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A study on the fundamental factors determining the efficacy of siRNAs with high C/G contents

Cellular & Molecular Biology Letters volume 13, pages 283–302 (2008)Cite this article

Abstract

Although there are many reports about the efficacy of siRNAs, it is not clear whether those siRNAs with high C/G contents can be used to silence their target mRNAs efficiently. In this study, we investigated the structure and function of a group of siRNAs with high C/G contents. The results showed that single siRNAs against the Calpain, Otoferlin and Her2 mRNAs could induce different silencing effects on their targets, suggesting that the accessibility to target sequences influences the efficacy of siRNA. Unexpectedly, a single siRNA could target its cognate sequence in the 3’UTR of EEF1D or the 5’UTR of hTRF2 or CDC6. Their interaction induced different modes of gene silencing. Furthermore, the introduction of mutations into the 3’ end of the passenger strand showed that the position and number of mutated nucleotides could exert some influence on the efficacy of siRNA. However, these mutations did not completely block the passenger strand from exerting its RNAi effect. Interestingly, our findings also indicated that the target mRNA might play essential roles in maintaining or discarding the guide strand in RISCs. Thus, the conclusion could be drawn that favorable siRNA sequences, accessible target structures and the fast cleavage mode are necessary and sufficient prerequisites for efficient RNAi.

Abbreviations

Ago:

argonaute

C:

cytosine

Calp:

calpain

ds:

double-strand

G:

guanine

Her2:

v-erb-b2 erythroblastic leukemia viral oncogene homolog 2

HRP:

peroxidase

Otof:

otoferin

RISC:

RNA-induced silencing complex

RNAi:

RNA interference

RNase:

ribonuclease

siRNA:

short interfering RNA

TMB:

3,3′,5,5′ tetramethylbenzidine

References

  1. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391 (1998) 806–811.

    Article  PubMed  CAS  Google Scholar 

  2. Sharp, P.A. RNAi and double-strand RNA. Genes Dev. 13 (2002) 139–141.

    Article  Google Scholar 

  3. Kim, V.N. Small RNAs just got bigger: Piwi-interacting RNAs (piRNAs) in mammalian testes. Genes Dev. 20 (2006) 1993–1997.

    Article  PubMed  CAS  Google Scholar 

  4. Carmell, M.A. and Hannon, G.J. RNase III enzymes and the initiation of gene silencing. Nat. Struct. Mol. Biol. 11 (2004) 214–218.

    Article  PubMed  CAS  Google Scholar 

  5. Elbashir, S.M., Lendeckel, W. and Tuschl, T. RNA interference is mediated by 21-and 23-nucleotide RNAs. Genes Dev. 15 (2001) 188–200.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  7. Parker, J.S., Roe, SM. and Barford, D. Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex. Nature 434 (2005) 663–666.

    Article  PubMed  CAS  Google Scholar 

  8. Lingel, A., Simon, B., Izaurralde, E. and Sattler, M. Nucleic acid 3′-end recognition by the Argonaute2 PAZ domain. Nat. Struct. Mol. Biol. 11 (2004) 576–577.

    Article  PubMed  CAS  Google Scholar 

  9. Ma, J.B., Yuan, Y.R., Meister, G., Pei, Y., Tuschl, T. and Patel, D.J. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434 (2005) 666–670.

    Article  PubMed  CAS  Google Scholar 

  10. Valencia-Sanchez, M.A., Liu, J., Hannon G.J. and Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20 (2006) 515–524.

    Article  PubMed  CAS  Google Scholar 

  11. Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson, J.M., Song, J.J., Hammond, S.M., Joshua-Tor, L, and Hannon, G.J. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305 (2004) 1437–1441.

    Article  PubMed  CAS  Google Scholar 

  12. Dykxhoorn, D.M. and Lieberman, J. Running interference: Prospects and obstacles to using small interfering RNAs as small molecule drugs. Annu. Rev. Biomed. Eng. 8 (2006) 377–402.

    Article  PubMed  CAS  Google Scholar 

  13. Liu T.G., Yin, J.Q., Shang, B.Y., Min, Z., He, H.W., Jiang, J.M., Chen, F., Zhen, Y.S. and Shao, R.G. Silencing of hdm2 oncogene by siRNA inhibits p53-dependent human breast cancer. Cancer Gene Ther. 11 (2004) 748–756.

    Article  PubMed  CAS  Google Scholar 

  14. Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda, R. and Saigo, K. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 32 (2004) 936–948.

    Article  PubMed  CAS  Google Scholar 

  15. Schwarz, D.S., Hutvagner, G., Du, T., Xu, Z., Aronin, N. and Zamore, P.D. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115 (2003) 199–208.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  18. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S. and Khvorova, A. Rational siRNA design for RNA interference. Nat. Biotechnol. 22 (2004) 326–330.

    Article  PubMed  CAS  Google Scholar 

  19. Jagla, B., Aulner, N., Kelly, P.D., Song, D., Volchuk, A., Zatorski, A., Shum, D., Mayer, T., De Angelis, D.A. and Ouerfelli, O. Sequence characteristics of functional siRNAs. RNA 11 (2005) 864–872.

    Article  PubMed  CAS  Google Scholar 

  20. Vickers, T.A., Koo, S., Bennett, C.F., Crooke, S.T., Dean, N.M. and Baker, B.F. Efficient reduction of target RNAs by small interfering RNA and Rnase H-dependent antisense agents. J. Biol. Chem. 278 (2003) 7108–7118.

    Article  PubMed  CAS  Google Scholar 

  21. Kretschmer-Kazemi, Far., R. and Sczakiel, G. The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res. 31 (2003) 4417–4424.

    Article  CAS  Google Scholar 

  22. Clote, P. RNALOSS: a web server for RNA locally optimal secondary structures. Nucleic Acids Res. 33 (2005) W600–664.

    Article  PubMed  CAS  Google Scholar 

  23. Takasaki, S., Kotani, S. and Konagaya, A. An effective method for selecting siRNA target sequences in mammalian cells. Cell Cycle 3 (2004) 790–795.

    PubMed  CAS  Google Scholar 

  24. Heale, B.S., Soifer, H.S., Bowers, C. and Rossi, J.J. siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res. 33 (2005) e30.

    Article  PubMed  CAS  Google Scholar 

  25. Schubert, S. Grunweller, A., Erdmann, V.A. and Kurreck, J. Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions. J. Mol. Biol. 348 (2005) 883–893.

    Article  PubMed  CAS  Google Scholar 

  26. Westerhout, E.M., Ooms, M., Vink, M., Das, A.T. and Berkhout, B. HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res. 33 (2005) 796–804.

    Article  PubMed  CAS  Google Scholar 

  27. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31 (2003) 3406–3415.

    Article  PubMed  CAS  Google Scholar 

  28. Ding, Y., Chan, C.Y. and Lawrence, C.E. Sfold web server for statistical folding and rational design of nucleic acids. Nucleic Acids Res. 32 (2004) 135–141.

    Article  CAS  Google Scholar 

  29. Pei Y. and Tuschl, T. On the art of identifying effective and specific siRNAs. Nat. Methods 3 (2006) 670–676.

    Article  PubMed  CAS  Google Scholar 

  30. Hsieh AC, Bo R, Manola J, Vazquez F, Bare O, Khvorova A, Scaringe S, Sellers WR. A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens. NucleicAcids Res. 32 (2004) 893–901.

    Article  CAS  Google Scholar 

  31. Overhoff, M., Alken, M., Far, R.K., Lemaitre, M., Lebleu, B., Sczakiel, G. and Robbins, I. Local RNA target structure influences SiRNA efficacy: a systematic global analysis. J. Mol. Biol. 348 (2005) 871–881.

    Article  PubMed  CAS  Google Scholar 

  32. Yoshinari, K., Miyagishi, M. and Taira, K. Effects on RNAi of the tight structure, sequence and position of the targeted region. Nucleic Acids Res. 32 (2004) 691–699.

    Article  PubMed  CAS  Google Scholar 

  33. Hohjoh, H. Enhancement of RNAi activity by improved siRNA duplexes. FEBS Lett. 557 (2004) 193–198.

    Article  PubMed  CAS  Google Scholar 

  34. Amarzguioui, M. and Prydz, H. An algorithm for selection of functional siRNA sequences. Biochem. Biophys. Res. Commun. 316 (2004) 1050–1058.

    Article  PubMed  CAS  Google Scholar 

  35. Tomari, Y., Matranga, C., Haley, B., Martinez, N. and Zamore, P.D. A protein sensor for siRNA asymmetry. Science 306 (2004) 1377–1380.

    Article  PubMed  CAS  Google Scholar 

  36. Rose, S.D., Kim, D.H., Amarzguioui, M., Heidel, J.D., Collingwood, M.A., Davis, M.E., Rossi, J.J. and Behlke, M.A. Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res. 33 (2005) 4140–4156.

    Article  PubMed  CAS  Google Scholar 

  37. Matranga, C., Tomari, Y., Shin, C., Bartel, D.P. and Zamore, P.D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123 (2005) 607–620.

    Article  PubMed  CAS  Google Scholar 

  38. Ma, J.B., Yuan, Y.R., Meister, G., Pei, Y., Tuschl, T, and Patel, D.J. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434 (2005) 666–670.

    Article  PubMed  CAS  Google Scholar 

  39. Brown, K.M., Chu, C.Y. and Rana, T.M. Target accessibility dictates the potency of human RISC. Nat. Struct. Mol. Biol. 12 (2005) 469–470.

    Article  PubMed  CAS  Google Scholar 

  40. Bohula, E.A., Salisbury, A.J., Sohail, M., Playford, M.P., Riedemann, J., Southern, E.M. and Macaulay, V.M. The efficacy of small interfering RNAs targeted to the type 1 IGF receptor is influenced by secondary structure in the IGF1R transcript. J. Biol.Chem. 278 (2003) 15991–15997.

    Article  PubMed  CAS  Google Scholar 

  41. Luo, K.Q. and Chang, D.C. The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. Biochem. Biophys. Res. Commun. 318 (2004) 303–310.

    Article  PubMed  CAS  Google Scholar 

  42. Sohail, M., Akhtar, S. and Southern, E.M. The folding of large RNAs studied by hybridization to arrays of complementary oligonucleotides. RNA 5 (1999) 646–655.

    Article  PubMed  CAS  Google Scholar 

  43. Tomari, Y, Du, T and Zamore, P.D. Sorting of Drosophila small silencing RNAs. Cell 130 (2007) 299–308.

    Article  PubMed  CAS  Google Scholar 

  44. Okamura, K., Ishizuka, A., Siomi, H. and Siomi, M.C. Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18 (2004) 1655–1666.

    Article  PubMed  CAS  Google Scholar 

  45. Haley, B. and Zamore, P.D. Kinetic analysis of the RNAi enzyme complex. Nat. Struct. Mol. Biol. 11 (2004) 599–606.

    Article  PubMed  CAS  Google Scholar 

  46. Tang, G. siRNA and miRNA: an insight into RISCs. Trends Biochem. Sci. 30 (2005) 106–114.

    Article  PubMed  CAS  Google Scholar 

  47. Jing, Q., Huang, S., Guth, S., Zarubin, T., Motoyama, A., Chen, J., Di Padova, F., Lin, S.C., Gram, H. and Han J. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120 (2005) 623–634.

    Article  PubMed  CAS  Google Scholar 

  48. Morris, K.V., Chan, S.W., Jackbsen, S.E. and Looney, D.J. Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305 (2004) 1289–1292.

    Article  PubMed  CAS  Google Scholar 

  49. Jackson, A.L. and Linsley, P.S. Noise amidst the silence: off-target effects of siRNAs? Trends Genet. 20 (2004) 521–524.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Systems Biology Research Center, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing, 100101, P.R. China

    Jie-Ying Liao, James Q. Yin, Fang Chen & Jia-Chang Yue

  2. Molecular Oncology Research Institute, Tufts-NEMC, Boston, MA, 02111, USA

    Tie-Gang Liu

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  1. Jie-Ying Liao
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  2. James Q. Yin
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  3. Fang Chen
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Correspondence to James Q. Yin.

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Liao, JY., Yin, J.Q., Chen, F. et al. A study on the fundamental factors determining the efficacy of siRNAs with high C/G contents. Cell Mol Biol Lett 13, 283–302 (2008). https://doi.org/10.2478/s11658-008-0001-1

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  • DOI: https://doi.org/10.2478/s11658-008-0001-1

Keywords

Cellular & Molecular Biology Letters

ISSN: 1689-1392

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