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.

Synthesis of 4'-C-aminoalkyl-2'-O-methyl modified RNA and their biological properties.

In this paper, we describe the synthesis of 4'-C-aminoalkyl-2'-O-methylnucleosides and the properties of RNAs containing these analogs. Phosphoramidites of 4'-C-aminoethyl and 4'-C-aminopropyl-2'-O-methyluridines were prepared using glucose as starting material, and RNAs containing the analogs were synthesized using the phosphoramidites. Thermal denaturation studies revealed that these nucleoside analogs decreased the thermal stabilities of double-stranded RNAs (dsRNAs). Results of NMR, molecular modeling, and CD spectra measurements suggested that 4'-C-aminoalkyl-2'-O-methyluridine adopts an C2'-endo sugar puckering in dsRNA. The 4'-C-aminoalkyl modifications in the passenger strand and the guide strand outside the seed region were well tolerated for RNAi activity of siRNAs. Single-stranded RNAs (ssRNAs) and siRNAs containing the 4'-C-aminoethyl and 4'-C-aminopropyl analogs showed high stability in buffer containing bovine serum. Thus, siRNAs containing the 4'-C-aminoethyl and 4'-C-aminopropyl analogs are good candidates for the development of therapeutic siRNA molecules.