Generation of efficient miRNA inhibitors using tough decoy constructs

Over the last decade a previously unappreciated mechanism of gene regulation has been uncovered that is mediated by a large class of small noncoding RNAs known as microRNAs (miRNAs), and this mechanism is utilized by organisms ranging from plants to humans. MiRNAs are important downregulators of gene expression and are seen to be dysregulated in disease development. Thus inhibition of aberrantly upregulated miRNAs as a therapeutic approach has become a promising field. Many models of miRNA inhibitors currently exist, with decoy models being the most successful in current research. A promising inhibition model is the tough decoy (TuD) RNAs inhibitor, which uses antisense sequences to bind to target miRNAs, preventing them from binding to their endogenous targets. Since the TuD inhibitors have the ability to be successfully used in vitro and in vivo studies, this is a covetable inhibition method. In this chapter, we introduce how to design and generate miRNA tough decoy inhibitors with an adeno-associated viral construct. TuD inhibitors will have two miRNA binding sites. The TuD will include stem sequences, a miRNA binding site, and linkers. In vitro validation experiments to confirm the effectiveness of the TuD to inhibit miRNA are described. We also propose some practical approaches for making a TuD for miRNA of interest. We hope this chapter facilitates readers to create a simpler method to generate TuD that can be used for miRNA loss of function studies.