Rational siRNA design for RNA interference

Abstract

Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi)1,2,3,4. RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi5,6. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3′-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.

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Figure 1: The test panel of 180 siRNAs, targeting firefly luciferase (left) and human cyclophilin B (right).
Figure 2: Development of a multicomponent algorithm.
Figure 3: Validation of the multi-component rational design algorithm.

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References

  1. 1

    Sharp, P.A. RNA interference–2001. Genes Dev. 15, 485–490 (2001).

    CAS  Article  Google Scholar 

  2. 2

    Bernstein, E., Caudy, A.A., Hammond, S.M. & Hannon, G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Nykanen, A., Haley, B. & Zamore, P.D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309–321 (2001).

    CAS  Article  Google Scholar 

  4. 4

    Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Khvorova, A., Reynolds, A. & Jayasena, S. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Schwarz, D.S. et al. Unexpected asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Elbashir, S.M., Harborth, J., Weber, K. & Tuschl, T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, 199–213 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E. & Prydz, H. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 30, 1757–1766 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Ding, Y. & Lawrence, C.E. Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond. Nucleic Acids Res. 29, 1034–1046 (2001).

    CAS  Article  Google Scholar 

  10. 10

    Kirchner, R., Vogtherr, M., Limmer, S. & Sprinzl, M. Secondary structure dimorphism and interconversion between hairpin and duplex form of oligoribonucleotides. Antisense Nucleic Acid Drug. Dev. 8, 507–516 (1998).

    CAS  Article  Google Scholar 

  11. 11

    Groebe, D.R. & Uhlenbeck, O.C. Characterization of RNA hairpin loop stability. Nucleic Acids Res. 16, 11725–11735 (1988).

    CAS  Article  Google Scholar 

  12. 12

    Groebe, D.R. & Uhlenbeck, O.C. Thermal stability of RNA hairpins containing a four-membered loop and a bulge nucleotide. Biochemistry 28, 742–747 (1989).

    CAS  Article  Google Scholar 

  13. 13

    Semizarov, D. et al. Specificity of short interfering RNA determined through gene expression signatures. Proc. Natl. Acad. Sci. USA 100, 6347–6352 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Lau, N.C., Lim, L.P., Weinstein, E.G. & Bartel, D.P. An abundant class of tiny RNAs with probable roles in Caenorhabditis elegans. Science 294, 858–862 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Ambros, V. et al. A uniform system for microRNA annotation. RNA 9, 277–279 (2003).

    CAS  Article  Google Scholar 

  16. 16

    Lim, L.P., Glasner, M.E., Yekta, S., Burge, C.B. & Bartel, D.P. Vertebrate microRNA genes. Science 299, 1540 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Hutvagner, G. & Zamore, P.D. A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060 (2002).

    CAS  Article  Google Scholar 

  18. 18

    Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W. & Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888 (2001).

    CAS  Article  Google Scholar 

  19. 19

    Donis-Keller, H. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 7, 179–192 (1979).

    CAS  Article  Google Scholar 

  20. 20

    Scaringe, S.A. Advanced 5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis. Methods Enzymol. 317, 3–18 (2000).

    CAS  Article  Google Scholar 

  21. 21

    Wang, J. et al. Regulation of insulin preRNA splicing by glucose. Proc. Natl. Acad. Sci. USA 94, 4360–4365 (1997).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We would like to acknowledge Jason Spellberg and Julia Kendall for assistance with manuscript preparation and Carl Novina and Alexey Wolfson for helpful discussions. This work was supported in part by the National Science Foundation under grant no. 0320480.

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Correspondence to Anastasia Khvorova.

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Authors are employed by Dharmacon, which is involved in marketing RNAi-based technologies.

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Reynolds, A., Leake, D., Boese, Q. et al. Rational siRNA design for RNA interference. Nat Biotechnol 22, 326–330 (2004). https://doi.org/10.1038/nbt936

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