Amplification of 4‘-ThioDNA in the Presence of 4‘-Thio-dTTP and 4‘-Thio-dCTP, and 4‘-ThioDNA-Directed Transcription in Vitro and in Mammalian Cells

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.