[Show abstract][Hide abstract] ABSTRACT: Target prediction for animal microRNAs (miRNAs) has been hindered by the small number of verified targets available to evaluate the accuracy of predicted miRNA-target interactions. Recently, a dataset of 3,404 miRNA-associated mRNA transcripts was identified by immunoprecipitation of the RNA-induced silencing complex components AIN-1 and AIN-2. Our analysis of this AIN-IP dataset revealed enrichment for defining characteristics of functional miRNA-target interactions, including structural accessibility of target sequences, total free energy of miRNA-target hybridization and topology of base-pairing to the 5' seed region of the miRNA. We used these enriched characteristics as the basis for a quantitative miRNA target prediction method, miRNA targets by weighting immunoprecipitation-enriched parameters (mirWIP), which optimizes sensitivity to verified miRNA-target interactions and specificity to the AIN-IP dataset. MirWIP can be used to capture all known conserved miRNA-mRNA target relationships in Caenorhabditis elegans at a lower false-positive rate than can the current standard methods.