Small Interfering RNAs: A Revolutionary Tool for the Analysis of Gene Function and Gene Therapy

Methods for the delivery of siRNAs to somatic mammalian cells. (A) Synthetic 21-nt siRNA duplex prepared by chemical synthesis (23) aligned to a target mRNA. Target regions are selected such that siRNA sequences may contain uridine residues in the 2-nt overhangs. Uridine residues in the 2-nt 3′-overhang can be replaced by 2′-deoxythymidine without loss of activity, which significantly reduces costs of RNA synthesis and may also enhance nuclease resistance of siRNA duplexes when applied to mammalian cell (7). (B) Plasmid-based expression of short hairpin loops which give rise to siRNAs in vivo (11). The polymerase III promoter of H1 RNA (human RNase P RNA) drives the transcription of a nineteen-base-pair/nine-nucleotide-loop RNA hairpin. The transcription is terminated by the encounter of a polythymidine tract (T5) after the incorporation of two to three uridine residues encoded by the T5 element. Northern blot analysis showed that the hairpin RNAs were processed to siRNAs.