MicroRNA Central Loops Figure 3. Correlation and linear regression analysis of central loops of miRNA:MRE duplexes. A, Central loops (or bulges) of miRNA:MRE duplexes were divided into three categories: standard loop, type I decentered loop, and type II decentered loop. Standard loops start at between nt9 and nt11 of the miRNA:MRE duplex counting from the 59 end of the miRNAs. The type I decentered loop starts before nt9, with the type II decentered loop opening after nt11. The loop score is designated according to the algorithm introduced in the Methods. B–D, Relationship between miRNA repression efficiency and the central loop score (B), tail score (C), and minimum free energy (D). Compared with the tail score and minimum free energy, the central loop score has a closer correlative relationship with miRNA repression efficiency (r = 0.646, p,0.001). doi:10.1371/journal.pone.0001719.g003 structures of the miRNA:MRE duplexes by RNAcofold and screened by FindTar version 1.0 and FindTar version 2.0 (Table S4). FindTar version 1.0 uses the old criteria of seed homology plus free energy to predict MREs. FindTar version 2.0 uses new criteria, which adopts the central loop law as an additional criterion for MRE prediction. The MREs predicted by compu- tational algorithms with or without the loop score were compared with the Gold Standard, the results of luciferase activity assays (Table 1). The sensitivity, specificity, and Youden Index were calculated (Table 1). Addition of the central loop scores increased specificity, decreased false positive rates, and reached Youden Index 1. However, it increased false negative rate slightly. Youden Index is a measure of the accuracy of a test based on its false positive and false negative rates, with the ideal value being 1.0. The results confirmed the effect of the loop scores in reducing the false positives and increasing the accuracy and specificity of MRE prediction. Finally, we validated the importance of the loop score using published data[55,56]. Lim LP and coworkers introduced chemically synthesized miR-1, miR-124, miR-373, mir7, mir9, mir122a, mir128a, mir132, mir133a, mir142, mir148b, and mir181a duplexes into HeLa cells, resulting in down-regulation of many mRNAs. The majority of the down-regulated mRNAs exhibit seed homologies with the introduced miRNAs. The experiment also showed that there are many mRNAs which have seeds homologies but are not under the regulation of the transfected miRNAs (false MREs). We used our revised FindTar algorithm (FindTar version 2.0) to calculate loop scores for the miRNA:MREs for all seed- containing mRNAs which were up- or down-regulated by the miRNAs used, and re-analyzed their microarray data (Table 2). Genes down-regulated or up-regulated at a p-value ,0.05 at both 12hr and 24hr were scanned by FindTar for MREs of miR-1, miR- 124, miR-373, mir7, mir9, mir122a, mir128a, mir132, mir133a, mir142, mir148b, or mir181a. The MRE criteria for FindTar version PLoS ONE | www.plosone.org 5 March 2008 | Volume 3 | Issue 3 | e1719