Submitted to: Plant Cell Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2008
Publication Date: 2/26/2009
Citation: Park, W., Zhai, J., Lee, J. 2009. Highly efficient gene silencing using perfect complementary artificial miRNA targeting AP1 or heteromeric artificial miRNA targeting AP1 and CAL genes. Plant Cell Report. 28:469-480. Interpretive Summary: Gene silencing is a useful technique in plants and animals for elucidating biological function of genes by knocking down their expression. An example of a naturally occurring gene silencing technique is carried out by microRNAs (miRNAs). Under natural cell conditions, specific miRNAs are processed from a precursor RNA to target the expression of a specific gene. A recently developed artificial microRNA (amiRNA) exploits this natural endogenous gene silencing mechanism by substituting a miRNA sequence in a precursor RNA with a custom amiRNA sequence. It is reported that the overall success rate of amiRNA-based gene silencing is close to 75% and it suggests that the amiRNA design is not maximally optimized. Also, many important genes are expressed as multi copy genes that share sequence homology in which case a single amiRNA-mediated gene silencing is problematic. For these reasons, we investigated whether the effectiveness of an existing amiRNA approach could be improved by simple modifications in amiRNA design. In this study, we showed that an amiRNA with perfect sequence complementation to a target gene and designed with a mismatch in the starting point with its opposite strand acted as a highly potent gene silencing agent, achieving a desired null mutation effect. We also demonstrated that simultaneous gene silencing of members of a gene family were more consistently achieved by using heteromeric tandem amiRNAs than by using single amiRNAs that target conserved sequences. Our finding provides experimental evidence that designing amiRNAs with perfect complementarity to the target genes in combination with 5’ instability would be a new effective and specific silencing agent.
Technical Abstract: Gene silencing is a useful technique for elucidating biological function of genes by knocking down their expression. Recently developed artificial microRNAs (amiRNAs) exploit an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs that target transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed such that they have a few mismatching nucleotides with respect to their target sites as well as within mature amiRNA duplexes. In this study, we performed an analysis in which the conventional and modified form of an amiRNA was compared side by side. We showed that the amiRNA containing 5’ mismatch with its amiRNA* and perfect complementarity to its target gene acted as a highly potent gene silencing agent against AP1, achieving a desired null mutation effect. In addition, a simultaneous silencing of two independent genes, AP1 and CAL was tested by employing a multimeric form of amiRNAs. Advantages and potential disadvantages of using amiRNAs with perfect complementarity to the target gene are discussed. The results presented here should be helpful in designing more specific and effective gene silencing agents.