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 T-m value (16C 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 t(1/2) values of24h against S1 nuclease (an endonuclease) and 79.2min against SVPD (a 3-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50 human plasma (t(1/2)1631min) compared with FRNA (t(1/2)53.2min) and MeRNA (t(1/2)187min), 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.