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The synthesis and properties of fully modified 4′-thioDNAs, oligonucleotides consisting of 2′-deoxy-4′-thionucleosides, were examined. In addition to the known literature properties (preferable hybridization with RNA and resistance to endonuclease hydrolysis), we also observed higher resistance of 4′-thioDNA to 3′-exonuclease cleavage. Furthermore, we found that fully modified 4′-thioDNAs behaved like RNA molecules in their hybridization properties and structural aspect, at least in the case of the 4′-thioDNA duplex. This observation was confirmed by experiments using groove binders, in which a 4′-thioDNA duplex interacts with an RNA major groove binder, lividomycin A, but not with DNA groove binders, to give an increase in its thermal stability. Since a 4′-thioDNA duplex competitively inhibited the hydrolysis of an RNA duplex by RNase V1, it was not only the physical properties but also this biological data suggested that a 4′-thioDNA duplex has an RNA-like structure.

Herein we describe the synthesis of 4‘-thio-dTTP (1) and 4‘-thio-dCTP (2) and their susceptibility for PCR amplification. Double stranded 4‘-thioDNAs were amplified sufficiently by KOD dash DNA polymerase under appropriate conditions. Through this PCR study, it was shown that not only DNA-directed 4‘-thioDNA synthesis but also 4‘-thioDNA-directed 4‘-thioDNA synthesis occurred in the presence of 1 and/or 2. We then tested the ability of the resulting 4‘-thioDNAs to act as templates for transcription. In this experiment, the 4‘-thioDNAs as templates afforded RNA in 38−49% yield relative to the case of the natural DNA template. We further investigated whether transcription occurred in mammalian cells. As a result, not only natural DNA but also 4‘-thioDNAs were transcribed to afford a shRNA which showed gene-silencing effects via RNAi machinery.