siRNA design. Methods and protocols (Q351523)

RNA interference (RNAi) is a recently discovered methodology which can be used to understand the function of genes by controlled variations in expressions. Subsequent to intensive research of natural sRNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), a new field emerged, the design of (artificial) siRNAs that could mimic the mode of action of the natural sRNAs and add a valuable new angle to therapeutics. This book ' edited by D.J. Taxman is a valuable collection of 20 chapters covering aspects from values for parameters used in tools for designing siRNAs to screening methods for evaluating the efficiency of the designed siRNA. In the first chapter the author reviews several tools and the corresponding parameters, presenting in detail parameter impacts on the siRNA designs. The stability of the duplex, the frequency of occurrence or avoidance of motifs and the secondary structure of siRNA and/or target are discussed. The second chapter highlights statistical and analytical techniques that can make the selection of siRNAs more efficient. The authors review siRNA design guidelines and present new supervised and unsupervised machine learning methods such as decision tree learning, Bayes' theorem and average silencing probability used as a classifier trained on known effective siRNAs. The optimal siRNA candidates are then passed through a hidden Markov model which uses frequencies of combinations of dinucleotides as selection criteria. The third and fourth chapters change the focus from siRNA properties to the analysis of the target and off-target effects. The characteristics and mode of use of a new server siDirect 2.0 are presented in detail. In the fourth chapter the authors describe the issues derived from target site accessibility and present a bead based approach to design and screen siRNAs with a change of accessible target. The approach is exemplified on highly structured targets and positive sense RNA viruses. The fifth chapter proposes yet another method to avoid off target effects of designed siRNAs by incorporating beneficial chemical modifications into specified positions. The literature on this topic is extensively reviewed and a quick guide for this method is also included. In Chapter 6 the authors review other factors that affect the functionality of designed siRNAs, like length of available sequences for the siRNA targets and susceptibility to degradation in the cell. The authors also propose the incorporation of unlocked nucleobase analogs as a method to significantly reduce off target activity. The seventh chapter describes a technical approach to diminish the earlier presented factors for reduced efficiency of siRNAs, the asymmetric shorter duplex RNA (asiRNA). It is designed as an alternative structure that can target RNAi and is characterised by a shorter than the 19nt duplex region and an asymmetric 3' overhang. The authors also show that the asiRNAs are efficient for gene silencing through the AGO2 dependent pathway in mammals. Chapter 8 presents a chemical modified siRNA named fork like siRNA (fsiRNA) containing mis-matches at the 3' end region of the sense strand. In Chapter 9 the author presents a purely bioinformatics strategy for dual targeting siRNA duplexes, i.e., both strands of the duplex are active and down-regulate their respective target genes. Chapter 10 is built as a review on designing and validating siRNAs immunity activating properties. Another design method is reviewed in Chapter 11 where the authors present in detail the endoribonuclease prepared siRNAs (esiRNAs) as an efficient alternative to traditional siRNAs. Created using enzymatic generation of siRNA pools, these esiRNAs are built to target one transcript, thus reducing the off target effects mentioned earlier. Returning into the bioinformatics field, the authors of Chapter 12 review the current state of art of combinatorial RNAi (coRNAi), where multiple siRNA-target interactions are handled simultaneously. Continued to Chapter 13, with the description of monoRNAi, using siRNAs like short hairpin RNAs or artificial miRNAs as examples, the topic is completed with a well-documented description of extended shRNAs and long hairpin RNAs. Another approach to maximize the efficiency of siRNAs is proposed in Chapter 14 where the authors present bifunctional short hairpin RNAs (bishRNAs). These siRNAs can attract both cleavage dependent and cleavage independent RISC leading to both translational repression and mRNA degradation as modes of action. In Chapter 15 the authors present approaches to design specific siRNAs on the left and on the right hand loop, processed by the RNAi machinery with independent efficiencies. Chapter 16 summarises the different strategies and usages of long dsRNA which can be a trigger for the interferon pathway. In Chapter 17 the authors change the focus from the design of siRNAs to the analysis of RNAi reagents for invertebrate model organisms and human disease vectors. Using A. gambiae as a model, a bioinformatics pipeline to design genomic wide RNAi library is also presented. In Chapter 18 the author presents in detail a method to construct shRNA expression plasmids for silk worm cell lines. Chapter 19 reviews the design of effective amiRNA, multimeric amiRNA, against plant viruses. Using the wheat streak mosaic virus as example, the authors show that one policistronic amiRNA precursor is enough to prevent viral replication. The book concludes with Chapter 20 where the author describes the down-regulation of plant genes using miRNA induced gene silencing. Secondary siRNAs derived from a miRNA targeted transcript (miR173 is used as example) are shown to be efficient in regulating the gene expression. Through style and scientific information the book is aimed at researchers in the RNAi field, but its accessible delivery of contents makes it a good starting point for younger researchers in this topic.