Stephen Scaringe

Rational siRNA design for RNA interference.

Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3′-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.

Mechanistic insights aid computational short interfering RNA design.

RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.

[1] Advanced 5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis

Publisher Summary This chapter describes the synthesis of RNA oligonucleotide using 5´-silyl-2´-orthoester approach. For some applications, the RNA is of sufficient purity to use without further processing. After synthesis of an RNA oligonucleotide, the 2´-orthoester protected RNA is water soluble and significantly more stable for degradation than the final fully deprotected RNA product. These features of the 2´- orthoester group enable the RNA to be easily handled in aqueous solutions. The 2´-orthoester groups interrupt secondary structure. This property has made it possible to analyze and purify RNA oligonucleotides of every sequence regardless of secondary structure. This includes 10 to 15 base-long homopolymers of guanosine. Finally, when the RNA is ready for use, the 2´-orthoester groups are completely removed in less than 30 minutes under extremely mild conditions in common aqueous buffers. These unique properties of the 5´-silyl ether and 2´-orthoester protecting groups have made it possible to routinely synthesize high-quality RNA oligonucleotides.

Preparation of 5'-silyl-2'-orthoester ribonucleosides for use in oligoribonucleotide synthesis.

The recent discovery that small interfering RNAs (siRNAs) induce gene suppression in mammalian cells has sparked tremendous interest in using siRNA‐based assays and high‐throughput screens to study gene function. As a result, research programs at leading academic and commercial institutions have become a substantial and rapidly growing market for synthetic RNA. Important considerations in synthesizing RNA for biological gene function studies are sequence integrity, purity, scalability, and resistance to nucleases; ease of chemical modification, deprotection, and handling; and cost. Of the well‐established RNA synthesis methods, 2′‐ACE chemistry is the only one that meets all of these criteria. 2′‐ACE technology employs a unique class of silyl ethers to protect the 5′‐hydroxyl, in combination with an acid‐labile orthoester protecting group on the 2′‐hydroxyl (2′‐ACE). 2′‐ACE‐protected phosphoramidite monomers are joined using standard solid‐phase technology to achieve RNA synthesis at efficiencies rivaling those for DNA. This unit describes the synthesis of standard 5′‐silyl‐2′‐ACE‐protected phosphoramidites.

siRNA as a tool for streamlining functional genomic studies

Abstract RNA interference (RNAi) has the potential to accelerate greatly the pace of discovery biology. The active RNAi intermediate is the small interfering RNA (siRNA), a discrete nucleic acid duplex that can be generated by several methods and used to directly silence gene expression. The choice of methods employed depends largely on the research or therapeutic objective. In most cases, rational design offers several advantages over random design, including greater predictability of function, higher silencing potency and longer duration of suppression. Of the production methods, chemical synthesis provides the fastest production capability, the highest purity and the easiest scalability for high-throughput strategies. Effective coupling of several methods gives the greatest potential for the application of RNAi across functional genomic and target validation studies. Furthermore, the coupling of RNAi with cellular profiling technologies will provide opportunities to streamline drug discovery significantly.