The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene1,2,3. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.
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We thank Erik Bury for preliminary work on development of the algorithm. We also thank B. Csordas, V. Drephal, A. Garnier, S. Gfeller, D. Kirk, B. Pak, R. Theurillat, R. Widmer, S. Zhao and W. Zuercher for excellent technical support. We thank J. Mestan and F. Hofmann for E2 antibodies. We thank P. Weiss for helpful discussions and J. Hunziker and R. Haener for carefully reading the manuscript.
D.H., J.L., C.M., J.W., F.A., J.W., M.L., M.R., D.C., F.N. and J.H. are employees of Novartis. A.R., S.E. and B.M. are current or former employees of Compugen.
Dual reporter assay used to generate a data set for training of neural networks. (PDF 473 kb)
cDNA insert sequences used for siRNA design. (PDF 131 kb)
siRNA sequences and their normalized inhibitory activities used for training or testing of ANNs. (PDF 618 kb)
Single nucleotide (nt) motifs overrepresented with significance lower than 5% in the 200 most potent and 200 least potent siRNAs were taken from Supplementary Table 3. (PDF 56 kb)
siRNAs targeted to endogenous genes and used in experiments displayed in figures 3d-k. (PDF 51 kb)
Q-PCR primer sequences. (PDF 41 kb)
ABI Assays-on-demand. (PDF 43 kb)
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Huesken, D., Lange, J., Mickanin, C. et al. Design of a genome-wide siRNA library using an artificial neural network. Nat Biotechnol 23, 995–1001 (2005). https://doi.org/10.1038/nbt1118
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