leading to an enhanced immunological reaction. METHODS: We evaluated the assay performance (reproducibility, sensitivity, dilution linearity, interference) of the newly developed automated AFP PIA compared with the current AFP assay. RESULTS: Using pooled serum samples, the within-run reproducibility resulted in a correlation variation of 3.6-4.7%. The AFP assay detection limit was determined to be 2.5 microg/L. Linear sequential dilution was found up to nearly 700 microg/L. Even up to an AFP concentration of 1.0 g/L, the prozone phenomenon was not observed. Free and conjugated bilirubin, haemolytic haemoglobin, chyle and rheumatoid factor did not show any test interference. Using AFP-positive serum samples from 114 patients, the correlation between our PIA and a chemiluminescence immunoassay resulted in an excellent correlation coefficient of 0.994. CONCLUSIONS: The performance of AFP reagents in the PIA device shows that the system has excellent speed and equal sensitivity and specificity compared with the most highly sensitive conventional method. Our PIA system thus appears ready for use in the clinical diagnosis setting.

Novel fluorescent probe for analysis of hydroperoxides based on boron dipyrromethane fluorophore.

A new reagent, N-[2-(diphenylphosphino)ethyl]-4-(1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-8-yl)benzamide (DPPEA-BODIPY), was designed and synthesized for analysis of hydroperoxides. DPPEA-BODIPY fluoresces at low levels in the visible range (lambda(ex)/lambda(em) = 502 nm/515 nm) and reacts with hydroperoxides to produce DPPEA-BODIPY oxide, which fluoresces at high levels. The fluorescence intensity of the reaction mixture was observed to be linearly related to the methyl linoleate hydroperoxide (MeLOOH) concentration.

Effect of the polarization and incident angle of excitation light on the fluorescence enhancement observed with a multilayered substrate fabricated by Ag and Al2O3.

The fluorescence from a fluorophore on a multilayered substrate fabricated by a metal and a dielectric is known to be enhanced by more than 100-fold. In the course of this study, we prepared a multilayered substrate with Ag as the metal and Al(2)O(3) as the dielectric and then investigated the effects of the polarization of the excitation light on the enhancement of the multilayered substrate. It was found that the enhancement was attributed to an electric field oscillating parallel to the substrate. Maximum 200-fold enhancement could be achieved with 80 nm thick Al(2)O(3) when an unpolarized excitation light was used with an incident angle of 20 degrees.

Identification of G-quadruplex clusters by high-throughput sequencing of whole-genome amplified products with a G-quadruplex ligand.

G-quadruplex (G4) is a DNA secondary structure that has been found to play regulatory roles in the genome. The identification of G4-forming sequences is important to study the specific structure-function relationships of such regions. In the present study, we developed a method for identification of G4 clusters on genomic DNA by high-throughput sequencing of genomic DNA amplified via whole-genome amplification (WGA) in the presence of a G4 ligand. The G4 ligand specifically bound to G4 structures on genomic DNA; thus, DNA polymerase was arrested on the G4 structures stabilised by G4 ligand. We utilised the telomestatin derivative L1H1-7OTD as a G4 ligand and demonstrated that the efficiency of amplification of the G4 cluster regions was lower than that of the non-G4-forming regions. By high-throughput sequencing of the WGA products, 9,651 G4 clusters were identified on human genomic DNA. Among these clusters, 3,766 G4 clusters contained at least one transcriptional start site, suggesting that genes are regulated by G4 clusters rather than by one G4 structure.

Stopped-flow system with ozonizer for the estimation of low biochemical oxygen demand in environmental samples.

The stopped-flow system with an ozonizer was developed to estimate low biochemical oxygen demand (BOD) in rivers. Rivers contain many biopersistent organic compounds such as humic acid, lignin, and gum arabic. Free radicals generated by self-decomposition of ozone were used as powerful oxidants to split organic compounds. Ozonysis of the samples was carried out by 42.4 gN(-1)m(-3) ozone for 3 min at pH 7.0. Artificial wastewater (AWW) solutions were employed as standard solutions for the calibrations of the BOD sensor. At a BOD of 1 mgl(-1), the sensor response after ozonation was 1.6-fold higher than that before ozonation. The response time of the BOD sensor was only 5min, being independent of the concentrations, and the lower detection limit was 0.5 mgl(-1) BOD. The degradations of lignin and tannic acid by ozonation were 54.1 and 42.3%, respectively. In the biosensor responses by ozonation, lignin, gum arabic, and surfactant increased by double or more compared with previous responses. BOD in rivers was estimated using the stopped-flow system. Environmental samples pretreated with ozone gave high responses to the biosensor that were similar to those of the conventional BOD(5) method. Accordingly, a good correlation between the sensor and the conventional BOD(5) was obtained (r=0.989). The system has to evolve the highly sensitive BOD determination.

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance.

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.

Development of a self-sterilizing lancet coated with a titanium dioxide photocatalytic nano-layer for self-monitoring of blood glucose.

A photocatalyst was applied to a lancet for pricking the finger to obtain an antibacterial property. A photocatalytic and uniform nano-layer of titanium dioxide (TiO2) on the surface of the lancet (0.36 mm x 24.5 mm) was formed by sputtering and annealed for crystallization of the TiO2 layer. By elementary analysis of the TiO2 layer, titanium and oxygen were detected. Next, for the estimation of the antibacterial properties resulting from the photocatalytic effect, the lancet was packed into a capillary tube filled with a suspension of Escherichia coli K-12 (non-spore-forming bacterium), and was continuously rolled in a continuous UV-irradiation system under black-light irradiation. Distinct antibacterial effects after irradiation at 0.5 mW cm(-2) for 45 min were observed in the crystallized TiO2 layer on the lancet. Finally, lancing resistances obtained by pricking an artificial skin sheet were examined using control lancets, and lancets with an unannealed TiO2 layer or an annealed TiO2 layer. The results showed almost the same lancing resistances for the control (0.53+/-0 N, n=3) and the lancet with an annealed TiO2 layer (0.51+/-0.018 N), while the lancet with an unannealed TiO2 layer showed a high lancing resistance compared with the other lancets (0.62+/-0.05 N). In conclusion, the lancet coated with a crystallized, velvety nano-layer of TiO2 obtained by annealing had antibacterial properties and a similar lancing resistance compared with the bare lancet, and showed potential for application in monitoring blood glucose in diabetes.

Development of a package-free transparent disposable biosensor chip for simultaneous measurements of blood constituents and investigation of its storage stability.

A package-free transparent disposable biosensor chip was developed by a screen-printing technique. The biosensor chip was fabricated by stacking a substrate with two carbon electrodes on its surface, a spacer consisting of a resist layer and an adhesive layer, and a cover. The structure of the chip keeps the interior of the reaction-detecting section airtight until use. The chip is equipped with double electrochemical measuring elements for the simultaneous measurement of multiple items, and the reagent layer was developed in sample-feeding path. The sample-inlet port and air-discharge port are simultaneously opened by longitudinally folding in two biosensor units with a notch as a boundary. Then the shape of the chip is changed to a V-shape. The reaction-detecting section of the chip has a 1.0 microl sample volume for one biosensor unit. Excellent results were obtained with the chip in initial simultaneous chronoamperometric measurements of both glucose (r=1.00) and lactate (r=0.998) in the same samples. The stability of the enzyme sensor signals of the chip was estimated at ambient atmosphere on 8 testing days during a 6-month period. The results were compared with those obtained for an unpackaged chip used as a control. The package-free chip proved to be twice as good as the control chip in terms of the reproducibility of slopes from 16 calibration curves (one calibration curve: 0, 100, 300, 500 mg dl(-1) glucose; n=3) and 4.6 times better in terms of the reproducibility of correlation coefficients from the 16 calibration curves.

A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm.

Thrombin-inhibiting DNA aptamers have already been obtained through the systematic evolution of ligands by exponential enrichment (SELEX). However, SELEX is a method that screens DNA aptamers that bind to their target molecules, and it sometimes fails to screen good inhibitors. Therefore, it is necessary to develop a method of screening DNA aptamers based on their inhibitory effects on the target molecules. We developed a novel method of detecting aptamers using an evolution-mimicking algorithm, and we applied it to the search of new aptamers which inhibit thrombin. First, we randomly designed and synthesized ten 15mer oligonucleotides presumed to form G-quartet structures, and then measured their thrombin-inhibiting activities. The aptamers showing high inhibitory activity were selected, and we shuffled and mutated those sequences in silico to generate 10 new sequences of next-generation aptamers. After repeating the cycle five times, we successfully obtained the same aptamers reported previously, and they showed high inhibitory activity. In addition, we added 8mer oligonucleotides to both the 5' and the 3' end of the selected 15mer aptamers, and then repeated the evolution in silico. After two cycles, we were able to obtain aptamers with higher inhibitory activity than that of the 15mer aptamers.

Improving the efficiency of evolutionary de novo peptide design: strategies for probing configuration and parameter settings.

Evolutionary molecular design based on genetic algorithms (GAs) has been demonstrated to be a flexible and efficient optimization approach with potential for locating global optima. Its efficacy and efficiency are largely dependent on the operations and control parameters of the GAs. Accordingly, we have explored new operations and probed good parameter setting through simulations. The findings have been evaluated in a helical peptide design according to "Parameter setting by analogy" strategy; highly helical peptides have been successfully obtained with a population of only 16 peptides and 5 iterative cycles. The results indicate that new operations such as multi-step crossover-mutation are able to improve the explorative efficiency and to reduce the sensitivity to crossover and mutation rates (CR-MR). The efficiency of the peptide design has been furthermore improved by setting the GAs at the good CR-MR setting determined through simulation. These results suggest that probing the operations and parameter settings through simulation in combination with "Parameter setting by analogy" strategy provides an effective framework for improving the efficiency of the approach. Consequently, we conclude that this framework will be useful for contributing to practical peptide design, and gaining a better understanding of evolutionary molecular design.

A quantitative homogeneous assay for global DNA methylation levels using CpG-binding domain- and methyl-CpG-binding domain-fused luciferase.

Global DNA methylation levels have been considered as biomarkers for cancer diagnostics because transposable elements that constitute approximately 45% of the human genome are hypomethylated in cancer cells. We have previously reported a homogeneous assay for measuring methylated CpG content of genomic DNA based on bioluminescence resonance energy transfer (BRET) using methyl-CpG-binding domain (MBD)-fused luciferase (MBD-luciferase). In this study, a homogeneous assay for measuring unmethylated CpG content of genomic DNA in the same platform was developed using CXXC domain-fused luciferase (CXXC-luciferase) that specifically recognizes unmethylated CpG. In this assay, CXXC-luciferase recognizes unmethylated CpG on genomic DNA, whereby BRET between luciferase and the fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the genomic DNA concentration (R2 = 0.99) and unmethylated CpG content determined by the bisulfite method (R2 = 0.97). There was a significant negative correlation between the BRET signal of the CXXC-luciferase-based assay and that of the MBD-luciferase-based assay (R2 = 0.92). Moreover, we demonstrated that the global DNA methylation level determined using the bisulfite method was dependent on the ratio of the BRET signal in the MBD-luciferase-based assay to the total BRET signal in the MBD-luciferase- and CXXC-luciferase-based assays (R2 = 0.99, relative standard deviation < 2.2%, and analysis speed < 35 min). These results demonstrated that global DNA methylation levels can be quantified by calculating the BRET signal ratio without any calibration curve.

Development of photocatalytic biosensor for the evaluation of biochemical oxygen demand.

The photocatalytic biosensor of flow system using semiconductor TiO2 was developed to evaluate biochemical oxygen demand (BOD) levels in river water. Photocatalysis of sample was carried out in a photoreactor with TiO2 and a 6W black-light blue fluorescent tube as light source. Sample from a photoreactor outlet was measured by an oxygen electrode with a biofilm. The sensor response of photocatalytic biosensor was between 5 and 10 min depending on concentration of biochemical in the samples. At BOD of 1 mgl-1, the sensor response increased 1.33-fold in comparison with that without photocatalysis. The degradation of tannic acid and humic acid with photocatalysis were 51.8 and 38.4%, respectively. Gum arabic and linear alkylbenzene sulfonate (LAS) were degraded a little, but gave the responses of more than double to the sensor. Free radicals yielded by photocatalysis in a photoreactor did not affect the sensor response because their lifetime is extremely short. Fairly good correlation (r=0.983) between the sensor method and the conventional method was obtained for test samples. This biosensor using photocatalytic pretreatment improved the sensitivity.

Electron transfer mediator micro-biosensor fabrication by organic plasma process.

We propose a new strategy for constructing a mediator-type biosensor as a Bio-MicroElectroMechanical Systems (BioMEMS) application. A vinylferrocene plasma-polymerized film (PPF) was deposited directly onto the surface of an electrode under dry conditions. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly crosslinked network structure. This technique, capable of polymeric deposition of any kind of monomer, can also serve the purpose of anti-fouling coating, or layer-to-layer interface creation. With a subsequent plasma process, additional polymeric layer of hydrophilic acetonitrile was superimposed onto the existing vinylferrocene-PPF surface to offer crucial features such that the wettability could be adjusted for a better electron transfer, and amino functional groups could be attached to immobilize a large amount of enzyme. Based upon this scheme, the device fabrication could be designed in a manner that the whole procedure was made up of dry wafer-handling processes, which is compatible with mass production. A prototype device was fabricated to have an array of needle-shaped amperometric micro-biosensors. The resultant thin polymer layer carried a large number of the mediator molecules, accomplishing a lower overpotential (+410 mV) and a rapid response time (<5s). Stressing the advantages of the plasma polymerization process together with some additional features accomplished in our device fabrication, we would discuss new possibilities in the field of BioMEMS.

Exploration of structural features of monomeric helical peptides designed with a genetic algorithm.

A genetic algorithm (GA)-based strategy to dissect the determinants of peptide folding into alpha-helix was developed. The structural information of helical peptides was obtained with respect to patterns of sequence variability. In many previously reported studies the intrinsic alpha-helical propensities of amino acids although sequence-dependent are apparently independent of the amino acid position. In this research, monomeric helical peptides selected from possible sequences produced by a GA-chemical synthesis were analyzed to identify possible influential structural features. These hexadeca-peptides were obtained after four successive generations. A total of 128 synthetic peptides were evaluated via circular dichroism (CD) measurements in aqueous solution, while the mean ellipticity at 222 nm confirmed the monomeric state of the peptides. The results presented here show that our GA-based strategy may be useful in the design of proteins with increased alpha-helix content.

Rapid hybridization at high salt concentration and detection of bacterial DNA using fluorescence polarization.

The effects of NaCl concentration and temperature on the rate of hybridization of complementary single-stranded DNA (24-mers) were investigated. The single label of fluorescein was used for the probe DNA. The time courses of fluorescence polarization for the probe DNA were monitored. It was shown that detection of a specific DNA sequence (24-mer) was possible in less than 10 min using fluorescence polarization under the optimized conditions of 0.8 M NaCl at 46 degrees C in TE buffer. The effects of base-pair mismatches on DNA hybridization in the presence of NaCl or MgCl(2) were also investigated, and the specificity was considered by comparing the hybridization rate of the fluorescein-labeled probe. Determination of a specific DNA sequence was also possible in TE buffer containing 0.2 M MgCl(2). Moreover, in the presence of 0.2 M MgCl(2), there were no undesirable effects on hybridization and the presence of a single base pair mismatch could be identified. Rapid and specific determination of the DNA of enterohemorrhagic Escherichia coli, methicillin resistant Staphylococcus aureus and Legionella pneumophila, which had been multiplied by the asymmetric PCR, was performed under the optimized conditions for hybridization. It was confirmed that the conditions were also applicable to the hybridization between the probes and the amplified products of the actual bacterial genes. The combination of fluorescence polarization with the asymmetric PCR was quite effective. Moreover, the nested and asymmetric PCR product of bacterial gene could be detected effectively. The DNA detection method could also be used even if the specificity of the DNA amplification was not perfect and some unexpected bands were mixed with the target band during electrophoresis.