Sayda Elbashir

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate

Duplexes of 21–23 nucleotide (nt) RNAs are the sequence‐specific mediators of RNA interference (RNAi) and post‐transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3′ overhangs were the most efficient triggers of sequence‐specific mRNA degradation. Substitution of one or both siRNA strands by 2′‐deoxy or 2′‐O‐methyl oligonucleotides abolished RNAi, although multiple 2′‐deoxynucleotide substitutions at the 3′ end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5′ end of the guide siRNA rather than its 3′ end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.

Analysis of gene function in somatic mammalian cells using small interfering RNAs.

RNA interference (RNAi) is a highly conserved gene silencing mechanism that uses double-stranded RNA (dsRNA) as a signal to trigger the degradation of homologous mRNA. The mediators of sequence-specific mRNA degradation are 21- to 23-nt small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Twenty-one-nucleotide siRNA duplexes trigger specific gene silencing in mammalian somatic cells without activation of the unspecific interferon response. Here we provide a collection of protocols for siRNA-mediated knockdown of mammalian gene expression. Because of the robustness of the siRNA knockdown technology, genomewide analysis of human gene function in cultured cells has now become possible.