Longer 19-base pair short interfering RNA duplexes rather than shorter duplexes trigger RNA interference.
Short interfering RNAs (siRNAs) are valuable reagents for sequence-specific inhibition of gene expression via the RNA interference (RNAi) pathway. Recently, it was suggested that 16-bp siRNAs are effective RNAi triggers and superior to "classical" 19-bp siRNAs. This contradiction with generally accepted knowledge prompted us to reinvestigate this issue. Here, in a series of experiments performed with siRNA duplexes of various lengths (from 19 to 15 bp) designed to silence either overexpressed enhanced green fluorescent protein or endogenously expressed CDK9, we demonstrate that 19-bp siRNAs are more active silencers than shorter corresponding duplexes. The discrepancy between our results and those questioned appears to be due to different modes of shortening the duplex (either at the 3'-end or at the 5'-end, with respect to polarity of the guide strand). Importantly, duplexes with intact 5'-ends but shortened at their 3'-ends retain target site specificity, whereas those shortened at the 5'-end are complementary to different target sites located upstream.
Sierant
Malgorzata
M
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland.
Kazmierczak-Baranska
Julia
J
Paduszynska
Alina
A
Sobczak
Milena
M
Pietkiewicz
Aleksandra
A
Nawrot
Barbara
B
D020029:Base Pairing; D001483:Base Sequence; D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9