An algorithm for selection of functional siRNA sequences

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Abstract

Randomly designed siRNA targeting different positions within the same mRNA display widely differing activities. We have performed a statistical analysis of 46 siRNA, identifying various features of the 19 bp duplex that correlate significantly with functionality at the 70% knockdown level and verified these results against an independent data set of 34 siRNA recently reported by others. Features that consistently correlated positively with functionality across the two data sets included an asymmetry in the stability of the duplex ends (measured as the A/U differential of the three terminal basepairs at either end of the duplex) and the motifs S1, A6, and W19. The presence of the motifs U1 or G19 was associated with lack of functionality. A selection algorithm based on these findings strongly differentiated between the two functional groups of siRNA in both data sets and proved highly effective when used to design siRNA targeting new endogenous human genes.

Section snippets

Materials and methods

siRNA preparation and nomenclature. 21-Nucleotide RNAs were synthesized, quantified, and annealed as described [14]. SiRNA were targeted to four different genes: human tissue factor (hTF) (Accession No. M16553), murine tissue factor (mTF) (Accession No. M26071), human protein serine kinase H1 (PSK) (Accession No. AJ272212), and human c-src tyrosine kinase (CSK) (Accession No. NM_004383). Each siRNA duplex was identified according to the position of the first nucleotide of the sense strand

siRNA design

The siRNA included in this analysis were designed over a 2-year period using different criteria. All hTF siRNA and five PSK siRNA (PSK132, PSK314, PSK546, PSK566, and PSK739) contained TT overhangs and have been described previously [14]. Most of the hTF target sites were derivative in nature, i.e., they were derived either from sites previously targeted by hammerhead ribozymes (hTF167, hTF478, hTF372, and hTF562) or from an efficiently targeted site (i.e., hTF siRNA targeting the region

Discussion

We have performed a statistical analysis of 80 siRNA duplexes from two independent sets of siRNA for the purpose of identifying sequence features that are correlated with functionality. Since the overhangs in siRNA appear to have little or no effect on activity [7], [14], [21], [22], [23], [24], only the 19 bp duplex region was considered. The parameters that were evaluated for correlation with functionality at the 70% knockdown level included: (1) the GC content, (2) the potential for

Acknowledgements

This work was supported by grants from the Norwegian Cancer Society, Health and Rehabilitation, and the Research Council of Norway (RCN) to H.P. M.A. is a post-doctoral research fellow of RCN.

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    Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2004.02.157.

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    Present address: Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.

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