On-Demand Ligand-Base DNA Sensor with Electrochemical Impedance Spectroscopy.

We have developed a DNA sensor that can be finalized to detect a specific target on demand. The electrode surface was modified with 2,7-diamino-1,8-naphthyridine (DANP), a small molecule with nanomolar affinity for the cytosine bulge structure. The electrode was immersed in a solution of synthetic probe-DNA that had a cytosine bulge structure at one end and a complementary sequence to the target DNA at the other end. The strong binding between the cytosine bulge and DANP anchored the probe DNAs to the electrode surface, and the electrode became ready for target DNA sensing. The complementary sequence portion of the probe DNA can be changed as requested, allowing for the detection of a wide variety of targets. Electrochemical impedance spectroscopy (EIS) with the modified electrode detected target DNAs with a high sensitivity. The charge transfer resistance (Rct) extracted from EIS showed a logarithmic relationship with the concentration of target DNA. The limit of detection (LoD) was less than 0.01 µM. By this method, highly sensitive DNA sensors for various target sequences could be easily produced.

Ameliorating Role of Hydrogen-Rich Water Against NSAID-Induced Enteropathy via Reduction of ROS and Production of Short-Chain Fatty Acids.

BACKGROUND: Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy, the mechanism of which is involved in oxidative stress, can be lethal due to hemorrhage. Thus, we aimed to investigate the effect of hydrogen-rich water (HRW), in terms of oxidative stress, on intestinal mucosal damage as well as changes in the gut microbiome and the short-chain fatty acids (SCFAs) content in feces. METHODS: Hydrogen-rich water was orally administered for 5 days to investigate the effectiveness of indomethacin-induced enteropathy in mice. Small intestinal damage and luminal reactive oxygen species (ROS) were evaluated to investigate the ameliorating effects of hydrogen. Then, components of the gut microbiome were analyzed; fecal microbiota transplantation (FMT) was performed using the cecal contents obtained from mice drinking HRW. The cecal contents were analyzed for the SCFAs content. Finally, cells from the macrophage cell line RAW264 were co-cultured with the supernatants of cecal contents. RESULTS: Hydrogen-rich water significantly ameliorated IND-induced enteropathy histologically and reduced the expression of IND-induced inflammatory cytokines. Microscopic evaluation revealed that luminal ROS was significantly reduced and that HRW did not change the gut microbiota; however, FMT from HRW-treated animals ameliorated IND-induced enteropathy. The SCFA content in the cecal contents of HRW-treated animals was significantly higher than that in control animals. The supernatant had significantly increased interleukin-10 expression in RAW264 cells in vitro. CONCLUSION: Hydrogen-rich water ameliorated NSAID-induced enteropathy, not only via direct antioxidant effects but also via anti-inflammatory effects by increasing luminal SCFAs. These results suggest that hydrogen may have therapeutic potential in small intestinal inflammatory diseases.

Electrochemical Impedimetric Real-Time Polymerase Chain Reactions Using Anomalous Charge Transfer Enhancement.

We developed a new electrochemical impedimetric method for the real-time detection of polymerase chain reactions (PCR) based on our recent discovery that the DNA intercalator, [Ru(bpy)2DPPZ]2+, anomalously enhances charge transfer between redox mediators, K4[Fe(CN)6]/K3[Fe(CN)6], and a carbon electrode. Three mM [Fe(CN)6]3-/4- and 5 µM [Ru(bpy)2DPPZ]2+ were added to the PCR solution, and electrochemical impedance spectroscopy (EIS) measurements were performed at each elongation heat cycle. The charge transfer resistance (Rct) was initially low due to the presence of [Ru(bpy)2DPPZ]2+ in the solution. As PCR progressed, amplicon dsDNA was produced exponentially, and intercalated [Ru(bpy)2DPPZ]2+ ions, which could be detected as a steep Rct, increased at specific heat cycles depending on the amount of template DNA. The Rct increase per heat cycle, ΔRct, showed a peak at the same heat cycle as optical detection, proving that PCR can be accurately monitored in real time by impedance measurement. This simple method will enable a cost-effective and portable PCR device.

Protective Effect of Luminal Uric Acid Against Indomethacin-Induced Enteropathy: Role of Antioxidant Effect and Gut Microbiota.

BACKGROUND: Uric acid (UA) has anti- and pro-inflammatory properties. We previously revealed that elevated serum UA levels provide protection against murine small intestinal injury probably via luminal UA secreted in the small intestine. Luminal UA may act as an antioxidant, preventing microbiota vulnerability to oxidative stress. However, whether luminal UA is increased under hyperuricemia and plays a protective role in a dose-dependent manner as well as the mechanism by which luminal UA exerts its protective effects on enteropathy remains unknown. METHODS: Inosinic acid (IMP) (1000 mg/kg, i.p.) was administered to obtain high serum UA (HUA) and moderate serum UA (500 mg/kg IMP, i.p.) mice. UA concentrations and levels of oxidative stress markers in the serum and intestine were measured. Mice received indomethacin (20 mg/kg, i.p.) to evaluate the effects of UA on indomethacin-induced enteropathy. Reactive oxygen species (ROS) on the ileal mucosa were analyzed. The fecal microbiota of HUA mice was transplanted to investigate its effect on indomethacin-induced enteropathy. RESULTS: IMP increased luminal UA dose-dependently, with higher levels of luminal antioxidant markers. Indomethacin-induced enteropathy was significantly ameliorated in both UA-elevated groups, with decreased indomethacin-induced luminal ROS. The microbiota of HUA mice showed a significant increase in α-diversity and a significant difference in ß-diversity from the control. Fecal microbiota transplantation from HUA mice ameliorated indomethacin-induced enteropathy. CONCLUSIONS: The protective role of luminal UA in intestinal injury is likely exerted via oxidative stress elimination and microbiota composition modulation, preferably for gut immunity. Therefore, enhancing anaerobic conditions using antioxidants is a potential therapeutic target.

High allele discrimination in the typing of single nucleotide polymorphisms of miRNA.

MicroRNAs (miRNAs) belonging to the same family have similar sequences and are difficult to identify. Herein, we report the reverse transcription-hairpin-probe-polymerase chain reaction (RT-Hpro-PCR) technique, which utilises a reverse transcription (RT) primer containing a 5'-end deoxyribonucleic acid (DNA) tag, to detect miRNAs with similar sequences. This strategy follows a two-step RT-PCR method using 6-7-mer RT-primers with a ~ 10-mer tag sequence at the 5'-end and a probe with a hairpin structure (Hpro), including two C-bulges, attached. The findings demonstrate that the specificity of RT could be increased by shortening the complementary part of the RT primer containing a different base, wherein the PCR could successfully progress with the use of 5'-end DNA tag because of an increase in the length of the hybridised tagged primer. This study shows the potential of RT-Hpro-PCR to precisely detect miRNAs with similar sequences, which could help explore the roles of miRNAs in several biological processes.

RT-Hpro-PCR: A MicroRNA Detection System Using a Primer with a DNA Tag.

MicroRNAs (miRNAs) are short RNAs that regulate the expression of complementary messenger RNAs and are involved in numerous human diseases. However, current detection techniques lack the sensitivity to detect miRNAs of low abundance. Moreover, at a length of 20-25 bases, miRNAs are too short for the reverse transcription (RT) polymerase chain reaction (PCR). Here we have developed a new, rapid, and simple miRNA detection system utilizing an RT primer containing a DNA tag at the 5'-end to increase the length of the cDNA. This strategy increases the length of the hybridized tagged primer and the complementary template DNA, as well as the melting temperature of the primer⋅template DNA duplex. PCR efficiency is thus increased, thereby enhancing miRNA detection sensitivity.

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection.

A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP-primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a 'turn-on' system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world.

Synthesis and Photophysical Properties of Fluorescence Molecular Probe for Turn-ON-Type Detection of Cytosine Bulge DNA.

A fluorescent molecule DANP that binds to cytosine bulge DNA and emits characteristic fluorescence was conjugated to pyrene to give a new fluorescence probe PyDANP. Temperature- and solvent-dependent absorption changes showed that DANP and pyrene chromophores stacked at room temperature in an aqueous buffer solution and quenched fluorescence. Upon binding of DANP moiety in PyDANP to cytosine bulge DNA, the fluorescence from DANP bound to C-bulge increased by ∼12-fold, showing that PyDANP is a turn-ON probe for the detection of C-bulge DNA.

Detection of hepatitis C virus by single-step hairpin primer RT-PCR.

We developed a novel single-step virus detection system using the fluorescent molecule with a hairpin primer on the reverse transcription (RT)-polymerase chain reaction (PCR). Primers that are specific to Hepatitis C virus (HCV) having a hairpin tag at the 5'-end were used for RT and PCR. The HCV-RNA template was reverse transcribed by reverse transcriptase with the hairpin primer (HP), and the resulting cDNA was amplified directly by HP-PCR (RT-HP-PCR). Using the RT-HP-PCR, we succeeded in demonstrating the detection of HCV-RNA in single-step. The RT-HP-PCR selectively detected HCV-RNA extracted from patients' sera containing contaminant materials. The method could be applicable to the quantitative detection of HCV.

A novel DANP-coupled hairpin RT-PCR for rapid detection of Chikungunya virus.

Chikungunya has re-emerged as an important arboviral infection of global health significance. Because of lack of a vaccine and effective treatment, rapid diagnosis plays an important role in early clinical management of patients. In this study, we have developed a novel molecular diagnostic platform that ensures a rapid and cost-effective one-step RT-PCR assay, with high sensitivity and specificity, for the early detection of the Chikungunya virus (CHIKV). It uses 2,7-diamino-1,8-naphthyridine derivative (DANP)-labeled cytosine-bulge hairpin primers to amplify the nsP2 region of the CHIKV genome, followed by measurement of the fluorescence emitted from DANP-primer complexes after PCRs. The detection limit of our assay was 0.01 plaque-forming units per reaction of CHIKV. Furthermore, the HP-nsP2 primers were highly specific in detecting CHIKV, without any cross-reactivity with the panel of RNA viruses validated in this study. The feasibility of the DANP-coupled hairpin RT-PCR for clinical diagnosis was evaluated using clinical serum samples from CHIKV-infected patients, and the specificity and sensitivity were 100% (95% CI, 80.0% to 100%) and 95.5% (95% CI, 75.1% to 99.8%), respectively. These findings confirmed its potential as a point-of-care clinical molecular diagnostic assay for CHIKV in acute-phase patient serum samples.

Transformation of cytosine to uracil in single-stranded DNA via their oxime sulfonates.

The novel transformation of cytosine oxime sulfonate (C*) to uracil sulfonate (U*) proceeded by a Co(II)-assisted benzoyl peroxide reaction, eventually leading to the conversion of cytosine to uracil.

Reaction of cytosine with bisulfite and hydroxylamine.

The synthesis of DNA oligomer containing N(4)-hydroxy-5,6-dihydrocytosine-6-sulfonate by the simultaneous addition of bisulfite and hydroxylamine under mild conditions is reported. To the best of our knowledge, the reaction of the oxime into the ketone, N(4)-hydroxy-5,6-dihydrocytosine-6-sulfonate into 5,6-dihydrouracil-6-sulfonate or uracil has not been reported. Moreover, this issue contains the difficulty in specific reactivity of oxime derivative from DNA bases include (5m)C toward reagent; for example, T also reacts to potassium permanganate known as a reagent for oxime into ketone. Here we examined the transformation of DNA containing N(4)-hydroxy-5,6-dihydrocytosine-6-sulfonate into 5,6-dihydrouracil 6-sulfonate and the following elimination of sulfonate.

Synthesis and reaction of DNA oligomers containing modified cytosines related to bisulfite sequencing.

The synthesis of DNA oligomers containing N(4)-hydroxy-5,6-dihydrocytosine-6-sulfonate by using ligand-induced base flipping of cytosine followed by the simultaneous addition of bisulfite and hydroxylamine is reported. In contrast to C, the flipped-out 5-methylcytosine was selectively oxidized over thymines and cytosines in the duplex by potassium permanganate. Ligand-induced base flipping is a convenient and powerful strategy for the synthesis of modified cytosines and 5-methylcytosines related to bisulfite sequencing at the predetermined site of DNA.

The reaction of cytosine with bisulfite by base flipping from the duplex.

Methylation of cytosine at the C5-position in DNA plays a major role in epigenetic gene control. The detection of 5-methylcytosine was designed with the base flipping from the duplex using NC and CGG/CGG triad. Using bisulfite and hydroxylamine, the reaction of cytosine in 11-mer duplex produced the adduct of bisulfite and hydroxylamine, which was isolated by HPLC and identified by MALDI-TOF MS. The duplex containing (5m)C in (5m)CGG/(5m)CGG did not react with bisulfite and hydroxylamine under the same conditions. Further this method enables cytosine adjacent to mismatch guanine to react with bisulfite and hydroxylamine selectively.

Emission of characteristic fluorescence from the ligand-cytosine complex in U_A/ACU bulged RNA duplex.

Here we show that N,N-bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine (DANP) binds to the single cytosine bulge in RNA duplexes. When the base pairs flanking the C-bulge were A-U base pairs, a characteristic fluorescence was emitted from the DANP-C-bulge complex. The fluorescence would be useful for detecting the C-bulge in RNA secondary structures.

Allele specific C-bulge probes with one unique fluorescent molecule discriminate the single nucleotide polymorphism in DNA.

A combination of an allele specific C-bulge probe and the fluorescent molecule N,N'-bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine (DANP) that binds specifically to the C-bulge provides a method for single nucleotide polymorphism (SNP) typing with only one fluorescent molecule without covalent modification of the DNA probe. The allele specific C-bulge probe contains one additional cytosine and produces a C-bulge directly flanking the SNP site upon hybridization to the target DNA. The C-bulge is a scaffold to recruit and retain DANP directly neighboring the SNP site. The DANP fluorescent probe was selectively modulated by the flanking matched and mismatched base pairs. The mutation type could be discriminated by the modulated fluorescent intensity with respect to the allele specific C-bulge probes used for the assay.

Synthesis and redox properties of trinuclear ruthenium-acetylide complexes with tri(ethynylphenyl)amine bridge.

Novel trinuclear ruthenium complexes have been prepared by using tri(4-ethynylphenyl)amine as a bridging ligand. Cyclic voltammetry of the trinuclear ruthenium complexes revealed stepwise quasi-reversible redox behavior of three ruthenium-acetylide species and the central triphenylamine unit, whereas the mononuclear analog showed two sequential quasi-reversible redox waves. The spectroelectrochemical UV-VIS spectral studies suggested that the 1e- oxidized triruthenium species was stable and showed a characteristic absorption at lambda(max) = 505 nm. Chemical oxidation of the triruthenium complex with ferrocenium hexafluorophosphate led to the isolation of the 1e- oxidized complex, the near-IR spectrum of which revealed an intervalence charge transfer band due to the electronic coupling among three ruthenium species. The 1e(-) oxidized triruthenium complexes can be classified as class II mixed-valence compounds.

Convergent synthesis of platinum-acetylide dendrimers.

An efficient convergent route to the main chain type of organometallic dendrimers, in which platinum moieties are linked by 1,3,5-triethynylbenzene, has been developed. The synthesis of platinum-acetylide dendrons involved the use of two types of trialkylsilyl groups for protection of the terminal acetylene. The platinum-acetylide dendrimers were prepared up to the third generation by reacting dendrons with a triplatinum core and a tetraplatinum core. Spectroscopic characterization and trace experiments by gel permeation chromatography indicated that the dendrimer molecules have no structural defects. Although a pi-conjugated system was used as the bridging ligand, electronic and fluorescence spectra suggested that the interaction among the platinum-acetylide moieties in the dendrimers was small.