Synthesis and characterization of novel (S)-5′-C-aminopropyl-2′-fluorouridine modified oligonucleotides as therapeutic siRNAs

The lack of stability of natural nucleosides limits their application in small interfering RNA (siRNA)-mediated RNA interference (RNAi). Various chemical modifications have been reported to improve their pharmacokinetic behavior; however, the development of potential candidates is still underway. In this study, we designed and synthesized (S)-5′-C-aminopropyl-2′-fluorouridine (5′-AP-2′-FU) and evaluated the properties of siRNAs containing this analog. A comparative thermodynamic study revealed the enhanced thermal stability of double-stranded RNAs (dsRNAs) containing 5′-AP-2′-FU in a position-specific manner, whereas (S)-5′-C-aminopropyl-2′-O-methyluridine (5′-AP-2′-MoU)-modified dsRNAs exhibited lower melting temperatures. This improved thermal stability of RNA duplexes is attributed to favorable entropy loss, which induces the duplex into an N-type (C3′-endo) conformation and enhances duplex　binding in this case. The 5′-AP-2′-FU analog was also suitable for incorporation into the passenger strand to induce gene-silencing activity. Gene knockdown efficacy was comparable to that of unmodified siRNAs, and the best response was observed by introducing 5′-AP-2′-FU near the 3′-terminal end of the passenger strand. In addition, the single-stranded RNAs (ssRNAs) modified with 5′-AP-2′-FU showed strong resistance against decomposition by nucleases when treated with buffer containing bovine serum, which was similar to 5′-AP-2′-MoU.