Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway.
RNA interference (RNAi), mediated by either long double-stranded RNA (dsRNA) or short interfering RNA (siRNA), has become a routine tool for transient knockdown of gene expression in a wide range of organisms. The antisense strand of the siRNA duplex (antisense siRNA) was recently shown to have substantial mRNA depleting activity of its own. Here, targeting human Tissue Factor mRNA in HaCaT cells, we perform a systematic comparison of the activity of antisense siRNA and double-strand siRNA, and find almost identical target position effects, appearance of mRNA cleavage fragments and tolerance for mutational and chemical backbone modifications. These observations, together with the demonstration that excess inactive double-strand siRNA blocks antisense siRNA activity, i.e. shows sequence-independent competition, indicate that the two types of effector molecules share the same RNAi pathway. Interest ingly, both FITC-tagged and 3'-deoxy antisense siRNA display severely limited activity, despite having practically wild-type activity in a siRNA duplex. Finally, we find that maximum depletion of target mRNA expression occurs significantly faster with antisense siRNA than with double-strand siRNA, suggesting that the former enters the RNAi pathway at a later stage than double-strand siRNA, thereby requiring less time to exert its activity.
Holen
Torgeir
T
The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, N-0349 Oslo, Norway.
Amarzguioui
Mohammed
M
Babaie
Eshrat
E
Prydz
Hans
H
D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D034622:RNA Interference / Q000502:physiology*; D016372:RNA, Antisense / Q000235:genetics / Q000502:physiology; D012330:RNA, Double-Stranded / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology*; D013925:Thromboplastin / Q000235:genetics; D014162:Transfection
D016372:RNA, Antisense; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013925:Thromboplastin