Nucleic acids symposium series (2004)最新文献

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analyses. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Our present finding provides an interesting possibility that lamin-B1 may have an important role in regulating cell-death morphology.

Pyrene (donor) and perylene (acceptor) were assembled in a DNA duplex to increase the apparent Stokes' shift of perylene. Multiple donors were introduced in the vicinity of acceptors via D-threoninol and natural base pairs were inserted between donors and acceptors in order to suppress undesired interactions between them. When two pyrene moieties were located in proximity to one perylene with one base pair inserted between them, efficient FRET occurred within the duplex. Thus, strong emission at 495 nm was observed from perylene when excited at 345 nm where pyrene has its absorption. The apparent Stokes' shift became as large as 115 nm with a high FRET efficiency (Phi>1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short Förster radius (R(0)) of the donor pyrene.

Several human pathogens possess nucleoside or nucleotide kinases with large substrate specificity compared to their human counterparts. This phenomenon has been successfully exploited for the specific targeting of prodrugs such as Acyclovir against herpes virus. Combined structural and biochemical studies of these enzymes can thus provide essential information for the rational design of specific antimicrobial agents. Here we studied the structural basis for the specificity of a thymidylate kinase from the poxvirus family. Poxvirus thymidylate kinase has unusual substrate specificity and can accept bulky analogues such as 5-bromo-vinyl-dUMP (BVdUMP). The 2 A crystal structure of the thymidylate kinase bound to this compound now gives the structural basis for its specific molecular recognition.

We designed and synthesized dumbbell-shaped nanocircular RNAs for RNA interference applications, which consist of a stem and two loops(1). RNA dumbbells are specifically recognized and cleaved by the human Dicer enzyme, and are thus transformed into double-stranded RNA in cells, although this RNA is resistant to degradation in serum. The structure was optimized to maximize its RNAi activity. The most potent activity was achieved when the stem length was 23 base pairs. The RNAi activity is prolonged by the shape of the molecule, an endless structure, compared with that of normal siRNA.

We report the fluorescent polymer complex which can show fluorescence emission at 380 nm with the excitation of 520 nm in aqueous media. This photon upconversion based on triplet-triplet annihilation can efficiently take place via inter-molecular energy transfers between the Ru complex as a sensitizer and anthracene molecules as an emitter captured into the water-soluble network polymers. We performed the oxidation reaction of 2'-deoxyguanosine by riboflavin in the presence of the polymer complex with the visible light irradiation. It was clearly indicated that oxidative decomposition can be accelerated by UV light generation via upconversion based on triplet-triplet annihilation.

We chemically synthesized a series of aminooxy derivatives to develop novel probes for sensitive detection of abasic (AP) sites in DNA. The results of the conjugation reactions showed that the probes could efficiently react to AP sites by introducing an aromatic or a guanidino group in their structures. In particular, the probe having both functional groups showed the most effective reactivity, indicating that hydrophobic and electrostatic interactions cooperatively acted in the reaction of the probe to AP sites. We then synthesized a biotinylated probe and succeeded in more sensitive detection of AP sites in genomic DNA than with the conventional aldehyde reactive probe (ARP).

Recently, various microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified and play important roles in gene regulatory networks. However it is a little known about their biological functions. These small RNAs exhibit their function to form a ribonucleoprotein complex, the RISC (RNA-induced silencing complex), which modulates gene expression by translational repression. In this study, we developed a novel peptide antagonist to inhibit the RISC function. The peptide was conjugated to 2'-O-methyl oligoribonucleotides, which have a complementary sequence to the guide strand of siRNA, and regulatory effects of the peptide on RISC activity were examined. It was revealed that the peptide drastically enhanced inhibitory effects of the oligonucleotide on RISC activity. Here we demonstrate our peptide-oligonucleotide conjugate that can provide a powerful and specific way to regulate the small RNA function.

Thermostable RNA hairpins with specific loop sequences are critically important structural elements for proper folding of functional RNAs. Thermodynamic parameters of the structural elements are indispensable for secondary structure prediction. However when RNA folds into highly thermostable hairpin-loop structures, it is difficult to determine the melting temperature and other thermodynamic parameters from the normal UV melting analysis. In this study, we demonstrate a procedure to solve the problem using our software, Jfit. UV absorbance in the higher temperature region above 95 degrees C and the upper baseline were estimated using this analytical procedure. The ideal lines of -RlnK vs. 1/T were generated from the predicted thermodynamic parameters and the solution candidates were compared with the van't Hoff plot generated from the measured melting curve to judge their self-consistency. As a result, our analysis was able to reduce the number of the possible solution and to specify the best set of thermodynamic parameters for the stable hairpin formation.

Synthesis of 5'-fluoro-2'-beta-methylneplanocin analogues (5) was carried out. The cyclopentenone 16 was prepared from methyl mannopyranoside by using ring closing metathesis, stereoselective introduction of methyl group, and seleno cyclization as representative steps. Introduction of a fluorine atom was conducted by electrophilic fluorination. Antiviral activity of 5 will also be presented.

An acridone-labeled DNA aptamer was synthesized to produce an aptamer probe. This aptamer probe could detect a specific molecule based on conformational changes upon binding of the specific molecules and the quenching of acridone emission.