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Yoshihito Ueno

Publications and source records attributed to Yoshihito Ueno.

At least 19 recordsLinked to original sources
PubMed2005

Functional characterization of 2',5'-linked oligoadenylate binding determinant of human RNase L.

RNase L is activated by the binding of unusual 2',5'-linked oligoadenylates (2-5A) and acts as the effector enzyme of the 2-5A system, an interferon-induced anti-virus mechanism. Efforts have been made to understand the 2-5A binding mechanism, not only for scientific interests but also for the prospects that the understanding of such mechanisms lead to new remedies for viral diseases. We have recently elucidated the crystal structure of the 2-5A binding ankyrin repeat domain of human RNase L complexed with 2-5A. To determine the contributions of amino acid residues surrounding the 2-5A binding site, point mutants and a deletion mutant were designed based on the crystal structure. These mutant proteins were analyzed for their interaction with 2-5A using a steady-state fluorescence technique. In addition, full-length RNase L mutants were tested for their activation by 2-5A. The results reveal that pi-pi stacking interactions of Trp60 and Phe126, electrostatic interactions of Lys89 and Arg155, and hydrogen bonding by Glu131 make crucial contributions to 2-5A binding. It was also found that the crystal structure of the ankyrin repeat domain L.2-5A complex accurately portrays the 2-5A binding mode in full-length RNase L.

Adenine Nucleotides↗
PubMed2005

Synthesis of novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions and their ability to suppress human RNase L protein expression.

In order to examine the effect of modifications at the 3' overhang regions of short interfering RNAs (siRNAs) on their gene-silencing activities, we designed and synthesized novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions. Suppression of human RNase L protein expression by these siRNAs was analyzed by immunoblot with RNase L-specific antibody. It was found that, at 24 h post-transfection, the modified siRNAs having the thymidine dimers with the carbamate and urea linkage suppress the protein expression 78 and 37 times more efficiently than that with the natural phosphodiester linkage, respectively. Furthermore, the siRNA containing the carbamate linkage was 37 times more resistant to nucleolytic degradation by snake venom phosphodiesterase than the siRNA consisting of the natural phosphodiester linkage. Thus, the RNA duplexes having the thymidine dimers with the carbamate or urea linkage at their 3' overhang regions will be promising candidates for novel siRNA molecules to down-regulate protein expression.

Carbamates↗
PubMed2004

Thermal stability of triple helical DNAs containing 2'-deoxyinosine and 2'-deoxyxanthosine.

In this paper, we describe the synthesis and thermal stabilities of the triplexes containing either 2'-deoxyinosine (1) or 2'-deoxyxanthosine (3) in their second strands. It was found that the triplexes with the 2'-deoxy-5-methylcytidine(dM)*1:dC and dM*1:dA base triplets are thermally stable, but those containing the dM*1:T and dM*1:dG base triplets are unstable under both neutral and slightly acidic conditions. On the other hand, it was found that the oligonucleotide containing 3 could form thermally stable triplexes with the oligonucleotides that involve four natural bases opposite the sites of 3. The rank of the thermal stabilities of the triplexes was as follows: the triplex containing the dM*3:dC base triplet > that containing the dM*3:dA base triplet > that containing the dM*3:T base triplet > that containing the dM*3:dG base triplet.

Base Composition↗
PubMed2004

Synthesis of 2',5'-oligoadenylate analogs containing an adenine acyclonucleoside and their ability to activate human RNase L.

This paper described synthesis of 2',5'-oligoadenylate (2-5A) analogs containing the purine acyclonucleoside, 9-[(2'S,3'R)-2',3',4'-trihydroxybutyl]adenine (2). The ability of the analogs to activate recombinant human RNase L was evaluated using 5'-32P-r(C11U2C7)-3' as a substrate. The EC50 value (the concentration of the 2-5A required to cleave half of the RNA) of the parent 2-5A tetramer 13 was 1.0 nM, whereas those of the analog 14 incorporating 2 at the second position from the 5'-end and the analog 15 incorporating 2 at the third position from the 5'-end were 9.0 and 1.7 nM, respectively. The analogs 14 and 15 were only 9- and 1.7-fold less potent than the parent 2-5A 13 itself, in RNase L activation ability. Furthermore, the oligodeoxynucleotide containing 2 was more resistant to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3'-exonuclease) than the unmodified oligodeoxynucleotide. Thus, incorporation of an acyclonucleoside into 2-5A may be useful for developing an antiviral agent based on the 2-5A system.

Adenine↗
PubMed2004

Oligodeoxynucleotides having a loop consisting of 3'-deoxy-4'-C-(2-hydroxyethyl)thymidines form stable hairpins.

Components that form stable hairpin loops are highly useful for the development of functional DNA and RNA molecules. We have designed and synthesized a sugar-modified thymidine analogue, 3'-deoxy-4'-C-(2-hydroxyethyl)thymidine (X), as a nucleosidic loop component stabilizing the hairpin structure. The ODNs I-1-4, 5'-d[CGAACG-X(n)-CGTTCG]-3' (I-1, n = 1; I-2, n = 2; I-3, n = 3; I-4, n = 4), forming the hairpin loop structures, of which the loop moiety consisted of the analogue X, and also the corresponding unmodified ODNs II-1-4, 5'-d[CGAACG-T(n)-CGTTCG]-3' (II-1, n = 1; II-2, n = 2;II-3, n = 3; II-4, n = 4), having a thymidine loop, were synthesized by the phosphoramidite method. The melting temperatures (T(m)) of the ODNs I-1-4 containing X in the loop moiety at 5 microM were 67.1, 68.1, 73.0, and 69.3 degrees C, respectively, and those of the control natural ODNs II-1-4 were 65.3, 67.0, 69.2, and 68.8 degrees C, respectively. Thus, the ODNs I-1-4 formed a more thermally stable hairpin than the corresponding unmodified ODNs II-1-4 having an equal number of loop residues. The hairpin structures of the modified ODNs I-1-4 and the unmodified ODNs II-1-4 were investigated by CD spectroscopy and molecular mechanics calculations. These results showed that the 4'-branched nucleoside X can stabilize hairpin structures when it is present in the loop moiety, probably due to the flexibility of the one-carbon-elongated 4'-branched structure.

Base Pairing↗
PubMed2004

Nucleosides and nucleotides. Part 226: alternate-strand triple-helix formation by 3'-3'-linked oligodeoxynucleotides composed of asymmetrical sequences.

In this paper, we describe the synthesis of the 3'-3'-linked oligonucleotides connected with pentaerythritol composed of asymmetrical sequences. Stability of the triplexes between these oligonucleotides and the DNA targets involving the adjacent oligopurine domains on alternate strands was investigated using the electrophoretic mobility shift assay (EMSA) and DNase I footprinting experiment. It was found that the 3'-3'-linked oligonucleotides composed of asymmetrical sequences formed the stable antiparallel triplexes with the DNA targets as compared with the unlinked oligonucleotides. Thus, oligonucleotides linked with pentaerythritol would be useful as antigene oligonucleotides for DNA targets consisting of the alternating oligopyrimidine-oligopurine sequences.

Base Composition↗
PubMed2004

Contribution of Tyr712 and Phe716 to the activity of human RNase L.

Ribonuclease L (RNase L) is a key enzyme in the 2-5A host defense system, and its activity is strictly regulated by an unusual 2',5'-linked oligoadenylate (2-5A). A bipartite model, in which the N-terminal half of RNase L is responsible for the 2-5A binding and the C-terminal half alone is able to hydrolyse the substrate RNA, has been proposed on the basis of the results of deletion mutant analyses [Dong, B. & Silverman, R.H. (1997) J. Biol. Chem.272, 22236-22242]. Above all, the region between Glu711 and His720 was revealed to be essential for RNA binding and/or hydrolysis. To dissect the function of the region, we performed scanning mutagenesis over the 10 residues of glutathione S-transferase (GST)-fusion RNase L. Among the single amino acid mutants examined, Y712A and F716A resulted in a significant decrease of RNase activity with a reduced RNA binding acitivity. The losses of the RNase activity were not restored by its conservative mutation, whereas the RNA binding activity was enhanced in the case of Y712F. These results indicate that both Tyr712 and Phe716 provide the enzyme with a RNA binding activity and catalytic environment.

Amino Acid Sequence↗
PubMed2004

[Chloride].

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Acid-Base Imbalance↗
PubMed2003

A specific substrate-inhibitor, a 2'-deoxy-2'-fluorouridine-containing oligoribonucleotide, against human RNase L.

We examined the properties of RNA analogues containing 2'-deoxy-2'-alpha-fluorouridine (1) or 2'-O-methyluridine (2) as inhibitors against human RNase L, that cleaves a single-stranded RNA in the presence of 2',5'-linked oligoadenylate (2-5A). The RNA analogue, FF, containing two molecules of 1 in place of uridine efficiently inhibited the RNase L-catalyzed RNA cleavage reaction, whereas the analogue, MM, containing two molecules of 2 was found not to have affinity for the enzyme. The k(cat) value for FF was 1/100 of that for an unmodified RNA, UU, whereas the K(m) value of FF was only twice as great as that of UU. Thus, it was found that the analogue, FF, containing 1 is an efficient inhibitor against human RNase L.

Catalysis↗
PubMed2003

Synthesis of double-headed 2-5A-antisense chimeras and their ability to activate human RNase L.

The synthesis of a novel 2-5A-antisense chimera having two molecules of a 2-5A tetramer at the 5'-terminus of the antisense moiety with a 2-(hydroxymethyl)-1,3-propanediol linker is described. The ability of the synthesized 2-5A antisense chimeras to activate RNase L was estimated by monitoring the cleavage of a target RNA by the activated RNase L. The double-headed 2-5A-antisense chimera linked with two molecules of a butanediol linker more efficiently cleaved the target RNA as compared with the single-headed 2-5A-antisense chimera and the double-headed 2-5A-antisense chimera linked with a molecule of the butanediol linker.

Adenine Nucleotides↗
PubMed2003

New base pairing motifs. The synthesis and thermal stability of oligodeoxynucleotides containing imidazopyridopyrimidine nucleosides with the ability to form four hydrogen bonds.

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides 1-4 (N(N), O(O), N(O), and O(N), respectively) with the aim of developing two sets of new base pairing motifs consisting of four hydrogen bonds (H-bonds) is described. The proposed four tricyclic nucleosides 1-4 were synthesized through the Stille coupling reaction of a 5-iodoimidazole nucleoside with an appropriate 5-stannylpyrimidine derivative, followed by an intramolecular cyclization. These nucleosides were incorporated into ODNs to investigate the H-bonding ability. When one molecule of the tricyclic nucleosides was incorporated into the center of each ODN (ODN I and II, each 17mer), no apparent specificity of base pairing was observed, and all duplexes were less stable than the duplexes containing natural G:C and A:T pairs. On the other hand, when three molecules of the tricyclic nucleosides were consecutively incorporated into the center of each ODN (ODN III and IV, each 17mer), thermal and thermodynamic stabilization of the duplexes due to the specific base pairings was observed. The melting temperature (T(m)) of the duplex containing the N(O):O(N) pairs showed the highest T(m) of 84.0 degrees C, which was 18.2 and 23.5 degrees C higher than that of the duplexes containing G:C and A:T pairs, respectively. This result implies that N(O)and O(N) form base pairs with four H-bonds when they are incorporated into ODNs. The duplex containing N(O):O(N) pairs was markedly stabilized by the assistance of the stacking ability of the imidazopyridopyrimidine bases. Thus, we developed a thermally stable new base pairing motif, which should be useful for the stabilization and regulation of a variety of DNA structures.

Base Pairing↗
PubMed2003

Synthesis, thermal stability and resistance to enzymatic hydrolysis of the oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridines.

The synthesis of oligonucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridine (D) is described, and thermal stability and resistance to enzymatic hydrolysis of the ODNs are compared with ODNs containing 5-(N-aminohexyl)carbamoyl-2'-deoxyuridine (H). The ODNs containing D and the complementary RNA demonstrated a duplex thermal stabilization of 0.4-3.9 degrees C per modification depending on the position and the number, while the ODNs containing H with the RNA showed slightly less effective thermal stabilization. Further more, the ODNs containing D were found to be more resistant to nucleolytic hydrolysis, not only by snake venom phosphodiesterase (SVPD; a 3'-exonuclease) but also by DNase I (an endonuclease). The half-life of the 17mer containing five molecules of D against nucleolytic hydrolysis by SVPD was 240 times greater than the unmodified 17mer ODN, which is 1.8 times greater than the ODN containing 5Hs in the same sequence. Against DNase I, the same ODN containing 5Ds was 24 times greater stable than the unmodified 17mer and 15 times more stable than the ODN containing 5Hs. We also examined whether the duplexes formed by the ODNs containing D and the complementary RNAs could be a substrate of Escherichia coli RNase H. It was revealed that a minimum of five contiguous unmodified 2'-deoxyribonucleosides between Ds was required to constitute a substrate of E.coli RNase H. Thus, the ODN with Ds and at least five contiguous unmodified 2'-deoxyribonucleosides between Ds was found to be a candidate for a novel antisense molecule.

Base Sequence↗
PubMed2003

Synthesis of double-headed 2-5A-antisense chimeras and their ability to activate human RNase L.

The synthesis of a novel 2-5A-antisense chimera having two molecules of 2-5A tetramer at the 5'-terminus of the antisense moiety with an ethylene glycol linker is described. The ability of the synthesized 2-5A antisense chimeras to activate RNase L was estimated by monitoring the cleavage of a target RNA by the activated RNase L. It was found that the double-headed 2-5A-antisense chimera linked with two molecules of a butanediol linker more efficiently cleaved the target RNA as compared with the single-headed 2-5A-antisense chimera and the double-headed 2-5A-antisense chimera linked with a molecule of the butanediol linker.

Base Sequence↗
PubMed2002

Water mediated Dickerson-Drew-type crystal of DNA dodecamer containing 2'-deoxy-5-formyluridine.

To investigate the role of divalent cations in crystal packing, a Dickerson-Drew-type dodecamer with the sequence d(CGCGAATXCGCG), containing 2'-deoxy-5-formyluridine at X, was crystallized under several conditions with Ba(2+) ion instead of Mg(2+) ion. The crystal structure is isomorphous with the original Dickerson-type crystal containing Mg(2+) ion. In the Mg(2+)-free crystals, however, a five-membered ring of water molecules occupies the same position as the magnesium site found in the Mg(2+)-containing crystals, and connects the two duplexes similarly to the hydrated Mg(2+) ion. It has been concluded that the five-membered water molecules can take the place of the hydrated magnesium cation in crystallization. The 5-formyluracil residues form the canonical Watson-Crick pair with the opposite adenine residues.

Barium↗
PubMed2002

X-Ray analysis of d(CGCGAATTXGCG)(2) containing a 2' or minute-deoxy-N(4)-methoxycytosine residue at X: a characteristic pattern of sugar puckers in the crystalline state of the Dickerson-Drew type DNA dodecamers.

In a series of structural studies on damaged DNA, a modified Dickerson-Drew dodecamer with the sequence d(CGCGAATTmo(4)CGCG), where mo(4)C is 2'-deoxy-N(4)-methoxycytidine, was synthesized and its structure in a new crystal form has been determined by the X-ray diffraction method. The two dodecamers form a B-form duplex, in which the two mo(4)C residues, respectively, form a wobble pair and a Watson-Crick type pair with the guanine residues of the opposite strand. A comparison of the sugar conformations with those of the other related Dickerson-Drew dodecamers indicates a common feature of their puckering patterns. The sugar pucker of the third residue always adopts an intermediate state (C4'-exo-O4'-endo) between the A-form and B-form. This deviation is ascribed to the stacking interaction of the ribose ring at the third residue with the guanine base at the 12th residue, which is brought about by an extra G12:G2 interaction between two duplexes related by a crystallographic 2(1) symmetry.

Crystallization↗
PubMed2002

Induction of T --> G and T --> A transversions by 5-formyluracil in mammalian cells.

Oxidatively damaged thymine, 5-formyluracil (5-fU), was incorporated into a predetermined site of double-stranded shuttle vectors. The nucleotide sequences in which the modified base was incorporated were 5'-CFTAAG-3' and 5'-CTFAAG-3' (F represents 5-fU), the recognition site for the restriction enzyme AflII (5'-CTTAAG-3'). The 5-fU was incorporated into a template strand of either the leading or lagging strand of DNA replication. The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and were analyzed after the second transfection into Escherichia coli. The 5-fU did not block DNA replication in mammalian cells. The 5-fU residues were weakly mutagenic, and their mutation frequencies in double-stranded vectors were 0.01-0.04%. The T --> G and T --> A transversions were the mutations found most frequently, suggesting the formation of 5-fU.C and 5-fU.T base pairs, respectively. This is the first report that clearly shows the induction of transversion mutations by an oxidized pyrimidine base in DNA in mammalian cells.

Animals↗