Abstract
RNA interference (RNAi) is a highly efficient endogenous gene silencing mechanism mediated by short double-stranded RNAs termed small interfering RNAs (siRNAs). The current standard siRNA structure, which is used by most researchers to trigger sequence-specific target gene silencing, consists of a double strand region of 19 bp with 2 nt 3′-overhangs at both ends. However, in addition to the desired target gene silencing, this conventional siRNA structure also exhibits several unintended effects that constitute obstacles to the use of siRNA in gene function studies and therapeutics development. Here, we provide protocols for designing and preparing an alternative structure for RNAi trigger, termed asymmetric shorter-duplex RNA (asiRNA). The asiRNA structure has a duplex region shorter than 19 bp and has an asymmetric 3′-overhang structure. Importantly, the asiRNA structure not only triggers efficient target gene silencing comparable to that of the 19 bp standard siRNA structure but also significantly reduces nonspecific effects triggered by 19 bp siRNAs such as sense-strand-mediated off-target silencing and the saturation of RNAi machinery. Procedures are described for verifying that asiRNA activates gene silencing through an Ago2-dependent pathway and for assessing the miRNA pathway competition potency and specific and nonspecific silencing abilities of asiRNAs. We propose that asiRNA, an improved RNAi trigger that can overcome the nonspecific effects evoked by standard siRNA structures, can be developed as a precise and effective tool for both functional genomics and therapeutic applications.
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References
Hannon GJ (2002) RNA interference. Nature 418:244–251
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498
Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J 20:6877–6888
de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J (2007) Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 6:443–453
Lares MR, Rossi JJ, Ouellet DL (2010) RNAi and small interfering RNAs in human disease therapeutic applications. Trends Biotechnol 28:570–579
Scherer L, Rossi JJ, Weinberg MS (2007) Progress and prospects: RNA-based therapies for treatment of HIV infection. Gene Ther 14:1057–1064
Jackson AL, Linsley PS (2004) Noise amidst the silence: off-target effects of siRNAs? Trends Genet 20:521–524
Jackson AL et al (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21:635–637
Clark PR, Pober JS, Kluger MS (2008) Knockdown of TNFR1 by the sense strand of an ICAM-1 siRNA: dissection of an off-target effect. Nucleic Acids Res 36:1081–1097
Yoo JW, Kim S, Lee DK (2007) Competition potency of siRNA is specified by the 5′-half sequence of the guide strand. Biochem Biophys Res Commun 367:78–83
Koller E et al (2006) Competition for RISC binding predicts in vitro potency of siRNA. Nucleic Acids Res 34:4467–4476
Vickers TA, Lima WF, Nichols JG, Crooke ST (2007) Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells. Nucleic Acids Res 35:6598–6610
Grimm D et al (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441:537–541
Kleinman ME et al (2008) Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 452:591–597
Amarzguioui M, Holen T, Babaie E, Prydz H (2003) Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res 31:589–595
Chiu YL, Rana TM (2003) siRNA function in RNAi: a chemical modification analysis. RNA 9:1034–1048
Chang CI et al (2009) Asymmetric shorter-duplex siRNA structures trigger efficient gene silencing with reduced nonspecific effects. Mol Ther 17:725–732
Soutschek J et al (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432:173–178
Barik S (2006) RNAi in moderation. Nat Biotechnol 24:796–797
Marques JT, Williams BR (2005) Activation of the mammalian immune system by siRNAs. Nat Biotechnol 23:1399–1405
Choung S, Kim YJ, Kim S, Park HO, Choi YC (2006) Chemical modification of siRNAs to improve serum stability without loss of efficacy. Biochem Biophys Res Commun 342:919–927
Jo SG, Hong SW, Yoo JW, Lee CH, Kim S, Lee DK (2011) Selection and optimization of asymmetric siRNA targeting the human c-MET gene. Mol Cells 32:543–548
van Dongen S, Abreu-Goodger C, Enright AJ (2008) Detecting microRNA binding and siRNA off-target effects from expression data. Nat Methods 5:1023–1025
Acknowledgments
This work was supported by the Global Research Laboratory program by the Ministry of Education and Science and Technology in Korea (grant 2008–00582).
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Chang, C., Hong, S.W., Dua, P., Kim, S., Lee, Dk. (2013). The Design, Preparation, and Evaluation of Asymmetric Small Interfering RNA for Specific Gene Silencing in Mammalian Cells. In: Taxman, D. (eds) siRNA Design. Methods in Molecular Biology, vol 942. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-119-6_7
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DOI: https://doi.org/10.1007/978-1-62703-119-6_7
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-119-6
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