Synthesis and properties of vitamin E analog-conjugated neomycin for delivery of RNAi drugs to liver cells.

RNA interference (RNAi) is a promising tool to regulate gene expression by external double stranded RNAs (dsRNAs) such as siRNAs. As an efficient method to deliver siRNAs to liver cells, we propose a novel strategy using vitamin E (VE)-conjugated neomycin derivatives. With the aim of delivering RNAi-based drugs to liver cells, several tripod-type and prodrug-type neomycin derivatives were synthesized, all of which were thermodynamically stabilized RNA duplexes. The prodrug-type derivative 7 and the tripod-type derivative 10 were delivered to liver cancer cells and successfully induced RNAi activity. These results indicated the potential use of natural aminoglycosides as carriers of RNAi drugs.

Synthesis of oligodiaminomannoses and analysis of their RNA duplex binding properties and their potential application as siRNA-based drugs.

The synthesis of artificial cationic oligodiaminosaccharides, α-(1 → 4)-linked-2,6-diamino-2,6-dideoxy-d-mannopyranose oligomers (ODAMans), and their interactions with RNA duplexes are described. The monomer through the pentamer, all of which bear unnatural 2,6-diaminomannose moieties, were successfully prepared. UV melting and fluorescence anisotropy analyses revealed that the ODAMans bound and thermodynamically stabilized both 12mer RNA duplexes and an siRNA. Furthermore, it was clearly shown that the siRNA acquired substantial RNase A resistance due to its binding to the ODAMan 4mer.

Synthesis and properties of double-stranded RNA-bindable oligodiaminogalactose derivatives conjugated with vitamin E.

RNA interference (RNAi) is a gene-regulating system that is controlled by external short interfering RNAs (siRNAs). Sequence selective gene silencing by siRNA shows promise in clinical research. However, there have been few efficient methods for delivering siRNAs to target cells. In this study, we propose a novel type of RNA duplex-bindable molecule with an oligodiaminosaccharide structure. These 2,6-diamino-2,6-dideoxy-(1-4)-ß-D-galactopyranose oligomers (oligodiaminogalactoses; ODAGals) conjugated with α-tocopherol (vitamin E; VE) or a VE analog were designed as novel siRNA-bindable molecules that can be utilized to deliver RNAi drugs to the liver. Among these compounds, the VE analog-bound ODAGal was suggested to bind to RNA duplexes without inhibiting RNAi activity.

Synthesis and properties of cationic oligopeptides with different side chain lengths that bind to RNA duplexes.

A series of artificial peptides bearing cationic functional groups with different side chain lengths were designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated. The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relationship between the side chain length and the melting temperature (Tm) of the peptide-RNA complexes was observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain lengths, such as L-2-amino-4-guanidinobutyric acid (Agb) or L-arginine (Arg) oligomers, stabilized both RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of flexibility by introducing glycine (Gly) residues into the L-2-amino-3-guanidinopropionic acid (Agp) oligomers. It was found that insertion of Gly did not affect the thermal stability of the peptide-RNA complexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability. Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of RNA/RNA duplexes.

Synthesis of oligodiaminosaccharides having α-glycoside bonds and their interactions with oligonucleotide duplexes.

Syntheses of the novel oligodiaminosaccharides, α-(1→4)-linked-2,6-diamino-2,6-dideoxy-D-glucopyranose oligomers, and their interactions with nucleic acid duplexes DNA-DNA, RNA-RNA, and DNA-RNA are described. Monomers to tetramers of oligodiaminoglucose derivatives having α-glycosyl bonds were successfully synthesized using a chain elongation cycle including glycosylation reactions of a 6-phthalimide glycosyl donor. UV melting experiments for a variety of nucleic acid duplexes in the absence and presence of the oligodiaminosaccharides were performed. The synthesized oligodiaminosaccharides exhibited notable thermodynamic stabilization effects on A-type RNA-RNA and DNA-RNA duplexes, whereas B-type DNA-DNA duplexes were not stabilized by the synthesized oligodiaminosaccharides. Among the oligodiaminosaccharides, the tetramer exhibited the highest ability to stabilize A-type duplexes, and the increase in T(m) values induced by the tetramer were higher than those induced by neomycin B and tobramycin, which are known aminoglycosides having ability to bind and stabilize a variety of RNA molecules. CD spectrometry experiments revealed that the oligodiaminosaccharides caused small structural changes in RNA-RNA duplexes, whereas no appreciable changes were observed in the structure of DNA-DNA duplexes. ITC (isothermal titration calorimetry) experiments demonstrated that the amount of heat generated by the interaction between RNA-RNA duplexes and the tetradiaminosaccharides was approximately double that generated by that between DNA-DNA duplexes and the tetradiaminosaccharides. These results strongly suggested the existence of an A-type nucleic acid specific-binding mode of the oligodiaminosaccharides, which bind to these duplexes and cause small structural changes.

Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol.

We developed an efficient system for delivering short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. The α-tocopherol-conjugated siRNA was effective and safe for RNA interference-mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid)-DNA gapmer antisense oligonucleotide (ASO) was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5'-end of the ASO sequence by using 5'-α-tocopherol-conjugated 4- to 7-mers of unlocked nucleic acid (UNA) as a "second wing." Intravenous injection of mice with this α-tocopherol-conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol-conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS) molecules are bound to ASO with UNA sequences.

Stabilization of A-type nucleic acid duplexes by novel oligodiaminosaccharides.

Novel oligodiaminosaccharides, alpha-(1-->4)-linked-2,6-diamino-2,6-dideoxy-D-glucopyranose oligomers, were designed and synthesized to bind to A-type nucleic acid duplexes, such as RNA duplexes. Using properly designed glycosyl donors and glycosyl acceptors, an alpha-selective glycosylation was achieved. A chain elongation cycle was established and the oligodiaminosaccharides bearing the alpha-glycoside bonds (1-4mer) were synthesised. Analyses of their interactions with oligonucleotide duplexes were performed by using CD spectrometry and UV melting experiments. These experiments revealed that the 3mer and 4mer were found to remarkably stabilize RNA-RNA and RNA-DNA duplexes with small structural changes.