Small molecule-based detection of non-canonical RNA G-quadruplex structures that modulate protein translation.

Tandem repeats of guanine-rich sequences in RNA often form thermodynamically stable four-stranded RNA structures. Such RNA G-quadruplexes have long been considered to be linked to essential biological processes, yet their physiological significance in cells remains unclear. Here, we report a approach that permits the detection of RNA G-quadruplex structures that modulate protein translation in mammalian cells. The approach combines antibody arrays and RGB-1, a small molecule that selectively stabilizes RNA G-quadruplex structures. Analysis of the protein and mRNA products of 84 cancer-related human genes identified Nectin-4 and CapG as G-quadruplex-controlled genes whose mRNAs harbor non-canonical G-quadruplex structures on their 5'UTR region. Further investigations revealed that the RNA G-quadruplex of CapG exhibits a structural polymorphism, suggesting a possible mechanism that ensures the translation repression in a KCl concentration range of 25-100 mM. The approach described in the present study sets the stage for further discoveries of RNA G-quadruplexes.

G-quadruplexes in mRNA: A key structure for biological function.

The last several years have seen exciting advances in the understanding of the structure and function of higher-order structures of RNA. Expression levels of some specific genes were shown to be directly regulated by environmentally-responsive formation of certain secondary structures such as stem-loops and pseudoknots. Even among these noncanonical structures, RNA G-quadruplexes, which form on the regions of guanine-rich sequences in mRNA, are highly stable structures that are involved in a variety of biological processes. However, many questions regarding the biological significance of RNA G-quadruplexes remain unsettled, mainly because it is difficult to locate the structures in mRNA. This review focuses on emerging methods that locate RNA G-quadruplexes in mRNA by computational and biochemical techniques. In addition, recent reports on the biological functions of RNA G-quadruplexes are also covered to highlight their various roles in cells, such as in regulating mRNA processing and translation.

Generalized Preparation of Two-Dimensional Quasi-nanosheets via Self-assembly of Nanoparticles.

Two-dimensional (2D) nanomaterials are attracting increasing research interest because of their unique properties and promising applications. Here, we report a facile method to manipulate the assembly of nanoparticles (NPs) to fabricate free-standing 2D quasi-nanosheets. The as-generated 2D products are composed of few-layer NPs; that is, their thicknesses are only tens of nanometers but lateral dimensions could be up to several micrometers. Therefore, the novel structure was denoted as 2D "quasi-nanosheets (QNS)". Specifically, several types of building blocks could be assembled into 2D unary, binary, ternary, and even quaternary QNS by a universal procedure. The entire assembly process is carried out in solution and mediated simply by tuning the concentration of ligands surrounding the NPs. In contrast to traditional assembly techniques, even without any substrate or template, these QNS showed exceptionally high stability. They can remain intact for several days without any disassembly regardless of the solvent environment (e.g., water, ethanol, methanol, and hexane). In general, our method has effectively tackled several limitations associated with traditional assembly techniques and allows more freedom in manipulating assembly of NPs, which may hold great potential for future fabrication of 2D devices with rich functionalities.

Electrochemical Sensing of Neurotoxic Agents Based on Their Electron Transfer Promotion Effect on an Au Electrode.

An electrochemical molecular sensor based on a new principle is reported. Nereistoxin (NRT, 4-N,N-dimethylamino-1,2-dithiolane), a naturally occurring neurotoxin (nicotinic acetylcholine receptor agonist), was adsorbed on an Au electrode via Au-S covalent bonding and accelerated the electron transfer between the electrode and the marker, ferricyanide anion. The contrast between the electrochemical responses obtained with the bare and NRT-modified Au electrodes was more pronounced at a low ionic strength of the supporting electrolyte, KCl. In the presence of 1 mM KCl, almost a 0/1 contrast between the signals was obtained through electrostatic interaction between the protonated tertiary amino group of NRT and the anionic ferricyanide ion. No current was observed with an electrode modified with mercaptopropionic acid. An unusually low ionic strength thickened the electric double layer to the degree where current was not observed with the bare electrode. The effect of the electrostatic concentration of the marker ion becomes obvious under such conditions. Commercially available NRT-related pesticides such as Cartap and Bensultap were also detected using the same format after pretreatments by hydrolysis/reduction. The present sensing method was successfully applied to human serum with satisfactory sensitivity.

Rational design for cooperative recognition of specific nucleobases using ß-cyclodextrin-modified DNAs and fluorescent ligands on DNA and RNA scaffolds.

We propose a binary fluorimetric method for DNA and RNA analysis by the combined use of two probes rationally designed to work cooperatively. One probe is an oligonucleotide (ODN) conjugate bearing a ß-cyclodextrin (ß-CyD). The other probe is a small reporter ligand, which comprises linked molecules of a nucleobase-specific heterocycle and an environment-sensitive fluorophore. The heterocycle of the reporter ligand recognizes a single nucleobase displayed in a gap on the target labeled with the conjugate and, at the same time, the fluorophore moiety forms a luminous inclusion complex with nearby ß-CyD. Three reporter ligands, MNDS (naphthyridine-dansyl linked ligand), MNDB (naphthyridine-DBD), and DPDB (pyridine-DBD), were used for DNA and RNA probing with 3'-end or 5'-end modified ß-CyD-ODN conjugates. For the DNA target, the ß-CyD tethered to the 3'-end of the ODN facing into the gap interacted with the fluorophore sticking out into the major groove of the gap site (MNDS and DPDB). Meanwhile the ß-CyD on the 5'-end of the ODN interacted with the fluorophore in the minor groove (MNDB and DPDB). The results obtained by this study could be a guideline for the design of binary DNA/RNA probe systems based on controlling the proximity of functional molecules.

DNA molecular recognition and cellular selectivity of anticancer metal(II) complexes of ethylenediaminediacetate and phenanthroline: multiple targets.

By inhibiting only two or three of 12 restriction enzymes, the series of [M(phen)(edda)] complexes [M(II) is Cu, Co, Zn; phen is 1,10-phenanthroline; edda is N,N'-ethylenediaminediacetate] exhibit DNA binding specificity. The Cu(II) and Zn(II) complexes could differentiate the palindromic sequences 5'-CATATG-3' and 5'-GTATAC-3', whereas the Co(II) analogue could not. This and other differences in their biological properties may arise from distinct differences in their octahedral structures. The complexes could inhibit topoisomerase I, stabilize or destabilize G-quadruplex, and lower the mitochondrial membrane potential of MCF7 breast cells. The pronounced stabilization of G-quadruplex by the Zn(II) complex may account for the additional ability of only the Zn(II) complex to induce cell cycle arrest in S phase. On the basis of the known action of anticancer compounds against the above-mentioned individual targets, we suggest the mode of action of the present complexes could involve multiple targets. Cytotoxicity studies with MCF10A and cisplatin-resistant MCF7 suggest that these complexes exhibit selectivity towards breast cancer cells over normal ones.

Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts.

We found that both tetramethylammonium chloride (TMA-Cl) and tetra-ethylammonium chloride (TEA-Cl), which are used as monovalent cations for northern hybridization, drastically destabilized the tertiary structures of tRNAs and enhanced the formation of tRNA*oligoDNA hybrids. These effects are of great advantage for the hybridization-based method for purification of specific tRNAs from unfractionated tRNA mixtures through the use of an immobilized oligoDNA complementary to the target tRNA. Replacement of NaCl by TMA-Cl or TEA-Cl in the hybridization buffer greatly improved the recovery of a specific tRNA, even from unfractionated tRNAs derived from a thermophile. Since TEA-Cl destabilized tRNAs more strongly than TMA-Cl, it was necessary to lower the hybridization temperature at the sacrifice of the purity of the recovered tRNA when using TEA-Cl. Therefore, we propose two alternative protocols, depending on the desired properties of the tRNA to be purified. When the total recovery of the tRNA is important, hybridization should be carried out in the presence of TEA-Cl. However, if the purity of the recovered tRNA is important, TMA-Cl should be used for the hybridization. In principle, this procedure for tRNA purification should be applicable to any small-size RNA whose gene sequence is already known.

Metal Ion-Directed Specific DNA Structures and Their Functions.

Various DNA structures, including specific metal ion complexes, have been designed based on the knowledge of canonical base pairing as well as general coordination chemistry. The role of metal ions in these studies is quite broad and diverse. Metal ions can be targets themselves in analytical applications, essential building blocks of certain DNA structures that one wishes to construct, or they can be responsible for signal generation, such as luminescence or redox. Using DNA conjugates with metal chelators, one can more freely design DNA complexes with diverse structures and functions by following the simple HSAB rule. In this short review, the authors summarize a part of their DNA chemistries involving specific metal ion coordination. It consists of three topics: (1) significant stabilization of DNA triple helix by silver ion; (2) metal ion-directed dynamic sequence edition through global conformational change by intramolecular complexation; and (3) reconstruction of luminescent lanthanide complexes on DNA and their analytical applications.

Fabrication of Three-Dimensionally Deformable Metal Structures Using Precision Electroforming.

It is difficult to fabricate three-dimensional structures using semiconductor-process technology, because it is based on two-dimensional layered structure fabrication and the etching of thin films. In this study, we fabricated metal structures that can be dynamically deformed from two-dimensional to three-dimensional shapes by combining patterning using photolithography with electroforming technology. First, a resist structure was formed on a Cu substrate. Then, using a Ni sulfamate electroforming bath, a Ni structure was formed by electroforming the fabricated resist structure. Finally, the resist structure was removed to release the Ni structure fabricated on the substrate, and electroforming was used to Au-plate the entire surface. Scanning-electron microscopy revealed that the structure presented a high aspect ratio (thickness/resist width = 3.5), and metal structures could be fabricated without defects across the entire surface, including a high aspect ratio. The metallic structures had an average film thickness of 12.9 µm with σ = 0.49 µm, hardness of 600 HV, and slit width of 7.9 µm with σ = 0.25 µm. This microfabrication enables the fabrication of metal structures that deform dynamically in response to hydrodynamic forces in liquid and can be applied to fields such as environmental science, agriculture, and medicine.

Catalytic Amplification of Electrochemical Signal in Homogeneous Solution Using an Entropy-driven DNA Circuit.

The electrochemical signal from ferrocene on a DNA probe was successfully modulated in a homogeneous solution by the template-directed formation and dissociation of an inclusion complex with ß-cyclodextrin on another probe. The electrochemical response was amplified by combining with a DNA circuit, in which the target DNA served as a catalyst. This system did not require any modification of a complementary DNA with the ferrocene-modified probe on the electrode surface to separate the bound/free probe for the detection of 200 nM target DNA.

Detection of cancer cells in whole blood using a dynamic deformable microfilter and a nucleic acid aptamer.

Cancer cell count in the blood of cancer patients is extremely low. If these cells are easily detectable, cancer diagnosis may be possible by simply using a blood test, thus reducing patient burden. This study aimed to develop a cancer detection device by combining a microfilter that can be dynamically deformed and a nucleic acid aptamer that has a specific binding ability to cancer cells for easy detection. The cancer detection device was fabricated by photolithography, electroforming, and three-dimensional printing. The cancer cell detection ability of the fabricated device was evaluated using 1 mL of blood samples spiked with different concentrations of cancer cells. The lowest concentration of cancer cells in the blood was 5 cancer cells/1 mL blood. The fabricated microfilters specifically detected cancer cells in the blood successfully at exceedingly low concentrations. Moreover, the cancer detection experiment results using human whole blood revealed that cancer detection could be performed with higher accuracy using the fabricated cancer detection device compared to pre-existing cancer detection equipment (e.g., CellSearch system, Veridex). These findings provide important insights into the use of cancer cells in the blood as a diagnostic approach for cancer.

Cysteine Hydropersulfide Inactivates ß-Lactam Antibiotics with Formation of Ring-Opened Carbothioic S-Acids in Bacteria.

Hydrogen sulfide (H2S) formed during sulfur metabolism in bacteria has been implicated in the development of intrinsic resistance to antibacterial agents. Despite the conversion of H2S to hydropersulfides greatly enhancing the biochemical properties of H2S such as antioxidant activity, the effects of hydropersulfides on antibiotic resistance have remained unknown. In this work, we investigated the effects of H2S alone or together with cystine to form cysteine hydropersulfide (CysSSH) on the activities of antibacterial agents. By using the disc diffusion test, we found that CysSSH treatment effectively inactivated ß-lactams of the penicillin class (penicillin G and ampicillin) and the carbapenem class (meropenem). These ß-lactams were resistant to treatment with H2S alone or cystine alone. In contrast, cephalosporin class ß-lactams (cefaclor and cefoperazone) and non-ß-lactam antibiotics (tetracycline, kanamycin, erythromycin, and ofloxacin) were stable after CysSSH treatment. Chromatographic and mass spectrometric analyses revealed that CysSSH directly reacted with ß-lactams to form ß-lactam ring-opened carbothioic S-acids (BL-COSH). Furthermore, we demonstrated that certain bacteria (e.g., Escherichia coli and Staphylococcus aureus) efficiently decomposed ß-lactam antibiotics to form BL-COSH, which were transported to the extracellular space. These data suggest that CysSSH-mediated ß-lactam decomposition may contribute to intrinsic bacterial resistance to ß-lactams. BL-COSH may become useful biomarkers for CysSSH-mediated ß-lactam resistance and for investigation of potential antibacterial adjuvants that can enhance the antibacterial activity of ß-lactams by reducing the hydropersulfides in bacteria.

Catalytic formation of luminescent lanthanide complexes using an entropy-driven DNA circuit.

Two DNA conjugates modified with ethylenediaminetetraacetic acid and 1,10-phenanthroline were prepared as a pair of split probes. They were designed to form a duplex with their auxiliary groups facing each other, providing a microenvironment to accommodate lanthanide ions. The luminescent signal was amplified by catalytic duplex formation based on an entropy-driven DNA circuit.

Xylitol Separation from a Polyol Mixture Using Lanthanide Ion-loaded Resins.

Xylitol separation from a polyol mixture of the byproducts from bioethanol production processes was performed by liquid chromatography using short columns packed with lanthanide ion-loaded ion-exchange resins. Xylitol was successfully separated with sufficiently high resolution using adsorbents with medium rare-earth metal ions, such as Nd3+ and Sm3+. The adsorbents' specific nature is explained by the so-called "gadolinium break," which is known as a discontinuous behavior of thermodynamic parameters in complexation of the lanthanide series. From the observed behavior, the optimum lanthanide ions could be chosen to prepare appropriate adsorbents for ligand-exchange chromatography of given polyol mixtures.

Cooperative recognition of a repetitive sequence through consecutive formation of triplex and duplex structures.

Cooperative recognition of a repetitive sequence was performed with a short single DNA strand consisting of duplex- and triplex-forming regions modified with a ligand (benzoquinoquinoxaline) to stabilize a triplex structure. The former region was complementary with one unit of a repetitive sequence and the latter had a sequence that can bind with a cognate duplex formed by another DNA molecule bound on an adjacent site. The DNA binding to one unit of the repetitive sequence is expected to facilitate the second binding to an adjacent unit through cooperative triplex formation. The cooperativity was confirmed by evaluation of thermal stabilities of the complexes with a series of model repetitive sequences.

Detection of prostate-specific antigen in semen using DNA aptamers: an application of nucleic acid aptamers in forensic body fluid identification.

In forensics, body fluid identification plays an important role because it aids in reconstructing a crime scene. Therefore, it is essential to develop simple and reliable techniques for body fluid identification. Nucleic acid aptamers are useful tools in analytical chemistry that can be used to improve conventional forensic analytical techniques. They have numerous advantages over antibodies including their low cost, long shelf life, and applicability for chemical modification and PCR amplification. A DNA aptamer against a human prostate-specific antigen (PSA), which is a well-known protein marker for semen identification in forensics, has been reported previously. In this study, as a proof-of-concept for nucleic acid aptamer-based identification of body fluids, we developed a technique of aptamer-based PSA assays for semen identification that employed enzyme-linked oligonucleotide assay (ELONA) and real-time PCR. We evaluated their sensitivity and specificity for semen compared with those for blood, saliva, urine, sweat, and vaginal secretion. The assays have equivalent procedures compared to enzyme-linked immunosorbent assay; their results were consistent with those produced by the conventional immunochromatographic assay. The minimum volume of semen required for detection was 62.5 nL in ELONA and 5 nL in real-time PCR, making this assay applicable for semen detection in actual criminal investigation. Aptamers can be a cost-effective and versatile tool for forensic body fluid identification.

Electrochemical Molecular Beacon for Nucleic Acid Sensing in a Homogeneous Solution.

Ferrocene (Fc) and ß-cyclodextrin (ßCyD) were modified at each end of stem-loop structured DNA as an electrochemical signal generator and its quencher, respectively, to give an electrochemical molecular beacon (eMB). A relatively high efficiency of signal quenching was achieved by an inclusion complex (ßCyD ⊃ Fc) formation that was induced on the stem structure of the closed form (= stem-loop structure) of eMB. With the addition of target DNA, the structure of eMB opened to form a linear duplex, where the Fc dissociated from the ßCyD to restore its intrinsic electrochemical signal. The signal contrast of the electric current for this off/on-type sensor was high, ca. 95. This technique did not require any modification of the electrode surface, and it realized the detection of the target nucleic acids in a homogeneous solution with a high sensitivity using high-performance liquid chromatography (HPLC) equipped with electrochemical detector.

A RuO2 Nanosheet as a Novel Quencher-free Platform for the Detection of Nucleic Acids in a Homogeneous Solution.

A fluorescent dye-labeled DNA probe was adsorbed and quenched on the monolayer of RuO2 nanosheets. Significant fluorescent recovery was observed upon the addition of complementary DNA due to desorption of the probe from the surface of the RuO2 nanosheet through duplex formation. The efficiency of fluorescence recovery was higher than that for graphene oxide, which was known as a quencher-free platform for the detection of nucleic acids in a homogeneous solution.

A dynamically deformable microfilter for selective separation of specific substances in microfluidics.

To study an environmental or biological solution, it is essential to separate its constituents. In this study, a 3D-deformable dynamic microfilter was developed to selectively separate the target substance from a solution. This microfilter is a fine metallic nickel structure fabricated using photolithography and electroplating techniques. It is gold-coated across its entire surface with multiple slits of 10-20 µm in width. Its two-dimensional shape is deformed into a three-dimensional shape when used for fluid separation due to hydrodynamic forces. By adjusting the pressure applied to the microfilter, the size of the gap created by deformation can be changed. To effectively isolate the target substance, the relationship between the solution flow rate and the extent of microfilter deformation was investigated. The filtration experiments demonstrated the microfilter's ability to isolate the target substance with elastic deformation without undergoing plastic deformation. Additionally, modification of the microfilter surface with nucleic acid aptamers resulted in the selective isolation of the target cell, which further demonstrates the potential application of microfilters in the isolation of specific components of heterogeneous solutions.

Type of advanced airway and survival after pediatric out-of-hospital cardiac arrest.

BACKGROUND: There is a knowledge gap about advanced airway management (AAM) after pediatric out-of-hospital cardiac arrest (OHCA) in the prehospital setting. We assessed which AAM strategy would be associated with an increased chance of survival after pediatric OHCA. METHODS: A nationwide population-based observational study was conducted using the Japanese government-led registry data of OHCA. Pediatric OHCA patients (aged 1-17 years) who received prehospital AAM via endotracheal intubation (ETI) or supraglottic airway (SGA) insertion by emergency medical service (EMS) personnel from 2011 to 2017 were included. Patients who received ETI were compared with those who received SGA insertion. The primary outcome was one-month survival after OHCA. RESULTS: A total of 967 patients (mean [SD] age, 12.2 [5.1] years; 66.6% male) were included; 113 received ETI, and 854 received SGA insertion. Among the total cohort, 118 (12.2%) survived one month after OHCA. In the propensity score-matched cohort, no difference was observed in one-month survival between the ETI and SGA insertion groups: 13 of 113 patients (11.5%) vs 12 of 113 patients (10.6%); RR, 1.08; 95%CI, 0.52-2.27. This lack of association between AAM strategy and survival was observed across a variety of subgroup and sensitivity analyses, and also for neurologically favorable survival (P = 0.5611) in the propensity score-matched analysis. CONCLUSIONS: In Japan, among pediatric OHCA patients, there was no significant difference in one-month survival between prehospital ETI and SGA insertion by EMS personnel. Although an adequately powered randomized controlled trial is needed, EMS personnel may choose their familiar strategy when prehospital AAM was performed during pediatric OHCA.