Noriaki Minakawa

RNA interference induced by siRNAs modified with 4′‐thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4′‐thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense‐strand were well tolerated for RNAi activity except for full modification with 4′‐thioribonucleosides. However, the activity of siRNAs modified at the antisense‐strand was dependent on the position and the number of modifications with 4′‐thioribonucleosides. Since modifications of siRNAs with 4′‐thioribonucleosides were well tolerated in RNAi activity compared with that of 2′‐O‐methyl nucleosides, 4′‐thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.

Synthesis and characterization of 2′-modified-4′-thioRNA: a comprehensive comparison of nuclease stability

We report herein the synthesis and physical and physiological characterization of fully modified 2′-modified-4′-thioRNAs, i.e. 2′-fluoro-4′-thioRNA (F-SRNA) and 2′-O-Me-4′-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2′-modified oligonucleotides (ONs) and 4′-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest Tm value (+16°C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2′-fluoroRNA (FRNA), 2′-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t1/2 values of > 24 h against S1 nuclease (an endonuclease) and 79.2 min against SVPD (a 3′-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t1/2 = 1631 min) compared with FRNA (t1/2 = 53.2 min) and MeRNA (t1/2 = 187 min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2′-modified-4′-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.

Use of Cotton Rats to Evaluate the Efficacy of Antivirals in Treatment of Measles Virus Infections

ABSTRACT No practical animal models for the testing of chemotherapeutic or biologic agents identified in cell culture assays as being active against measles virus (MV) are currently available. Cotton rats may serve this purpose. To evaluate this possibility, 5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide (EICAR) and poly(acrylamidomethyl propanesulfonate) (PAMPS), two compounds that have been reported to inhibit MV in vitro, and ribavirin, an established antiviral drug with MV-inhibitory activity, were evaluated for their antiviral activities against MV and respiratory syncytial virus (RSV) in tissue culture and in hispid cotton rats. A single administration of PAMPS markedly inhibited pulmonary RSV or MV replication (>3 log10 reduction in pulmonary titer compared to that for controls), but only if this compound was administered intranasally at about the time of virus inoculation. Both EICAR and ribavirin exhibited therapeutic activity against RSV and MV in cotton rats when they were administered parenterally. However, both of these compounds were less effective against MV. On the basis of the pulmonary virus titers on day 4 after virus inoculation, the minimal efficacious dose of EICAR against MV (120 mg/kg of body weight/day when delivered intraperitoneally twice daily) appeared to be three times lower against this virus than that of ribavirin delivered at a similar dose (i.e., 360 mg/kg/day). These findings correlated with those obtained in vitro. The data obtained suggest that cotton rats may indeed be useful for the initial evaluation of the activities of antiviral agents against MV.

Study of Modification Pattern–RNAi Activity Relationships by Using siRNAs Modified with 4′-Thioribonucleosides†

A detailed study of the modification pattern–RNAi activity relationships by using siRNAs that are modified with 4′‐thioribonucleosides has been carried out against photinus luciferase and renilla luciferase genes in cultured mammalian NIH/3T3, HeLa, and MIA PaCa‐2 cell lines. When the photinus luciferase gene was targeted, all of the modified siRNAs showed activity equal to, or less than the unmodifed siRNA. In contrast, all modified siRNAs that have a similar modification pattern showed activity equal to or much higher than the unmodified siRNA when tested with the renilla luciferase gene. These results indicated that siRNAs such as RNA33 and RNA53, which each have four residues of the 4′‐thioribonucleoside unit on both ends of the sense strand and four residues on the 3′‐end of the antisense strand, were the most effective. Accordingly, we succeeded in developing modified siRNAs that have the greatest number of 4′‐thioribonucleosides without loss of RNAi activity, and that exhibit potent RNAi activity against two target genes in three different cell lines. Our findings also indicate the significance of target sequences and cell lines when RNAi activity is compared with that of the unmodified siRNA.

Intracellular stability of 2′-OMe-4′-thioribonucleoside modified siRNA leads to long-term RNAi effect

Chemically modified siRNAs are expected to have resistance toward nuclease degradation and good thermal stability in duplex formation for in vivo applications. We have recently found that 2′-OMe-4′-thioRNA, a hybrid chemical modification based on 2′-OMeRNA and 4′-thioRNA, has high hybridization affinity for complementary RNA and significant resistance toward degradation in human plasma. These results prompted us to develop chemically modified siRNAs using 2′-OMe-4′-thioribonucleosides for therapeutic application. Effective modification patterns were screened with a luciferase reporter assay. The best modification pattern of siRNA, which conferred duration of the gene-silencing effect without loss of RNAi activity, was identified. Quantification of the remaining siRNA in HeLa-luc cells using a Heat-in-Triton (HIT) qRT–PCR revealed that the intracellular stability of the siRNA modified with 2′-OMe-4′-thioribonucleosides contributed significantly to the duration of its RNAi activity.

Selective recognition of unnatural imidazopyridopyrimidine:naphthyridine base pairs consisting of four hydrogen bonds by the Klenow fragment.

In this work, we investigated how thermally stable ImO(N):NaN(O) and ImN(O):NaO(N) pairs are recognized by the Klenow fragment (KF). As a result, these complementary base pairs, especially the ImN(O):NaO(N) pair, were recognized selectively due to the four hydrogen bonds between the nucleobases and the shape complementarity of the Im:Na pair similar to the purine:pyrimidine base pair.

In vitro optimization of 2′-OMe-4′-thioribonucleoside–modified anti-microRNA oligonucleotides and its targeting delivery to mouse liver using a liposomal nanoparticle

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Previous studies, which characterized miRNA function, revealed their involvement in fundamental biological processes. Importantly, miRNA expression is deregulated in many human diseases. Specific inhibition of miRNAs using chemically modified anti-miRNA oligonucleotides (AMOs) can be a potential therapeutic strategy for diseases in which a specific miRNA is overexpressed. 2′-O-Methyl (2′-OMe)-4′-thioRNA is a hybrid type of chemically modified oligonucleotide, exhibiting high binding affinity to complementary RNAs and high resistance to nuclease degradation. Here, we evaluate 2′-OMe-4′-thioribonucleosides for chemical modification on AMOs. Optimization of the modification pattern using a variety of chemically modified AMOs that are perfectly complementary to mature miR-21 revealed that the uniformly 2′-OMe-4′-thioribonucleoside–modified AMO was most potent. Further investigation showed that phosphorothioate modification contributed to long-term miR-122 inhibition by the 2′-OMe-4′-thioribonucleoside–modified AMO. Moreover, systemically administrated AMOs to mouse using a liposomal delivery system, YSK05-MEND, showed delivery to the liver and efficient inhibition of miR-122 activity at a low dose in vivo.

Nucleosides and nucleotides. 127. A novel and convenient post-synthetic modification method for the synthesis of oligodeoxyribonucleotides carrying amino linkers at the 5-position of 2′-deoxyuridine ☆

5-Methoxycarbonyl-2′-deoxyuridine has been synthesized and incorporated in oligonucleotides. The fully protected oligonucleotides were treated with diaminoethane or diaminohexane and the oligonucleotides were deprotected and purified to give oligonucleotides carrying amino-linkers, which were further derivatized with an intercalator. Properties of these oligonucleotides are described.

Nucleosides and nucleotides. 116. Convenient syntheses of 3-deazaadenosine, 3-deazaguanosine, and 3-deazainosine via ring closure of 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide or -carbonitrile☆

Abstract An easy chemical synthesis of 3-deazapurine nucleosides, 3-deazainosine [1-β- D -ribofuranosylimidazo[4,5-c]pyridin-4(5H)-one (8)], 3-deazaguanosine [6-amino-1-β- D -ribofuranosylimidazo[4,5-c]pyridin-4(5H)-one (23)], and 3-deazaadenosine [4-amino-1-β- D -ribofuranosylimidazo[4,5-c]pyridine (29)] is described. The approach consists of ring closure between substituents at 4- and 5-positions of the imidazole ring. Treatment of 5-ethynyl-1-β- D -ribofuranosylimidazole-4-carboxamide (2), which was readily obtained from AICA-riboside (1), with aqueous dimethylamine, followed by aqueous acetic acid gave 8 in 64% yield. 5-(2-Hydroxyiminoethyl)-1-(2,3,5-tri-O-tert-butyldimethylsilyl-β- D -ribofuranosyl)imidazole-4-carboxamide (19) was synthesized from 3 by treatment of aqueous dimethylamine, followed by hydroxylamine hydrochloride. Dehydration of 19 was achieved by phenyl isocyanate to give 5-cyanomethyl derivative 21, from which 3-deazaguanosine (23) was easily obtained. 3-Deazaadenosine (29) was synthesized from 5-ethynyl-1-(2,3,5-tri-O-tert-butyldimethylsilyl-β- D -ribofuranosyl)imidazole-4-carbonitrile (25).

Synthesis and Characterization of Oligodeoxynucleotides Containing Naphthyridine:Imidazopyridopyrimidine Base Pairs at their Sticky Ends. Application as Thermally Stabilized Decoy Molecules†

We describe the synthesis and properties of oligodeoxynucleotides (ODNs) containing 1,8‐naphthyridine C‐nucleoside (Na‐NO) and imidazo[5′,4′:4,5]pyrido[2,3‐d]pyrimidine nucleoside (Im‐ON) at the termini. The modified ODNs were more resistant (6 to 40 times) than natural DNA to snake venom phosphodiesterase (SVPD). Although incorporation of one pair each of Na‐NO:Im‐ON on the sticky ends of the duplex was insufficient for thermal stabilization (+2.5 °C per pair relative to the G:C pair), the duplex containing two consecutive Na‐NO:Im‐ON pairs at its sticky ends was markedly stabilized thermally. The stabilizing effect of the incorporation of additional Na‐NO:Im‐ON pairs is estimated to be +7.8 °C per pair. Application as thermally stabilized decoy molecules to NF‐κB (p50) was also demonstrated. The DNA duplexes containing the Na‐NO:Im‐ON pairs (ODN I:ODN II and ODN III:ODN IV) acted as competitors to the natural NF‐κB‐binding duplex (ODN V: ODN VI), and the calculated IC50 values of ODN I:ODN II and ODN III:ODN IV were 20.1±13.3 and 10.9±4.8 nM, respectively, greater than that of ODN V:ODN VI.