Current Approaches on Viral Infection: Proteomics and Functional Validations

Zheng, Jie; Tan, Boon Huan; Sugrue, Richard; Tang, Kai

Nucleic acid-based antisense agents have been widely used to inhibit or knockdown targeted gene expression. These antisense technologies constitute antisense oligonucleotides (ODNs), ribozymes, DNAzymes, and RNA interference (RNAi). Each approach has its own strengths and weaknesses. For instance, although there are some off-target effects, RNAi is regarded as a potent and efficient tool for gene silencing even that its sequence target is in low concentration. Also, it could implement in vivo and in vitro systems, escape the immune responses, and involve in network pathways (Scherer and Rossi, 2003) . RNAi is based on the posttranscriptional gene silencing mechanism, which is induced and mediated by small interfering 21-23 nucleotide dsRNA (siRNA; Novina and Sharp, 2004) . It targets specific sequence and results in degradation and knockdown of this gene expression. Specifically, natural dsRNAs from the cytoplasm are recognized by RNAi DEfective family member-4 (RDE-4), resulting in dicer-mediated cleavage into 21-23 nucleotide siRNA. These cleaved siRNAs are then recruited into a RNA-inducing silencing complex (RISC), which consists of helicase, exonuclease, endonuclease, and homology searching domains. On one hand, the duplex siRNAs are unwound by helicase. On the other hand, one antisense single strand associated with RISC directs its binding with complementary mRNA. And this binding induces and stimulates the ATP-dependent activities of exonuclease and endonuclease, which could cleave the targeted homologous transcript mRNA (Lee and Sinko, 2006; Pushparaj et al., 2008) . Besides natural siRNA, chemically synthesized siRNAs, short hairpin RNA (shRNA), and microRNA (miRNA) could also induce gene silencing in similar mechanisms (Dykxhoorn et al., 2003) . A pioneering trial of siRNA was applied to tomato cell lines and antisense 25 nucleotides specific to 1-aminocyclopro-pane-1-carboxylate oxidase (ACO) mRNA were detected (Hamilton and Baulcombe, 1999) . In the following 2 years, Elbashir et al. (2001) firstly utilized duplexes of 21-nucleotide siRNAs to mediate the degradation of corresponding mRNAs in mammalian cells. It was also regarded as a breakthrough approach in the year 2002 by Science (Couzin, 2002) .