The Guideline of the Design and Validation of MiRNA Mimics

  • Zhiguo Wang
Part of the Methods in Molecular Biology book series (MIMB, volume 676)


The miRNA mimic technology (miR-Mimic) is an innovative approach for gene silencing. This approach is to generate nonnatural double-stranded miRNA-like RNA fragments. Such an RNA fragment is designed to have its 5′-end bearing a partially complementary motif to the selected sequence in the 3′UTR unique to the target gene. Once introduced into cells, this RNA fragment, mimicking an endogenous miRNA, can bind specifically to its target gene and produce posttranscriptional repression, more specifically translational inhibition, of the gene. Unlike endogenous miRNAs, miR-Mimics act in a gene-specific fashion. The miR-Mimic approach belongs to the “miRNA-targeting” and “miRNA-gain-of-function” strategy and is primarily used as an exogenous tool to study gene function by targeting mRNA through miRNA-like actions in mammalian cells. The technology was developed by my research group (Department of Medicine, Montreal Heart Institute, University of Montreal) in 2007 (Xiao, et al. J Cell Physiol 212:285–292, 2007; Xiao et al. Nat Cell Biol, in review).

Key words

miRNAs Gene expression miR-Mimic miRNA interference (miRNAi) RNA interference (RNAi) 



This work was supported in part by the Canadian Institute of Health Research, Heart and Stroke Foundation of Quebec, and Fonds de la Recherche de l’Institut de Cardiologie de Montreal. Dr. Z. Wang is a Changjiang Scholar Professor of the Ministry of Education of China and a Longjiang Scholar Professor of Heilongjiang, China. The authors thank XiaoFan Yang for her excellent technical supports.


  1. 1.
    Xiao J, Yang B, Lin H, Lu Y, Luo X, Wang Z (2007) Novel approaches for gene-specific interference via manipulating actions of microRNAs: examination on the pacemaker channel genes HCN2 and HCN4. J Cell Physiol 212:285–292.PubMedCrossRefGoogle Scholar
  2. 2.
    Xiao J, Lin H, Luo X, Chen G, Wang Z (2009) microRNA-605 joins p53:Mdm2 network to form a positive feedback loop in cell-fate decision. EMBO J (accepted).Google Scholar
  3. 3.
    Xia H, Mao Q, Paulson HL, Davidson BL (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 20:1006–1010.PubMedCrossRefGoogle Scholar
  4. 4.
    Golden DE, Gerbasi VR, Sontheimer EJ (2008) An inside job for siRNAs. Mol Cell 31:309–312.PubMedCrossRefGoogle Scholar
  5. 5.
    Pushparaj PN, Aarthi JJ, Manikandan J, Kumar SD (2008) siRNA, miRNA, and shRNA: in vivo applications. J Dent Res 87:992–1003.PubMedCrossRefGoogle Scholar
  6. 6.
    Wang Z, Luo X, Lu Y, Yang B (2008) miRNAs at the heart of the matter. J Mol Med 86:771–783.PubMedCrossRefGoogle Scholar
  7. 7.
    Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB (2003) Prediction of mammalian microRNA targets. Cell 115:787–798.PubMedCrossRefGoogle Scholar
  8. 8.
    Doench JG, Sharp PA (2004) Specificity of microRNA target selection in translational repression. Genes Dev 18:504–511.PubMedCrossRefGoogle Scholar
  9. 9.
    Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, Mello CC (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23–34.PubMedCrossRefGoogle Scholar
  10. 10.
    Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99:123–132.PubMedCrossRefGoogle Scholar
  11. 11.
    Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17:438–442.PubMedCrossRefGoogle Scholar
  12. 12.
    Hutvágner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science 297:2056–2060.PubMedCrossRefGoogle Scholar
  13. 13.
    Zeng Y, Yi R, Cullen BR (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci USA 100:9779–9784.PubMedCrossRefGoogle Scholar
  14. 14.
    Brennecke J, Stark A, Russell RB, Cohen SM (2005) Principles of microRNA–target recognition. PLoS Biol 3:404–418.CrossRefGoogle Scholar
  15. 15.
    Kiriakidou M, Tan GS, Lamprinaki S, De Planell-Saguer M, Nelson PT, Mourelatos Z (2007) An mRNA m(7)G cap binding-like motif within human Ago2 represses translation. Cell 129:1141–1151.PubMedCrossRefGoogle Scholar
  16. 16.
    Kloosterman WP, Wienholds E, Ketting RF, Plasterk RH (2004) Substrate requirements for let-7 function in the developing zebrafish embryo. Nucleic Acids Res 32:6284–6291.PubMedCrossRefGoogle Scholar
  17. 17.
    Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200.PubMedCrossRefGoogle Scholar
  18. 18.
    Haley B, Zamore PD (2004) Kinetic analysis of the RNAi enzyme complex. Nat Struct Mol Biol 11:599–606.PubMedCrossRefGoogle Scholar
  19. 19.
    Martinez J, Tuschl T (2004) RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev 18:975–980.PubMedCrossRefGoogle Scholar
  20. 20.
    Kiriakidou M, Nelson PT, Kouranov A, Fitziev P, Bouyioukos C, Mourelatos Z, Hatzigeorgiou A (2004) A combined computational–experimental approach predicts human microRNA targets. Genes Dev 18:1165–1178.PubMedCrossRefGoogle Scholar
  21. 21.
    Ha I, Wightman B, Ruvkun G (1996) A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation. Genes Dev 10:3041–3050.PubMedCrossRefGoogle Scholar
  22. 22.
    Chiu YL, Rana TM (2003) siRNA function in RNAi: a chemical modification analysis. RNA 9:1034–1048.PubMedCrossRefGoogle Scholar
  23. 23.
    Czauderna F, Fechtner M, Dames S, Aygun H, Klippel A, Pronk GJ, Giese K, Kaufmann J (2003) Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res 31:2705–2716.PubMedCrossRefGoogle Scholar
  24. 24.
    Prakash TP, Allerson CR, Dande P, Vickers TA, Sioufi N, Jarres R, Baker BF, Swayze EE, Griffey RH, Bhat B (2005) Positional effect of chemical modifications on short interference RNA activity in mammalian cells. J Med Chem 48:4247–4253.PubMedCrossRefGoogle Scholar
  25. 25.
    Kraynack BA, Baker BF (2006) Small interfering RNAs containing full 2′-Omethylribonucle-otide-modified sense strands display Argonaute2/eIF2C2-dependent activity. RNA 12:163–176.PubMedCrossRefGoogle Scholar
  26. 26.
    Allerson CR, Sioufi N, Jarres R, Prakash TP, Naik N, Berdeja A, Wanders L, Griffey RH, Swayze EE, Bhat B (2005) Fully 2′-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. J Med Chem 48:901–904.PubMedCrossRefGoogle Scholar
  27. 27.
    Layzer JM, McCaffrey AP, Tanner AK, Huang Z, Kay MA, Sullenger BA (2004) In vivo activity of nuclease-resistant siRNAs. RNA 10:766–771.PubMedCrossRefGoogle Scholar
  28. 28.
    Harborth J, Elbashir SM, Vandenburgh K, Manninga H, Scaringe SA, Weber K, Tuschl T (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev 13:83–105.PubMedCrossRefGoogle Scholar
  29. 29.
    Braasch DA, Jensen S, Liu Y, Kaur K, Arar K, White MA, Corey DR (2003) RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 42:7967–7975.PubMedCrossRefGoogle Scholar
  30. 30.
    Morrissey DV, Blanchard K, Shaw L, Jensen K, Lockridge JA, Dickinson B, McSwiggen JA, Vargeese C, Bowman K, Shaffer CS, Polisky BA, Zinnen S (2005) Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication. Hepatology 41:1349–1356.PubMedCrossRefGoogle Scholar
  31. 31.
    Amarzguioui M, Holen T, Babaie E, Prydz H (2003) Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res 31:589–595.PubMedCrossRefGoogle Scholar
  32. 32.
    Altmann KH, Dean NM, Fabbro D, Freier SM, Geiger T, Haener R, Huesken D, Martin P, Monia BP, Muller M, Natt F, Nicklin P, Phillips J, Pieles U, Sasmor H, Moser H (1996) Second generation of antisense oligonucleotides. From nuclease resistance to biological efficacy in animals. Chimia 50:168–176.Google Scholar
  33. 33.
    Hoke GD, Draper K, Freier SM, Gonzalez C, Driver VB, Zounes MC, Ecker DJ (1991) Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection. Nucleic Acids Res 19:5743–5748.PubMedCrossRefGoogle Scholar
  34. 34.
    Braasch DA, Paroo Z, Constantinescu A, Ren G, Oz OK, Mason RP, Corey DR (2004) Biodistribution of phosphodiester and phosphorothioate siRNA. Bioorg Med Chem Lett 14:1139–1143.PubMedCrossRefGoogle Scholar
  35. 35.
    McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA (2002) RNA interference in adult mice. Nature 418:38–39.PubMedCrossRefGoogle Scholar
  36. 36.
    Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN, Harborth J, Heyes JA, Jeffs LB, John M, Judge AD, Lam K, McClintock K, Nechev LV, Palmer LR, Racie T, Rohl I, Seiffert S, Shanmugam S, Sood V, Soutschek J, Toudjarska I, Wheat AJ, Yaworski E, Zedalis W, Koteliansky V, Manoharan M, Vornlocher HP, MacLachlan I (2006) RNAi-mediated gene silencing in non-human primates. Nature 441:111–114.PubMedCrossRefGoogle Scholar
  37. 37.
    Morrissey DV, Lockridge J.A, Shaw L, Blanchard K, Jensen K, Breen W, Hartsough K, Machemer L, Radka S, Jadhav V, Vaish N, Zinnen S, Vargeese C, Bowman K, Shaffer CS, Jeffs LB, Judge A, MacLachlan I, Polisky B (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat Biotechnol 23:1002–1007.PubMedCrossRefGoogle Scholar
  38. 38.
    Ge Q, Filip L, Bai A, Nguyen T, Eisen HN, Chen J (2004) Inhibition of influenza virus production in virus-infected mice by RNA interference. Proc Natl Acad Sci USA 101:8676–8681.PubMedCrossRefGoogle Scholar
  39. 39.
    Yang B, Lin H, Xiao J, Luo X, Li B, Lu Y, Wang H, Wang Z (2007) The muscle-specific microRNA miR-1 causes cardiac arrhythmias by targeting GJA1 and KCNJ2 genes. Nat Med 13:486–491.PubMedCrossRefGoogle Scholar
  40. 40.
    Xiao J, Luo X, Lin H, Xu C, Gao H, Wang H, Yang B, Wang Z (2007) MicroRNA miR-133 represses HERG K+ channel expression contributing to QT prolongation in diabetic hearts. J Biol Chem 282:12363–12367.PubMedCrossRefGoogle Scholar
  41. 41.
    Luo X, Lin H, Lu Y, Li B, Xiao J, Yang B, Wang Z (2007) Transcriptional activation by stimulating protein 1 and post-transcriptional repression by muscle-specific microRNAs of IKs-encoding genes and potential implications in regional heterogeneity of their expressions. J Cell Physiol 212:358–367.PubMedCrossRefGoogle Scholar
  42. 42.
    Luo X, Lin H, Pan Z, Xiao J, Zhang Y, Lu Y, Yang B, Wang Z (2008) Overexpression of Sp1 and downregulation of miR-1/miR-133 activates re-expression of pacemaker channel genes HCN2 and HCN4 in hypertrophic heart. J Biol Chem 283:20045–20052.PubMedCrossRefGoogle Scholar
  43. 43.
    Xu C, Lu Y, Lin H, Xiao J, Wang H, Luo X, Li B, Yang B, Wang Z (2007) The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis via targeting HSP60/HSP70 and caspase-9 in cardiomyocytes. J Cell Sci 120:3045–3052.PubMedCrossRefGoogle Scholar
  44. 44.
    Lu Y, Xiao J, Lin H, Bai Y, Luo X, Wang Z, Yang B (2009) Complex antisense inhibitors offer a superior approach for microRNA research and therapy. Nucleic Acids Res 37:e24–e33.PubMedCrossRefGoogle Scholar
  45. 45.
    Xiao L, Xiao J, Luo X, Lin H, Wang Z, Nattel S (2008) Feedback remodeling of cardiac potassium current expression. A novel potential mechanism for control of repolarization reserve. Circulation 118:983–992.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Zhiguo Wang
    • 1
  1. 1.Department of Medicine, Montreal Heart InstituteUniversity of MontrealMontrealCanada

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