Book contents
- Frontmatter
- Contents
- Foreword by Andrew Fire
- Foreword by Marshall Nirenberg
- List of Contributors
- Introduction
- Section one Basic RNAi, siRNA, microRNAs and gene-silencing mechanisms
- Section two Design, synthesis of siRNAs
- 6 Design and synthesis of small interfering RNA (siRNA)
- 7 Automated design and high throughput chemical synthesis of siRNA
- 8 Rational design of siRNAs with the Sfold software
- 9 Enzymatic production of small interfering RNAs
- Section three Vector development and in vivo, in vitro and in ovo delivery methods
- Section four Gene silencing in model organisms
- Section five Drug target validation
- Section six Therapeutic and drug development
- Section seven High-throughput genome-wide RNAi analysis
- Index
- Plate section
- References
7 - Automated design and high throughput chemical synthesis of siRNA
Published online by Cambridge University Press: 31 July 2009
Edited by
Book contents
- Frontmatter
- Contents
- Foreword by Andrew Fire
- Foreword by Marshall Nirenberg
- List of Contributors
- Introduction
- Section one Basic RNAi, siRNA, microRNAs and gene-silencing mechanisms
- Section two Design, synthesis of siRNAs
- 6 Design and synthesis of small interfering RNA (siRNA)
- 7 Automated design and high throughput chemical synthesis of siRNA
- 8 Rational design of siRNAs with the Sfold software
- 9 Enzymatic production of small interfering RNAs
- Section three Vector development and in vivo, in vitro and in ovo delivery methods
- Section four Gene silencing in model organisms
- Section five Drug target validation
- Section six Therapeutic and drug development
- Section seven High-throughput genome-wide RNAi analysis
- Index
- Plate section
- References
Summary
Introduction
The application of RNA interference (RNAi) to mammalian cells (Caplen et al., 2001; Elbashir et al., 2001a) has the potential to revolutionize the field of functional genomics and target validation in drug discovery research (Harborth et al., 2001; Tuschl and Borkhardt, 2002). In addition RNAi is also viewed as potential means for nucleic acid-based therapeutic applications. The ability to simply, effectively, and specifically down-regulate the expression of genes in mammalian cells holds enormous scientific, commercial, and therapeutic potential. To ensure success in gene silencing experiments, the design and synthesis of the targeting molecule (the siRNA) must be carefully optimized. The present chapter describes the design criteria for successful gene silencing, followed by a description of recent improvements in high-throughput chemical synthesis of siRNAs, and transcriptome wide gene silencing studies.
Advantages of using RNAi over other technologies for functional genomics
A variety of methods and technologies are available for studying gene function such as gene knockouts and transgenic animal models (Capecchi, 1989; Feil et al., 1996; Jaenisch, 1998; Le and Sauer, 2001; Perkins, 2002), the majority of which are time-consuming and expensive. To facilitate functional genomics in mammalian cells at a transcriptome-wide level, an efficient approach that enables rapid and high throughput analysis is required. Use of synthetic siRNA for RNA interference offers a rapid and cost-effective alternative to previous methods, and can be used to silence expression of several genes simultaneously (Harborth et al., 2001; Kamath et al., 2003; Kamath and Ahringer, 2003).
- Type
- Chapter
- Information
- RNA Interference TechnologyFrom Basic Science to Drug Development, pp. 118 - 128Publisher: Cambridge University PressPrint publication year: 2005
References
(1989). Altering the Genome by homologous recombination. Science, 244, 1288–1292CrossRefGoogle ScholarPubMed
, , , and (2001). Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proceedings of the National Academy of Sciences USA, 98, 9742–9747CrossRefGoogle ScholarPubMed
, , , , and (2003). Genomewide view of gene silencing by small interfering RNAs. Proceedings of the National Academy of Sciences USA, 100, 6343–6346CrossRefGoogle ScholarPubMed
and (2002). RNAi in human cells: Basic structural and functional features of small interfering RNA. Molecular Cell, 10, 549–561CrossRefGoogle ScholarPubMed
, , , , and (2001). Antisense DNAs as multisite genomic modulators identified by DNA microarray. Proceedings of the National Academy of Sciences USA, 98, 9819–9823CrossRefGoogle ScholarPubMed
, and (2003). siRNAs can function as miRNAs. Genes & Development, 17, 438–442CrossRefGoogle ScholarPubMed
, and (2003). Killing the messenger: Short RNAs that silence the gene expression. Nature Reviews Molecular Cell Biology, 4, 457–467CrossRefGoogle ScholarPubMed
, , , , and (2001a). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498CrossRefGoogle Scholar
, and (2001b). RNA interference is mediated by 21 and 22 nt RNAs. Genes & Development, 15, 188–200CrossRefGoogle Scholar
, , , and (2001c). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. European Molecular Biology Organization Journal, 20, 6877–6888CrossRefGoogle Scholar
, , and (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 26, 199–213CrossRefGoogle ScholarPubMed
, , , , and (1996). Ligand activated site specific recombination in mice. Proceedings of the National Academy of Sciences USA, 93, 10887–90CrossRefGoogle ScholarPubMed
, , , , and (2002). Evaluating the specificity of Antisense oligonucleotide conjugates: A DNA array analysis. Journal of Biological Chemistry, 277, 22980–22984CrossRefGoogle ScholarPubMed
, , , , , , , and (2002). Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3′-ends of siRNAs. Antisense and Nucleic Acid Drug Development, 12, 301–309CrossRefGoogle ScholarPubMed
, , , and (2001). Identification of essential genes in cultured mammalian cells using small interfering RNAs. Journal of Cell Science, 114, 4557–4565Google ScholarPubMed
, , , and (2002). Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Research, 30, 1757–1766CrossRefGoogle ScholarPubMed
, , , , , , , and (2003). Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology, 21, 635–637CrossRefGoogle ScholarPubMed
, , , , , , , , , , , and (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, 421, 231–237CrossRefGoogle ScholarPubMed
and (2003). Genome-wide RNAi screening in Caenorhabditis elegans. Methods, 4, 313–321CrossRefGoogle Scholar
, and (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell, 115, 209–216CrossRefGoogle ScholarPubMed
and (2003). The activity of siRNA in mammalian cells is related to structural target accessibility: A comparison with antisense oligonucleotides, Nucleic Acids Research, 31, 4417–4424CrossRefGoogle ScholarPubMed
, and (2003). High-throughput selection of effective RNAi probes for gene silencing. Genome Research, 13, 2333–2340CrossRefGoogle ScholarPubMed
and (2001). Conditional gene knockout using Cre Recombinase. Molecular Biotechnology, 3, 269–275CrossRefGoogle Scholar
, and (2003). Comparison of the suppressive effects of Antisense oligonulceotides and siRNAs directed against the same targets in mammalian cells. Antisense and Nucleic Acid Drug Development, 13, 1–7CrossRefGoogle Scholar
, and (2001). ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell, 107, 309–321CrossRefGoogle ScholarPubMed
(2002). Functional Genomics in mouse. Functional & Integrative Genomics, 3, 81–91CrossRefGoogle Scholar
, , , , (2001). Reliable chemical synthesis of oligoribonucleotides (RNA) with 2′-O-[(Triisopropylsilyl)oxy]methyl (2′-O-tom) protected phosphoramidites. Helvetica Chimica Acta, 84, 3773–37953.0.CO;2-E>CrossRefGoogle Scholar
Pitsch, S., Weiss, P. A., Jenny, L. (1999a). US Patent 5, 986,084
, , , and (1999b). Fast and reliable automated synthesis of RNA and partially 2′-O-Protected precursors (“caged RNA”) based on two novel, orthogonal 2′-O-protecting groups. Helvetica Chimica Acta, 82, 1753–17613.0.CO;2-Y>CrossRefGoogle Scholar
Pitsch, S. and Weiss, P. A. (2001). Chemical Synthesis of RNA-Sequences with 2′-O-[(Triisopropylsilyl)oxy]methyl-protected Ribonucleoside Phosphoramidites (=TOM-Phosphoramidites) (Unit; 3.9). In: Current Protocols of Nucleic Acid Chemistry Ed. S. Beaucage, John Wiley & Sons, Inc., New York, NY
, and (2003). Small RNAs with imperfect match to endogenous mRNA repress translation: Implications for off-target activity of small inhibitory RNA in mammalian cells. Journal of Biological Chemistry, 278, 44312–44319CrossRefGoogle ScholarPubMed
, , , , and (2003). Asymmetry in the assembly of the RNAi Enzyme Complex. Cell, 115, 199–208CrossRefGoogle ScholarPubMed
, , , , and (2003). Specificity of short interfering RNA determined through gene expression signatures. Proceedings of the National Academy of Sciences USA, 100, 6347–6352CrossRefGoogle ScholarPubMed
, and (1999). RNA Aptamers as effective protein antagonists in a multicellular organism. Proceedings of the National Academy of Sciences USA, 96, 10033–10038CrossRefGoogle Scholar
and (1981). Identification of common molecular subsequences. Journal of Molecular Biology, 147:195–197CrossRefGoogle ScholarPubMed
and (2002). Small interfering RNAs: A revolutionary tool for the analysis of gene function and gene therapy. Molecular Interventions, 2, 158–167CrossRefGoogle ScholarPubMed
, and (2003). microRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proceedings of the National Academy of Sciences USA, 100, 9779–84CrossRefGoogle ScholarPubMed