ShRNAs design

Moore, C.B., Guthrie, E.H., Huang, M.T., Taxman, D.J., 2010. Short hairpin RNA (shRNA) design, delivery, and assessment of gene knockdown. Methods Mol. Biol. 629, 141-158. 

Select five shRNA sequences for your target gene by using Web resources (see Subheading 3.1.1, step 1). As the HI promoter is used, siRNA sequences may start with any nucleotide. Design a pair of shA and shB complementary oligonucleotides for each siRNA sequence. 

The design of shRNAs most commonly features a 19-21 bp duplex that is joined by a 4-9 nt loop sequence. The transcript is typically expressed by an RNA polymerase III (pol III) promoter, usually U6 or HI. Pol III promoters are ideal for this application for several reasons ... 

They can be designed to start and stop transcription at specific locations to create the requisite shRNA. 

An alternative vector-driven RNAi tool is patterned after the endogenously expressed miRNAs. Engineered miRNA transcripts can be driven by pol II promoters and therefore have the potential to be expressed in a tissue- or temporal-specific manner. As with shRNAs, miRNA-based constructs can be delivered to cells via viral vectors. The miRNAs are designed to perfectly match their targets so that the mechanism of gene... 

Compared to 66.5% of gene therapy clinical trials aiming at the treatment of cancer, only 2 of the 12 siRNA clinical trials are designed for cancer therapy, indicating the siRNA cancer therapy is still in its infancy. Same as the therapeutic application of pDNA, the major bottleneck for a successful siRNA therapy is the efficient delivery. Scientists have gained extensive experiences in the delivery of pDNA and these experiences can be utilized for the delivery of plasmid-based shRNA. However, the same experience cannot be transferred to siRNA directly since siRNA is very different from pDNA in terms of the molecular weight, molecular topography, and in vivo stability. In addition, compared to pDNA, siRNA is more difficult to be condensed into nanosized complex. Nevertheless, many nonviral vectors have been developed for siRNA delivery and one of them was recently approved for phase I clinical trial. 

Fig. 1. Model of asymmetric RISC activation. RNAi transgenes produce shRNA molecules as shown. The strand antisense to the target mRNA is black, and the sense strand is gray. Dicer processes shRNA to siRNA, a duplex with 5 phosphorylated ends and 2-nt 3 overhangs. RISC may interact with siRNA at either end, but incorporates the strand it reads only in a 5 to 3 direction. For effective RNAi, RISC should be biased to acquire the antisense strand. This can be accomplished by designing the 5 end of the antisense strand to be A/U rich, while simultaneously making the other end of the siRNA molecule G/C rich. This tips the balance toward preferential uptake of the antisense strand and results in knockdown of the targeted gene.

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