Identification of microRNA-RNA interactions using tethered RNAs and streptavidin aptamers

The molecular mechanisms of cell differentiation, organogenesis and tumorigenesis are directed by the gene regulatory network (GRN). Genes regulating all of these events are primarily post-transcriptionally inhibited by microRNAs (miRNAs), which are short non-coding RNAs that function mainly via binding to the 3' untranslated regions (3'UTRs) of the target mRNAs. Identification of the miRNAs that bind to the genes in the GRN is crucial for understanding the GRN at the transcriptional level. Previously reported biomedical methods of identification for miRNA-RNA interactions primarily focused on the miRNA response elements (MRE) within the target RNA sequences, but the cognate miRNAs cannot identify all the miRNAs regulating a single target gene and cannot be used for in vivo experiments. In this study, we report a novel and efficient way to identify miRNAs that bind to a specific RNA sequence utilizing tethered RNA and streptavidin aptamers, which were previously employed for protein-RNA interaction research. We developed an optimized RNA pull-down assay with a combination of UV-crosslinking, streptavidin aptamers and magnetic beads, and with this method we identified the miRNAs binding to the 3'UTR of krüpple-like factor 4 (KLF4) in mouse dental papilla cells, as predicted by bioinformatic analysis, as well as the enrichment of the members in the let-7 miRNA family in the same cells. Furthermore, we developed an optimized method for the staining of streptavidin aptamers in vivo. Our method, which takes advantage of RNA aptamers, can be a powerful tool for direct and high-throughput identification of all of the miRNAs interacting with the target RNAs.