Variables and Strategies in Development of Therapeutic Post-Transcriptional Gene Silencing Agents

Figure 3

RNAi mechanism. An expressed RNA hairpin (shRNA) is cleaved first by Dicer III to a double-stranded RNA of 21 nt with 5′ phosphorylated ends. A pri-miRNA is processed in the nucleus into a pre-miRNA by Drosha, leaves the nucleus, and is further processed by Dicer in the cytoplasm or as part of RISC. Or a transfected or transduced siRNA is phosphorylated at each 5′ end. The short dsRNAs are incorporated into the RISC complex, and the antisense strand (guide strand) is selected on the basis of engineering weaker 5′ energy than 3′ energy. The passenger strand is displaced. The guide strand is organized into RISC as an A-form α-helix within Ago2, which is the RNA endonuclease of RISC. By diffusion limitations, loaded RISC searches for a complimentary partner to its antisense element in the transcriptosome. Upon collision, kissing complex formation and full annealing, the target RNA is positioned for endonuclease cleavage by Ago2. After cleavage, it is thought that ATP hydrolysis occurs, which provides helicase energy to strip the products from the Ago2 cavity in order to prevent product inhibition on RNAi. Product release then frees the charged RISC to seek other target mRNAs for subsequent rounds of Michaelis-Menten turnover.