MicroRNA Central Loops Figure 4. Mutation assays. Site-directed mutagenesis of miR-17-5p (A) , miR-372 (B), pRL-VEGF-Con1 (D), and pRL-VEGF-Con2 (E) caused movement of the standard loops of miRNA:MRE duplexes, forming Type I and Type II decentered loops instead, while the free energy of miRNA:MREs was kept at similar levels. COS-7 cells were transfected with wild type or mutated miRNAs and different report vectors. The levels of luciferase activity decreased significantly due to the change in loop location (C and F). doi:10.1371/journal.pone.0001719.g004 1.0 includes seed homologies and free energy ,215, and FindTar version 2.0 includes seed homologies, free energy ,215, and loop score $20. The total number of bases in the 39-UTR of the seed- containing mRNAs which were up- or down-regulated by the miRNAs used, were counted. The ratio of bases per MRE-Up/ bases per MRE-D was used to evaluate the specificity of miRNA- mediated gene down regulation. Bases per MRE were calculated by the total number of bases in the 39-UTR sequence of all affected mRNAs divided by the number of total MREs in these mRNAs. If all the mRNAs had seed homologies with the introduced miRNAs just by chance (the extreme case), bases per MRE-Up should be equal to bases per MRE-D. In this case, the ratio of bases per MRE-Up/bases per MRE-D would be close to 1, and the down-regulation of the messages would not have a relationship with the miRNAs. Otherwise, the ratio would be over 1 and the down-regulation of the messages would be considered at least partially mediated by the miRNAs used. This would be due to the presence of more MREs in the down-regulated mRNAs than in the up-regulated mRNAs, because of the specificity of the MREs. In this report, all the ratios are over 1.5, as shown in Table II. Furthermore, the ratios in group screened with the loop scores (L) are higher than those in unscreened group (NL). These results suggest that, besides seed sequence, loop scores also affect the specificity of miRNA-mediated gene regulation. Discussion In the past few years, complementarity between miRNA and the 39-UTR of target mRNAs, the conservation of target 39-UTR sequences across different species, and the thermodynamics of the miRNA:MREs were widely used as the criteria to predict MREs in different computational algorithms, including miRanda [34,37], TargetScan [31], DIANA-microT [32], and RNAhybrid [35]. The number of binding sites, the distance between sites, the cost of disruption to existing mRNA UTR secondary structure [57] and target-site accessibility [58] are also included as factors which might affect the accuracy of MRE prediction. However, the high levels of false positive prediction and the poor overlap of the predicted MREs between different computational algorithms were still common problems. We believed this was because some PLoS ONE | www.plosone.org 6 March 2008 | Volume 3 | Issue 3 | e1719