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Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers

DOI: 10.4061/2011/131579

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RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future. 1. Introduction RNA interference (RNAi) is an evolutionarily conserved, gene-silencing mechanism that is triggered by double-stranded RNA (dsRNA). Two types of small RNA—namely, small interfering RNA (siRNA) and microRNA (miRNA)—are central players in RNAi. Both siRNAs and miRNAs regulate gene expression by annealing to mRNA sequence elements that are fully or partially complementary [1, 2]. Since transfected synthetic siRNAs were shown to induce RNAi in mammalian cells [3], they have been widely used to decipher gene function through suppression of gene expression, and they have also attracted attention because of their potential for therapeutic applications [4, 5]. miRNAs are a phylogenetically conserved family of endogenous small RNAs that play important roles in a wide variety of biological functions, including cell differentiation, tumor genesis, apoptosis, and metabolism [1, 2, 6, 7]. miRNAs are initially generated as long primary transcripts (pri-miRNA) that are processed in the nucleus by the enzyme complexes Drosha and DiGeorge Critical Region 8 (DGCR8) to a 70–90?nt stem-loop structure called pre-miRNA. The pre-miRNA is then exported to the cytoplasm. There, the exported pre-miRNA or exogenous dsRNA is cleaved by the enzyme Dicer into a ~22-nucleotide (nt) duplex known as miRNA or siRNA, respectively. The duplex is then incorporated into the RNA-induced silencing complex (RISC). After removing one strand called the passenger strand, the remaining strand,


[1]  M. Ghildiyal and P. D. Zamore, “Small silencing RNAs: an expanding universe,” Nature Reviews Genetics, vol. 10, no. 2, pp. 94–108, 2009.
[2]  R. W. Carthew and E. J. Sontheimer, “Origins and Mechanisms of miRNAs and siRNAs,” Cell, vol. 136, no. 4, pp. 642–655, 2009.
[3]  S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl, “Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells,” Nature, vol. 411, no. 6836, pp. 494–498, 2001.
[4]  P. D. Zamore, “RNA interference: big applause for silencing in Stockholm,” Cell, vol. 127, no. 6, pp. 1083–1086, 2006.
[5]  D. Castanotto and J. J. Rossi, “The promises and pitfalls of RNA-interference-based therapeutics,” Nature, vol. 457, no. 7228, pp. 426–433, 2009.
[6]  V. Ambros, “The functions of animal microRNAs,” Nature, vol. 431, no. 7006, pp. 350–355, 2004.
[7]  S. Tsuchiya, Y. Okuno, and G. Tsujimoto, “MicroRNA: biogenetic and functional mechanisms and involvements in cell differentiation and cancer,” Journal of Pharmacological Sciences, vol. 101, no. 4, pp. 267–270, 2006.
[8]  P. J. Paddison, A. A. Caudy, E. Bernstein, G. J. Hannon, and D. S. Conklin, “Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells,” Genes and Development, vol. 16, no. 8, pp. 948–958, 2002.
[9]  Y. Zeng, E. J. Wagner, and B. R. Cullen, “Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells,” Molecular Cell, vol. 9, no. 6, pp. 1327–1333, 2002.
[10]  M. T. McManus, C. P. Petersen, B. B. Haines, J. Chen, and P. A. Sharp, “Gene silencing using micro-RNA designed hairpins,” RNA, vol. 8, no. 6, pp. 842–850, 2002.
[11]  D. Siolas, C. Lerner, J. Burchard et al., “Synthetic shRNAs as potent RNAi triggers,” Nature Biotechnology, vol. 23, no. 2, pp. 227–231, 2005.
[12]  A. V. Vlassov, B. Korba, K. Farrar et al., “shRNAs targeting hepatitis C: effects of sequence and structural features, and comparison with siRNA,” Oligonucleotides, vol. 17, no. 2, pp. 223–236, 2007.
[13]  T. Ohgi, Y. Masutomi, K. Ishiyama, H. Kitagawa, Y. Shiba, and J. Yano, “A new RNA synthetic method with a -O-(2-cyanoethoxymethyl) protecting group,” Organic Letters, vol. 7, no. 16, pp. 3477–3480, 2005.
[14]  Y. Shiba, H. Masuda, N. Watanabe et al., “Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the -O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate,” Nucleic Acids Research, vol. 35, no. 10, pp. 3287–3296, 2007.
[15]  K. H. Chung, C. C. Hart, S. Al-Bassam et al., “Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155,” Nucleic Acids Research, vol. 34, no. 7, article e53, 2006.
[16]  J. Wu, A. N. Bonsra, and G. Du, “pSM155 and pSM30 vectors for miRNA and shRNA expression,” Methods in Molecular Biology, vol. 487, pp. 205–219, 2009.
[17]  H. Zhang, F. A. Kolb, L. Jaskiewicz, E. Westhof, and W. Filipowicz, “Single processing center models for human Dicer and bacterial RNase III,” Cell, vol. 118, no. 1, pp. 57–68, 2004.
[18]  Y. Lee, C. Ahn, J. Han et al., “The nuclear RNase III Drosha initiates microRNA processing,” Nature, vol. 425, no. 6956, pp. 415–419, 2003.
[19]  D. H. Kim, M. A. Behlke, S. D. Rose, M. S. Chang, S. Choi, and J. J. Rossi, “Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy,” Nature Biotechnology, vol. 23, no. 2, pp. 222–226, 2005.
[20]  L. Peters and G. Meister, “Argonaute proteins: mediators of RNA silencing,” Molecular Cell, vol. 26, no. 5, pp. 611–623, 2007.
[21]  A. L. Jackson and P. S. Linsley, “Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application,” Nature Reviews Drug Discovery, vol. 9, no. 1, pp. 57–67, 2010.
[22]  G. Geiss, G. Jin, J. Guo, R. Bumgarner, M. G. Katze, and G. C. Sen, “A comprehensive view of regulation of gene expression by double-stranded RNA-mediated cell signaling,” Journal of Biological Chemistry, vol. 276, no. 32, pp. 30178–30182, 2001.
[23]  S. A. Veals, C. Schindler, D. Leonard et al., “Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins,” Molecular and Cellular Biology, vol. 12, no. 8, pp. 3315–3324, 1992.
[24]  J. M. Silva, M. Z. Li, K. Chang et al., “Second-generation shRNA libraries covering the mouse and human genomes,” Nature Genetics, vol. 37, no. 11, pp. 1281–1288, 2005.
[25]  B. R. Cullen, “RNAi the natural way,” Nature Genetics, vol. 37, no. 11, pp. 1163–1165, 2005.
[26]  L. M. Li, X. Y. Lin, A. Khvorova, S. W. Fesik, and Y. Shen, “Defining the optimal parameters for hairpin-based knockdown constructs,” RNA, vol. 13, no. 10, pp. 1765–1774, 2007.
[27]  Q. Ge, H. Ilves, A. Dallas et al., “Minimal-length short hairpin RNAs: the relationship of structure and RNAi activity,” RNA, vol. 16, no. 1, pp. 106–117, 2010.
[28]  T. R. Brummelkamp, R. Bernards, and R. Agami, “A system for stable expression of short interfering RNAs in mammalian cells,” Science, vol. 296, no. 5567, pp. 550–553, 2002.


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