Stereocontrolled solid-phase synthesis of phosphorothioate oligoribonucleotides using 2'-O-(2-cyanoethoxymethyl)-nucleoside 3'-O-oxazaphospholidine monomers.

A method for the synthesis of P-stereodefined phosphorothioate oligoribonucleotides (PS-ORNs) was developed. PS-ORNs of mixed sequence (up to 12mers) were successfully synthesized by this method with sufficient coupling efficiency (94-99%) and diastereoselectivity (≥98:2). The coupling efficiency was greatly improved by the use of 2-cyanoethoxymethyl (CEM) groups in place of the conventional TBS groups for the 2'-O-protection of nucleoside 3'-O-oxazaphospholidine monomers. The resultant diastereopure PS-ORNs allowed us to clearly demonstrate that an ORN containing an all-(Rp)-PS-backbone stabilizes its duplex with the complementary ORN, whereas its all-(Sp)-counterpart has a destabilizing effect.

Trimethylsilyl trifluoromethanesulfonate-promoted reductive 2'-O-arylmethylation of ribonucleoside derivatives.

Arylmethyl groups such as benzyl, p-methoxybenzyl, and 1-pyrenylmethyl groups were introduced to the 2'-O-position of nucleosides by reductive etherification. Combining corresponding aromatic aldehydes with 2'-O-trimethylsilylnucleoside derivatives in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) resulted in moderate to good yields of the 2'-O-arylmethyluridine derivatives, whereas the corresponding cytidine and adenosine derivatives were obtained in low yields. The reaction of ribonucleosides with aliphatic aldehydes did not proceed smoothly. Anomerization of the uridine derivatives by TMSOTf was observed in CH(2)Cl(2), toluene, and CH(3)CN, but was completely suppressed when the reactions were conducted in 1,4-dioxane.

Agmatine-conjugated cytidine in a tRNA anticodon is essential for AUA decoding in archaea.

A modified base at the first (wobble) position of some tRNA anticodons is critical for deciphering the genetic code. In eukaryotes and eubacteria, AUA codons are decoded by tRNAsIle with modified bases pseudouridine (and/or inosine) and lysidine, respectively. The mechanism by which archaeal species translate AUA codons is unclear. We describe a polyamine-conjugated modified base, 2-agmatinylcytidine (agm(2)C or agmatidine), at the wobble position of archaeal tRNA(Ile) that decodes AUA codons specifically. We demonstrate that archaeal cells use agmatine to synthesize agm(2)C of tRNA(Ile). We also identified a new enzyme, tRNA(Ile)-agm(2)C synthetase (TiaS), that catalyzes agm(2)C formation in the presence of agmatine and ATP. Although agm(2)C is chemically similar to lysidine, TiaS constitutes a distinct class of enzyme from tRNA(Ile)-lysidine synthetase (TilS), suggesting that the decoding systems evolved convergently across domains.

Chemical synthesis and properties of 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine.

Unique taurine-containing uridine derivatives, 5-taurinomethyluridine (tau m5U) and 5-taurinomethyl-2-thiouridine (tau m5s2U), which were discovered in mammalian mitochondrial tRNAs, exist at the first position of the anticodon. In this paper, we report the first efficient synthesis of tau m5U and tau m5s2U and describe their physicochemical properties. These modified ribonucleosides were synthesized by the reaction of 5-substituted uridine derivatives with a tetrabutylammonium salt of taurine that is highly reactive and well-soluble in common organic solvents. UV and 1H NMR spectrometric studies revealed the structural properties of the taurine-containing base moieties and the sugar conformations of these modified ribonucleosides.

Chemical synthesis of RNA including 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine.

Recently, novel base-modified uridine derivatives, 5-taurinomethyluridine (tau m(5)U) and 5-taurinomethyl-2-thiouridine (tau m(5)s(2)U) have been discovered from mammalian mitochondrial tRNAs, and these modified ribonucleosides were found to exist at the first position of the anticodon. We herein report efficient reactions for the synthesis of RNA oligomers including these base-modified ribonucleosides.

Chemical synthesis of RNA including 5-taurinomethyluridine.

Recently, a novel base-modified uridine derivative, 5-taurinomethyluridine (taum(5)U) was discovered from bovine and human mitochondrial tRNAs, and this modified ribonucleoside was found to existed at the first position of the anti-codon. We report efficient reactions for the synthesis of RNA oligomers including this base-modified ribonucleotide.

Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA.

In latent adeno-associated virus (AAV) infection, the viral genome is integrated preferentially into the human chromosome 19 q arm at a specific region designated AAVS1, which has an open chromatin conformation as indicated by the presence of a DNase I-hypersensitive site (DHS-S1). We examined whether an insulator, which defines the domain of gene expression by directionally blocking the action of enhancers and by preventing the spread of heterochomatin, is present near the DHS-S1 in the middle of a 2.6-kbp AAVS1-related DNA fragment used in this study. The fragment, cloned into an Epstein-Barr virus (EBV)-based eukaryotic episomal plasmid, was introduced into HEK293 cells. The DHS-S1 on the plasmid replicating in the nuclei was hypersensitive to DNase I digestion, and thus, the EBV plasmid system was used in an enhancer-blocking assay with the 2.6-kbp DNA and two shortened DNAs, of 1.6 kbp and 336 bp, containing DHS-S1. The three DNA fragments, when inserted in the proper direction between the cytomegalovirus immediate-early enhancer and minimal promoter, repressed the expression of a reporter gene. Thus, the enhancer-blocking activity was located within the 336-bp DNA containing the entire region (300 bp) of DHS-S1. To investigate the prevention of repression caused by heterochromatin, a transgene-expressing cassette flanked by the two 336-bp DNAs placed in the enhancer-blocking direction was introduced into HEK293 and HeLa cells. All the cell clones examined with the cassette integrated into cell DNA continued to express the transgene, which indicates that the pair of 336-bp DNA apparently prevented the spread of heterochromatin. The results show that an insulator lies between nucleotides 17 and 354 near the DHS-S1 in AAVS1. In a gel shift test, the 336-bp DNA did not bind an in vitro-prepared CCCTC-binding factor that binds to the chicken beta-globin insulator, suggesting that the AAVS1 insulator requires an as yet unidentified binding protein. The newly identified AAVS1 insulator is likely to contribute to the maintenance of an open chromatin conformation that affects the life cycle of AAV.

Chromatin remodeling factor encoded by ini1 induces G1 arrest and apoptosis in ini1-deficient cells.

Ini1/hsnf5 gene encodes INI1 protein, a chromatin remodeling factor associated with the SWI/SNF complex. In yeast, this complex modifies chromatin condensation to coactivate various transcriptional factors. However, in human, little is known about the SWI/SNF complex and INI1. To elucidate cellular functions of ini1, we constructed a recombinant adenovirus (AdexHA-INI1) capable of overexpressing INI1 in ini1-deficient cells. AdexHA-INI1 produced intranuclear INI1 in three ini1-deficient cell lines, changed their morphology, and decreased the proportion of viable cells. Flow cytometry and a BrdU incorporation assay showed that after the infection, growth of these cells was partially arrested at G1. In two of the three ini1-deficient cell lines, apoptosis was found to occur after the infection, as detected by the presence of cleaved poly (ADP-ribose) polymerase. To determine functional domains of INI1, we constructed plasmids expressing INI1 and its deletion mutants, which were used for a colony formation assay. Repeats 1 and 2 of INI1 were found to be required to suppress the growth of the three ini1-deficient cell lines. The results support the hypothesis that ini1 is a tumor suppressor gene and suggest a novel link between human SWI/SNF chromatin remodeling complex and apoptosis.