**2. Mechanisms of RNAi**

The RNAi phenomenon was first found in antisense hybridization of antisense RNA and mRNA. However, further studies showed that the sense strand could also result in reduced gene expression (Guo& Kemphues,1995). Subsequent studies demonstrated that each strand

RNA Interference for Tumor Therapy 63

small groove of dsRBD N-terminal α-helix connect with five nucleotides of RNA. Region 2: a stem loop between β1and β2 of dsRBD connects with 2-5 nucleotides of RNA in the second small groove through 2'-OH. Region 3: the α-helix at the C-terminal of dsRBD travels across the large groove of RNA and connects with the phosphodiester bond in RNA (Ryter &

The long dsRNA that enters cells is processed by Dicer into double-stranded siRNA, and then bind with Dicer, trans-activitor response region RNA-binding protein (TRBP), and protein kinase R (PKR)-activating protein (PACT) to form a RNA-induced silencing complex

RNAi induced by completely processed single-stranded siRNA (mostly exogenous) can be directly installed into RISC without the participation of TRBP/PACT-Dicer complex. However, in other RNAi processes, RNAi cannot be installed into RISC without the participation of TRBP/PACT-Dicer complex. Through TRBP/PACT, siRNA-TRBP/PACT-Dicer complex binds with Argonaute 2 (Ago2/EIF2C2) to form RISC loading complex (RLC) (Robb&Rana,2007). RLC initiates disentanglement of siRNA and distinguishs the guide strand and the passenger strand. The guide strand 5'-end has the property of a low thermodynamic stability and can be bound into RISC by priority(Tomari *et al,* 2004). The passenger strand is cleaved by Ago 2 and separated from RLC (Matranga *et al,* 2005). The specific sequence for binding between the RISC-mediated RNA guide strand and target mRNA is mainly located in the "seed" or "core" region of 2-8 nucleotides of siRNA 5'-end (Ma *et al,* 2005). mRNA oligonucleotide is cleaved from the middle in the presence of Ago2

Ago 2 is the only protein that has the endonuclease function in RLC, mainly consisting of an N-terminal domain, a PAZ domain, a middle domain and a PIWI domain, of which the Nterminal domain, middle domain and PIWI domain form a crescent basin structure, with one "arm" supporting the PAZ domain above the "basin" (Song *et al,* 2004;Yuan *et al,* 2005). This structure forms a groove carrying positive charge. The N-terminal domain and PIWI domain form the small groove, which can bind with nucleic acid. The PAZ domain and the crescent "basin" form the large groove, which can contain double strands and locate the easily broken phosphate bond directly to the catalytic site of Rnase H sheet on the PIWI domain. Ago 2 can melt siRNA, forming single-stranded antisense siRNA complementary to target mRNA. The Rnase H PIWI domain of Ago 2 is located in the 5'-end, and the PAZ domain can recognize siRNA3'-end. When siRNA binds with complementary target mRNA, Ago 2 begins digesting mRNA, forming RNA fragments with 5'-carboxylate and 3' phosphate to degrade mRNA of the endogenous target gene, thus preventing gene expression. During the process of degradation, mRNA comes in between the N-terminal and the PAZ domain and goes out between the PAZ domain and the middle domain. When mRNA moves between the PAZ domain and the "crescent basin", it is cleaved by the active

RISC can be recycled. When a target mRNA is degraded, RISC can continue to degrade other target mRNA, thus amplifying the PTGS effect of the target gene (Gregory *et al,* 2005) and this amplification can maintain for few days in dividing cells. Obviously, it is advantageous over other therapeutic methods, and is a potent method for specific inhibition of gene expression. In addition, a few studies(Meister *et al,* 2005) also found that RISC also

and loses the expression function (Schwarz *et al,* 2004; Hammond,2005).

Schultz, 1998).

(RISC) (Hammond *et al,* 2000).

site of the PIWI domain.

of dsRNA can induce RNAi, but only few types of dsRNA can lead to potent interference effect (Fire *et al,* 1998). The molecules that can trigger RNAi include small interference RNA (siRNA), microRNA (miRNA) and small hairpin RNA (shRNA). The mechanisms of RNAi triggered by these molecules are not exactly the same.
