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• A proximity-enabled miRNA inhibition approach was reported. • The approach relies on the enzymatic inactivation of Dicer by bifunctional small molecules. • miR-21 inhibitor was successfully developed using this method. • This approach could be applied to generate bifunctional inhibitors for other miRNAs. microRNAs (miRNAs) are considered as master regulators of biological processes. Dysregulation of miRNA expression has been implicated in many human diseases. Driven by the key biological roles and the therapeutic potential, developing methods for miRNA regulation has become an intense research area. Due to favorable pharmacological properties, small molecule-based miRNA inhibition emerges as a promising strategy and significant progresses have been made. However, it remains challenging to regulate miRNA using small molecules because of the inherent difficulty in RNA targeting and inhibition. Herein we outline the workflow of generating bifunctional small molecule inhibitors blocking miRNA biogenesis through proximity-enabled inactivation of Dicer, an enzyme required for the processing of precursor miRNA (pre-miRNA) into mature miRNA. By conjugating a weak Dicer inhibitor with a pre-miRNA binder, the inhibitor can be delivered to the Dicer processing site associated with the targeted pre-miRNA, and as a result inhibiting Dicer-mediated pre-miRNA processing. This protocol can be applicable in producing bifunctional inhibitors for different miRNAs. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We describe here a straightforward method to generate retroviral miRNA sponge constructs using a single directional ligation reaction. This approach allows generation of sponges containing more than 20 miRNA binding sites. We provide a basis for the design of the sponge constructs with respect to the sequence of the miRNA binding site and the sequences flanking the miRNA binding sites. In-silico validation approaches are presented to test the predicted efficiencies of the sponges in comparison to known target genes. In addition, we describe in vitro validation experiments to confirm the effectiveness of the miRNA sponges. Finally, we describe how the here described procedure can be adapted to easily generate sponges that target multiple miRNAs simultaneously. In summary, our approach allows rapid generation of single or combination miRNA sponges that can be used for long-term miRNA loss-of-function studies. [Copyright &y& Elsevier]