Nucleic acids symposium series (2004)最新文献

I have constructed a novel aptazyme-based biosensor system for detecting cofactors of the aptazymes using a cell-free luciferase synthesis in wheat germ extract. In this system, the activity of the aptazyme that is fused to a 5'-untranslated region of a luciferase gene can be detected as luciferase expression. In translating the aptazyme-fused mRNA as-is using a wheat germ cell-free translation system, the luciferase is almost not expressed because of the following triple suppression effects: (1) 5'-terminal three bases and (2) 5'-terminal duplex prevent the ribosome from binding to own mRNA; (3) if the ribosome binds, translation of a mimic gene in the aptazyme inhibits that of the downstream luciferase gene (OFF state). In contrast, in the presence of the aptazyme cofactor, the aptazyme in mRNA is self-cleaved to produce an aptazyme-free luciferase gene, which is translated efficiently (ON state). The ON/OFF efficiency and the detection limit of the aptazyme-based biosensor for theophylline are much higher and lower, respectively, compared to those of previously-reported one that utilizes a prokaryotic translation system.

The novel multi-arm DNA structures were designed using 2D-DNA Origami method, and these structures were folded into 3D hollow prism structures by introduction of connection strands into the arms. The opening of the prism structures were examined by a high-speed AFM, which showed the dissociation events of the connecting arms in the 3D-structures.

We have successfully developed a new method for photoregulation of G-quadruplex formation using cis-trans photoisomerization of the photochromic nucleobase (8FV)G. Our photo-controllable quadruplexes can be switched between a very stable quadruplex state and a non-structured state in a straightforward and reversible fashion. We also demonstrated reversibly control binding of a G-quadruplex aptamer to thrombin.

A 2'-deoxyuridylate dimer cyclized via cross-linkage by an ethylene (U(et)(p)U) or a propylene (U(pr)(p)U) linker at the 5-position was incorporated into DNA oligomers. Fluorescence resonance energy transfer (FRET) experiments showed that they bent at approximately 90 degrees . We investigated binding abilities of U(et)(p)U and U(pr)(p)U DNA oligomers to HMGB1 A-box protein, which specifically binds to bent DNA, using nuclear magnetic resonance (NMR) spectroscopy. Both DNA oligomers bind to HMGB1 A-box protein, however, the U(et)(p)U DNA oligomer has higher affinity than the U(pr)(p)U DNA oligomer. In order to explain this difference, we studied the solution structures of the U(et)(p)U and U(pr)(p)U DNA oligomers using NMR. Most (1)H signals except for 4', 5' and 5'' were assigned. Cross-peak patterns of (1)H-(1)H NOESY spectra indicate that both oligomers have right-handed B-form like structures and the cyclization in 2'-deoxyuridylates does not break Watson-Crick base pairs. Chemical shift differences between these two DNA oligomers suggest the presence of the local structural differences in the region of 2'-deoxyuridylate dimer and its 3' side between the U(et)(p)U and U(pr)(p)U DNA oligomers.

Evanescent-field-coupled (EFC) waveguide-mode sensors recently been shown to be suitable for detecting various biomolecules. In the present studies, we demonstrated that both nucleic acids hybridization and nucleic acids-protein interactions can be analyzed using perforated evanescent-field-coupled waveguide-mode nanobio-sensors.

Several 9-(2-C-cyano-2-deoxy-l-beta-D-arabino-pentofuranosyl)purine derivatives were tested against Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cells. The guanine derivative (2, CNDAG) as well as the 2-amino-6-substituted-purine derivatives 3, 4 and 5 inhibited KSHV-positive cell growth but showed no cytotoxicity against KSHV-negative cells at >15 muM concentrations. Therefore, it was found that compounds 2, 3, 4 and 5 showed selective cytotoxicity against PEL cells infected with KSHV.

5-Formyl-2'-deoxyuridine (fdUrd) is a product of the oxidation of thymidine and is known to induce mutation (A:T to G:C) in DNA. Therefore, a selective detection method for fdUrd is needed, but convenient methods have not been developed. We planned to develop a novel selective method to detect fdUrd in damaged DNA based on a specific fluorogenic derivatization of fdUrd using 2-aminothiophenol derivatives (ATs) as fluorogenic reagents. To achieve this goal, we first investigated the synthesis and fluorescence properties of some 5-(benzothiazol-2-yl)-2'-deoxyuridine derivatives (btdUrds). We also report the reaction between oligodeoxynucleotides (ODNs) containing fdUrd and ATs.

Influenza viruses A and B cause widespread infections of the human respiratory tract; however, existing vaccines and drug therapy are of limited value for their treatment. Here we show that bispecific short hairpin small-interfering RNA constructs containing an eight-nucleotide intervening spacer, targeted against influenza virus A or influenza virus B, can inhibit the production of both types of virus in infected cell lines. This multiple vector showed remarkable ability to cope with both influenza virus A or B. Furthermore, the Autographa californica multiple nuclear polyhedrosis virus can infect a range of mammalian cells, facilitating its use as a baculovirus vector for gene delivery into cells. In this study, baculovirus-mediated bispecific short-hairpin RNA expression markedly inhibited both influenza virus A and B production.

Modified DNA containing silylated pyrene derivative at the 5'-terminus showed the obvious discrimination ability of the duplex formation, that the modified DNA showed the strong fluorescence emission in the presence of the complementary DNA, in spite of the very weak emission in the absence of the complementary DNA. The intensity of the fluorescence emission of the double strand found to increase about 10 times compared with that of the single strand. In this paper, we tried to disclose the detailed mechanism of such a discrimination ability of the silylated pyrene-modified DNA.

The excitonic interaction of thiazole orange dyes is known to suppress fluorescence emission. We have developed hybridization-sensitive fluorescent probes utilizing the excitonic interaction of two thiazole orange dyes connected to the probes. Here, we report nuclease-resistant hybridization-sensitive probes for long-term intracellular RNA imaging.