Defining the optimal parameters for hairpin-based knockdown constructs.

Induction of gene silencing using intracellularly expressed silencing triggers has been explored for large-scale loss-of-function screening, creation of knockdown cell lines or knockdown animals, and disease intervention. In all of these applications, the use of highly potent silencing constructs can maximize the possibility of obtaining target knockdown and thereby is intrinsically important for the chance of success. Several attempts have been made to improve the potency of a silencing construct. Results published in high profile journals such as Nature Biotechnology and Nature Genetics suggest that shRNAs with a 29-nucleotide (nt) stem is much more potent than shRNAs with a 19-nt stem, and miR30-based silencing constructs are much more potent than shRNA-based constructs. In this study, we systematically investigated several parameters, including the use of shRNA- or miR30-based scaffolds, the length of shRNA, and the selection of shRNA sequences for their impact on the knockdown efficiency of a silencing construct. Our studies revealed that the optimal configurations for a potent silencing trigger could be an shRNA with a 19-nt stem and a 9-nt loop. By comparing properties that favor the functional shRNAs and siRNAs using a set of 190 shRNAs against 19 targets and 360 siRNAs against four targets, we found that the functional shRNAs and siRNAs displayed similar but not identical nucleotide preferences. Based on the characteristic nucleotide preferences in the functional versus the nonfunctional shRNAs, we developed a computer program that outperforms an advanced siRNA selection algorithm for the enrichment of highly functional shRNAs.

[1]  John J Rossi,et al.  Long-term inhibition of HIV-1 infection in primary hematopoietic cells by lentiviral vector delivery of a triple combination of anti-HIV shRNA, anti-CCR5 ribozyme, and a nucleolar-localizing TAR decoy. , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[2]  Thomas Tuschl,et al.  siRNAs: applications in functional genomics and potential as therapeutics , 2004, Nature Reviews Drug Discovery.

[3]  A. Gunasekera,et al.  Development of a tightly regulated U6 promoter for shRNA expression , 2004, FEBS letters.

[4]  Haibin Xia,et al.  siRNA-mediated gene silencing in vitro and in vivo , 2002, Nature Biotechnology.

[5]  H. Paulson,et al.  RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia , 2004, Nature Medicine.

[6]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[7]  M. Staver,et al.  Evaluating hypoxia-inducible factor-1alpha as a cancer therapeutic target via inducible RNA interference in vivo. , 2005, Cancer research.

[8]  G. Hannon,et al.  Germline transmission of RNAi in mice , 2003, Nature Structural Biology.

[9]  Patrick J. Paddison,et al.  Second-generation shRNA libraries covering the mouse and human genomes , 2005, Nature Genetics.

[10]  Michael T. McManus,et al.  A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference , 2003, Nature Genetics.

[11]  Dieter Huesken,et al.  Design of a genome-wide siRNA library using an artificial neural network , 2005, Nature Biotechnology.

[12]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[13]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[14]  B. Cullen.  Transcription and processing of human microRNA precursors. , 2004, Molecular cell.

[15]  K. Taira,et al.  U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells , 2002, Nature Biotechnology.

[16]  Wei Ge,et al.  Synthetic shRNAs as potent RNAi triggers , 2005, Nature Biotechnology.

[17]  A. Fire,et al.  RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[18]  W. Forrester,et al.  A DNA vector-based RNAi technology to suppress gene expression in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Reuven Agami,et al.  A large-scale RNAi screen in human cells identifies new components of the p53 pathway , 2004, Nature.

[20]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[21]  B. Cullen,et al.  Use of RNA polymerase II to transcribe artificial microRNAs. , 2005, Methods in enzymology.

[22]  S. Lowe,et al.  Probing tumor phenotypes using stable and regulated synthetic microRNA precursors , 2005, Nature Genetics.

[23]  A. Reynolds,et al.  Rational siRNA design for RNA interference , 2004, Nature Biotechnology.

[24]  René Bernards,et al.  Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB , 2003, Nature.

[25]  S. Elledge,et al.  A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Anastasia Khvorova,et al.  Functional siRNAs and miRNAs Exhibit Strand Bias , 2003, Cell.

[27]  Theresa A. Storm,et al.  Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways , 2006, Nature.

[28]  B. Cullen,et al.  Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha , 2005, The EMBO journal.

[29]  G. Hannon,et al.  Unlocking the potential of the human genome with RNA interference , 2004, Nature.

[30]  Patrick J. Paddison,et al.  A resource for large-scale RNA-interference-based screens in mammals , 2004, Nature.