Synthesis, gene silencing activity, thermal stability, and serum stability of siRNA containing four (S)-5'-C-aminopropyl-2'-O-methylnucleosides (A, adenosine; U, uridine; G, guanosine; and C, cytidine).

Herein, we report the synthesis of (S)-5'-C-aminopropyl-2'-O-methyladenosine and (S)-5'-C-aminopropyl-2'-O-methylguanosine phosphoramidites and the properties of small interfering RNAs (siRNAs) containing four (S)-5'-C-aminopropyl-2'-O-methylnucleosides (A, adenosine; U, uridine; G, guanosine; and C, cytidine). The siRNAs containing (S)-5'-C-aminopropyl-nucleosides at the 3'- and 5'-regions of the passenger strand were well tolerated for RNA interference (RNAi) activity. Conversely, the (S)-5'-C-aminopropyl modification in the central region of the passenger strand decreased the RNAi activity. Furthermore, the siRNAs containing three or four consecutive (S)-5'-C-aminopropyl-2'-O-methylnucleosides at the 3'- and 5'-regions of the passenger strand exhibited RNAi activity similar to that of the corresponding 2'-O-methyl-modified siRNAs. Finally, it was observed that (S)-5'-C-aminopropyl modifications effectively improved the serum stability of the siRNAs, compared with 2'-O-methyl modifications. Therefore, (S)-5'-C-aminopropyl-2'-O-methylnucleosides would be useful for improving the serum stability of therapeutic siRNA molecules without affecting their RNAi activities.

(S)-5'-C-Aminopropyl-2'-O-methyl nucleosides enhance antisense activity in cultured cells and binding affinity to complementary single-stranded RNA.

Antisense oligonucleotides (ASOs) are a promising clinical tool that could be applied for unmet medical needs, but there are several limitations for their therapeutic application. Here, we designed and synthesized (S)-5'-C-aminopropyl-2'-O-methylcytidine, and oligonucleotides containing (S)-5'-C-aminopropyl-2'-O-methyluridine and -methylcytidine. We then investigated the properties of ASOs containing these nucleoside analogs. (S)-5'-C-Aminopropyl modifications enhanced the thermal stability of DNA/RNA duplexes when compared to other commercially available 2'-O-methyl modifications. This suggested that the terminal ammonium cation on the alkyl side chains neutralized the negative charge of the phosphates in the duplex. Additionally, the overall conformation of ASO/RNA duplexes was retained with the modified ASOs. Thus, these duplexes exhibited the ability to elicit RNase H activity. Furthermore, we found that ASOs containing the (S)-5'-C-aminopropyl modification exhibited higher antisense potency than those containing the 2'-O-methyl modification in cultured cells. Therefore, the (S)-5'-C-aminopropyl-2'-O-methyl nucleosides synthesized in this study are promising candidates for developing antisense therapeutics.

4'-C-Aminomethyl-2'-deoxy-2'-fluoroarabinonucleoside increases the nuclease resistance of DNA without inhibiting the ability of a DNA/RNA duplex to activate RNase H.

An antisense oligonucleotide is expected as an innovative drug for cancer and hereditary diseases. In this paper, we designed and synthesized DNAs containing a novel nucleoside analog, 1-(4-C-aminomethyl-2-deoxy-2-fluoro-ß-d-arabinofuranosyl)thymine, and evaluated their properties. It was revealed that the analog slightly decreases the thermal stability of the DNA/RNA duplex but significantly increases the stability of DNA in a buffer containing bovine serum. Furthermore, it turned out that the DNA/RNA duplex containing the analog is a good substrate for Escherichia coli RNase H. Thus, DNAs containing the nucleoside analog would be good candidates for the development of therapeutic antisense oligonucleotides.

Synthesis of siRNAs incorporated with cationic peptides R8G7 and R8A7 and the effect of the modifications on siRNA properties.

Small interfering RNA (siRNA) can be used as an innovative next-generation drug. However, there are several challenges in the therapeutic application of siRNAs, including their low cell membrane permeability. In this study, we designed and synthesized siRNAs, incorporating the cationic peptides R8G7 and R8A7 to improve cell membrane permeability of siRNAs. Thermal denaturation studies revealed that R8G7 and R8A7 modifications increased the thermal stability of the siRNA duplexes. Incorporating these peptides at the 3'-ends of the siRNA passenger strands increased the stability of the siRNAs in a buffer containing bovine serum. Further, we found that the peptide-siRNA conjugates did not show sufficient RNA interference (RNAi) activity in the absence of the transfection reagent; however, when the transfection reagent was used, the peptide-siRNA conjugates preserved their RNAi activity.

Synthesis and evaluation of (S)-5'-C-aminopropyl and (S)-5'-C-aminopropyl-2'-arabinofluoro modified DNA oligomers for novel RNase H-dependent antisense oligonucleotides.

We designed and synthesized two novel thymidine analogs: (S)-5'-C-aminopropyl-thymidine and (S)-5'-C-aminopropyl-2'-ß-fluoro-thymidine. Then, DNA oligomers containing these analogs were synthesized, and their functional properties were evaluated. Compared with the naturally occurring thymidine, it was revealed that (S)-5'-C-aminopropyl-2'-arabinofluoro-thymidine was sufficiently thermally stable, while (S)-5'-C-aminopropyl-thymidine featured thermal destabilization. The difference in thermal stability resulted from a moderate change in the secondary structure of the DNA/RNA duplexes and a molecular fluctuation in monomers derived from the (S)-5'-C-aminopropyl side chain, as well as from a variation in sugar puckering derived from the 2'-arabinofluoro modification. Meanwhile, the incorporation of these analogs significantly enhanced the nuclease resistance of the DNA oligomers. Moreover, the (S)-5'-C-aminopropyl-2'-arabinofluoro-modified DNA/RNA duplexes showed a superior ability to activate RNase H-mediated cleavage of the RNA strand compared to the (S)-5'-C-aminopropyl-modified DNA/RNA duplexes.

Synthesis and characterization of 4'-C-guanidinomethyl-2'-O-methyl-modified RNA oligomers.

This study investigated the synthesis and properties of 4'-C-guanidinomethyl-2'-O-methyluridine and RNAs containing the analog. Thermal and thermodynamic stabilities of double-stranded RNAs (dsRNAs) containing the nucleoside analog were examined. It was found that although the analog decreased the thermal and thermodynamic stabilities of dsRNA, it had base-discrimination ability. The 4'-C-guanidinomethyl modification increased stability of RNAs in a buffer containing serum. Furthermore, small interference RNAs incorporating one analog at the passenger strand still preserved their RNA interference activities. It was suggested that the 4'-guanidinomethyl modification significantly improved cell membrane permeability of RNA. Thus, 4'-C-guanidinomethyl-2'-O-methyl analogs may be useful in improving the properties of therapeutic siRNA molecules.

Synthesis and Biophysical Characterization of RNAs Containing ( R)- and ( S)-5'- C-Aminopropyl-2'- O-methyluridines.

We designed and synthesized ( R)-5'- C-aminopropyl-2'- O-methyluridine and ( S)-5'- C-aminopropyl-2'- O-methyluridine, which are applicable to small interfering RNAs (siRNAs). We have evaluated the properties of siRNAs containing ( R)-5'- C-aminopropyl-2'- O-methyl and ( S)-5'- C-aminopropyl-2'- O-methyl modifications and have compared them to that of the 4'- C-aminopropyl-2'- O-methyl modification. Although these modifications decreased the thermal stability of double-stranded RNAs (dsRNAs) and siRNAs, the dsRNA containing the ( S)-5'- C-aminopropyl-2'- O-methyl modification showed the highest melting temperature ( Tm) among them. Silencing activity of the modified siRNAs was assessed by a dual luciferase reporter assay using HeLa cells. Incorporation of the ( R)-5'- C-aminopropyl-2'- O-methyl and ( S)-5'- C-aminopropyl-2'- O-methyl modifications on a passenger and guide strand was found to be tolerated for the silencing activity of siRNAs except for in the seed region on the guide strand. Furthermore, these modifications significantly increased the stability of single-stranded RNAs (ssRNAs) and siRNAs in a buffer containing bovine serum.