Kazuyuki Takai

Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides.

The inhibition of specific transcription regulatory proteins is a new approach to control gene expression. The transcriptional activities of DNA‐binding proteins can be inhibited by the use of double‐stranded oligonucleotides that compete for the binding to their specific target sequences in promoters and enhancers. We used nicked (NDODN‐κB) and circular (CDODN‐κB) dumbbell DNA oligonucleotides containing a NF‐κB binding site to analyze the inhibition of the NF‐κB‐dependent activation of the human immunodeficiency virus type‐1 (HIV‐1) enhancer. The dumbbell DNA oligonucleotides are stable, short segments of double‐stranded DNA with closed nucleotide loops on each end, which confer resistance to exonucleases. The dumbbell and other oligonucleotides (decoys) with the NF‐κB sequence were found to compete with the native strand for NF‐κB binding. The circular dumbbell and double‐stranded phosphorothioate oligonucleotides competed with the native strand for binding to the NF‐κB binding proteins, while the nicked NF‐κB dumbbell was a less effective competitor. In Jurkat T‐cells, the dumbbell and other oligonucleotides were tested for their ability to block the activation of the plasmid HIV‐NL4‐3 Luc. The CDODN‐κB strongly inhibits the specific transcriptional regulatory proteins, as compared with the NDODN‐κB and the double stranded phosphodiester oligonucleotides. On the other hand, the double stranded phosphorothioate oligonucleotides could also block this activation, but the effect was non‐specific. The circular (CDODN) dumbbell oligonucleotides may efficiently compete for the binding of specific transcription factors within cells, thus providing anti‐HIV‐1 or other therapeutic effects.

Specific inhibition of influenza virus RNA polymerase and nucleoprotein gene expression by circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides

We have designed a new class of oligonucleotides, `dumbbell RNA/DNA chimeric oligonucleotides, consisting of a sense RNA sequence and its complementary antisense DNA sequence, with two hairpin loop structures. The reaction of the nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNase H gave the corresponding antisense phosphodiester oligodeoxynucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target RNA, which gave RNA cleavage products by treatment with RNase H. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligonucleotide (anti‐ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide. The CDRDON with four target sites (influenza virus A RNA polymerases (PB1, PB2, PA) and nucleoprotein (NP)) was synthesized and tested for inhibitory effects by a CAT‐ELISA assay using the clone 76 cell line. The circular dumbbell DNA/RNA chimeric oligonucleotide (CDRDON‐PB2‐as) containing an AUG initiation codon sequence as the target of PB2 showed highly inhibitory effects.

Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis

Nonnatural amino acids with photofunctional groups were incorporated site‐specifically into a polypeptide by using in vitro protein synthesizing system. The nonnatural amino acids were attached to tRNACCU through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon. l‐p‐Phenylazophenylalanine, l‐2‐anthrylalanine, l‐1‐naphthylalanine, l‐2‐naphthylalanine and l‐p‐biphenylalanine were successfully incorporated into a polypeptide, but l‐1‐pyrenylalanine was not. The polypeptides containing the nonnatural amino acids showed photofunctionalities.

Site-specific cleavage of tRNA by imidazole and/or primary amine groups bound at the 5′-end of oligodeoxyribonucleotides

Sequence specific RNA cleaving molecules were synthesized by attaching novel polyamine derivatives bearing imidazole and/or primary amine groups to the 5-end of DNA oligonucleotides as the sequence-recognizing moieties. The actions of the molecules on a half-tRNA(Asp) were investigated. The oligonucleotides directed the nuclease activity (the imidazole and the primary amine are the catalytic groups) of the enzyme to the nucleotides directly adjacent to the complementary target sequence on the substrate RNA. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0, indicating the participation of protonated and non-protonated imidazoles residues in the process. The specificity of these hybrid enzymes can be easily altered, and they should prove to be useful tools for probing RNA structures in solution and as potential reactive groups in antisense oligonucleotide derivatives. We also describe the site-specific cleavage of tRNA(Asp) by the cleaving reagents bearing imidazole and/or primary amine groups at the 5-end of oligodeoxyribonucleotides.

Antisense therapy of influenza.

The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides (S-ODNs) with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with those directed to the PB2 target sites. The liposomally encapsulated antisense phosphorothioate oligonucleotides exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas the free antisense phosphorothioate oligonucleotides were observed to inhibit viral absorption to MDCK cells. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. Balb/c mice exposed to the influenza virus A (A/PR/8/34) strain at dose of 100 LD(50)s were treated i.v. with various doses (5-40 mg/kg) of liposomally (Tfx-10) encapsulated PB2-AUG or PA-AUG before virus infection and 1 and 3 days postinfection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in days (MDS) and increased the survival rates with a dose-dependent manner. We demonstrate the first successful in vivo antiviral activity of antisense administered i.v. in experimental respiratory tract infections induced with influenza virus A.

A single uridine modification at the wobble position of an artificial tRNA enhances wobbling in an Escherichia coli cell-free translation system

5‐Methoxyuridine was introduced into the first position of the anticodon of the unmodified form of tRNA1 Ser from Escherichia coli. The codon reading efficiencies of this tRNA (tRNA(5‐methoxyuridine UGA)) relative to those of the unmodified counterpart (tRNA(UGA)) were measured in a cell‐free translation system. tRNA(5‐methoxyuridine UGA) was more efficient than tRNA(UGA) in the reading of the UCU and UCG codons and was less efficient in the reading of the UCA codon. Thus, the single modification of U to 5‐methoxyuridine can enhance the wobble readings.

Adaptability of nonnatural aromatic amino acids to the active center of the E. coli ribosomal A site

3‐N‐Aminoacyl analogs of puromycin with nonnatural aromatic amino acids were synthesized and their inhibitory activity in E.coli in vitro protein synthesizing system was evaluated. The analogs with l‐2‐naphthylalanine, l‐p‐biphenylalanine, l‐2‐anthrylalanine and trans‐l‐p‐phenylazophenylalanine were found to inhibit the protein synthesis with high efficiency. The inhibition suggests that these nonnatural amino acids are accepted by the active center of the E. coli ribosomal A site. A model for the adaptability of nonnatural aromatic amino acids to the active center is proposed.

SPECIFIC INHIBITION OF HUMAN TELOMERASE ACTIVITY BY TRANSFECTION REAGENT FUGENE6-ANTISENSE PHOSPHOROTHIOATE OLIGONUCLEOTIDE COMPLEX IN HELA CELLS

Human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphodiester (ODNs) and phosphorothioate (S-ODNs) oligonucleotides were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The ODNs and S-ODNs were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 was used to enhance the cellular uptake of oligonucleotides in cell cultures. The results showed that S-ODN-3 (19-mer) encapsulated with FuGENE6 clearly inhibited the telomerase activity in HeLa cells, and the inhibitory efficiency increased with an increase in the S-ODN-3. However, free S-ODN-3 showed no inhibitory activity. On the other hand, ODN-3 encapsulated with FuGENE6 had no detectable inhibitory activity. The encapsulated S-ODNs exhibited higher inhibitory activities than the free S-ODNs, and showed sequence specific inhibition. Thus, the activities of the S-ODNs were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, and is appropriate for use in vitro and in vivo.

Effects of anticodon 2'-O-methylations on tRNA codon recognition in an Escherichia coli cell-free translation.

The methylation of 2-hydroxyl groups is one of the most common posttranscriptional modifications of naturally occurring stable RNA molecules. Some tRNA species have a 2-O-methyl nucleoside at the first position of the anticodon, and it was suggested that this modification stabilizes the codon-anticodon duplex. However, no tRNA species have been found to have the modification at the second or third position of the anticodon. In the present study, we measured the effects of anticodon 2-O-methylation on the codon-reading efficiencies of the anticodon variants of the unmodified forms of Escherichia coli tRNA1(Ser), using a cell-free protein synthesis assay. The modification of C in the first position of the anticodon into 2-O-methylcytidine increased the efficiency of reading the G-ending codon. On the other hand, the modifications of the second and/or third positions were detrimental to the codon-reading activity. Thus, 2-hydroxyl groups at the second and third positions of the anticodon may have some role in the translation reaction, and this may be the reason why 2-O-methyl nucleosides are not found in these positions within natural tRNA species.

Specific Inhibition of Influenza Virus RNA Polymerase and Nucleoprotein Gene Expression by Liposomally Encapsulated Antisense Phosphorothioate Oligonucleotides in MDCK Cells

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza A virus replication in MDCK cells. Liposomally encapsulated and free antisense S-ODNs with four target sites (PB1, PB2, PA and NP genes) were tested for their abilities to inhibit virus-induced cytopathogenic effects in a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the site around the PB2 AUG initiation codon showed highly inhibitory effects. In contrast, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with that directed to the PB2 target site. The liposomally encapsulated antisense S-ODNs exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas free antisense S-ODNs were observed to inhibit viral adsorption to MDCK cells. Liposomal preparations of oligonucleotides facilitated their release from endocytic vesicles, and thus cytoplasmic and nuclear localization was observed. The activities of the antisense S-ODNs were effectively enhanced by using the liposomal carrier. Interestingly, the liposomally encapsulated FITC-S-ODN-PB2–as accumulated in the nuclear region of MDCK cells. However, weak fluorescence was observed within the endosomes and the cytoplasm of MDCK cells treated with the free antisense S-ODNs. The cationic lipid particles may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in vitro or in vivo.